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Ex vivo bone marrow purging using BPD-mediated photodynamic therapy Yip, Stephen 1998

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EX  VIVO B O N E M A R R O W P U R G I N G USING BPD- M E D I A T E D PHOTODYNAMIC THERAPY by Stephen Y i p B. Sc. T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 1990  A THESIS SUBMITTED INPARTIAL FULFILMENT O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OFPHILOSOPHY  ,  in THE FACULTY OF GRADUATE DEPARTMENT OFMICROBIOLOGY  STUDIES  &  IMMUNOLOGY  W e accept this thesis as c o n f o r m i n g to the r e q u i r e d standard  T H E TJNIVERSITY OF BRITISH September, 1998 © Stephen Y i p , 1998  COLUMBIA  In presenting this thesis in partial fulfilment o f the requirements f o r an advanced degree at T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree that the L i b r a r y shall make it freely available for reference and study. I further agree that p e r m i s s i o n f o r extensive c o p y i n g o f this thesis f o r scholarly purposes may be granted b y the H e a d o f m y Department or by his or her representatives. It is u n d e r s t o o d that c o p y i n g or publication o f this thesis f o r financial gain shall not be a l l o w e d without m y written permission.  Department c f M i c r o b i o l o g y & I m m u n o l o g y The University of British C o l u m b i a 6174 University B l v d Vancouver, Canada V 6 T 1Z3  Date: 18 September 1998  A B S T R A C T  P h o t o d y n a m i c therapy ( P D T ) u s i n g the second generation photosensitiser b e n z o p o r p h y r i n derivative m o n o a c i d ring- A ( B P D , Verteporfin®) offers an attractive alternative to p u r g i n g o f contaminating neoplastic cells d u r i n g autologous haematopoietic stem c e l l transplantation. Enhancement o f P D T c y t o t o x i c i t y was attempted u s i n g t w o independent approaches: c o m b i n a t i o n treatment w i t h d o x o r u b i c i n ( D o x ) and selective protection o f n o r m a l haematopoietic cells using the tetrapeptide N - A c S D K P . T h e m u r i n e leukaemic c e l l line L 1 2 1 0 was 45 x more susceptible to the sequenced c o m b i n a t i o n regimen o f 1 h 2.5 u M D o x incubation f o l l o w e d by P D T mediated b y 5.0 ng/ml B P D and 15 J / c m red light (Dox-> P D T ) than n o r m a l m u r i n e haematopoietic cells. T h e 2  significant enhancement i n cytotoxicity was dependent on the concentration o f B P D used as w e l l as on the sequence o f treatment. S p e c i f i c a l l y , it was observed w i t h 5.0 ng/ml but not w i t h 2.5 ng/ml B P D and o n l y w h e n D o x was used before P D T . T h e reverse sequence o f P D T - > D o x and simultaneous D o x / P D T treatment were not associated w i t h enhanced k i l l i n g . Interestingly, L 1 2 1 0 cells were m u c h more susceptible to the c o m b i n a t i o n therapy than normal D B A / 2 haematopoietic progenitor cells w h i c h offered interesting therapeutic i m p l i c a t i o n s . B P D uptake and c e l l u l a r G S H content, appeared not to be responsible for the potentiation o f L 1 2 1 0 k i l l i n g i n the D o x - > P D T sequence. N e x t , the potential o f selective stem cell protection in B P D - mediated P D T was investigated. Preincubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M N - A c S D K P for 1.5 h significantly protected resultant c o l o n y f o r m a t i o n f r o m P D T b y a factor o f 1.5- 2 over cells that were incubated w i t h control peptides or w i t h tissue culture m e d i u m . Interestingly, L 1 2 1 0 cells were not protected by N - A c S D K P and the c o n t r o l peptides. H o w e v e r , N - A c S D K P mediated photoprotection d i d not appear to extend to earlier murine haematopoietic cells and stem cells as demonstrated b y the long- term bone m a r r o w culture ( L T B M C ) assay. T h e same f i n d i n g s o f differential photoprotection were also demonstrated in h u m a n haematopoietic cells. T h e m e c h a n i s m o f protection appeared to be mediated by i n h i b i t i o n o f progression to S phase o f the c e l l c y c l e since depletion o f c y c l i n g D B A / 2 bone m a r r o w cells w i t h 50 p M cytosine arabinoside (ara- C ) resulted i n cells more tolerant to subsequent P D T cytotoxicity. T h e above two approaches enhanced selective B P D - mediated P D T cytotoxicity and therefore m a y b e o f merit in the c l i n i c a l use o f P D T i n p u r g i n g .  ii  ABSTRACT  II  LIST O F T A B L E S  X  LIST O F F I G U R E S  XII  LIST O F A B B R E V I A T I O N S  XV  ACKNOWLEDGEMENTS  XIX  INTRODUCTION  1  1.1 L E U K A E M I A : HISTORY  1  a. C l i n i c a l observations  1  b. D i a g n o s t i c s and Therapeutics  .2  c. S u p p o r t i v e therapy  4  d. B o n e m a r r o w transplantation.  6  1.2 L E U K A E M I A : T H E S T A T E O F T H E A R T  10  a. C l i n i c a l advances  10  b. D i a g n o s t i c s and therapeutics  10  i. E p i d e m i o l o g y & aetiology  10  i i . N o v e l chemotherapeutics  11  i i i . A n t i b o d y - based diagnostics & therapeutics  13  iv. C e l l - mediated immunotherapy  15  v. M u r i n e models o f h u m a n l e u k a e m i a  16  v i . Gene- based therapeutics  17  c. S u p p o r t i v e therapy  18  i. G- C S F and G M - C S F support f o r chemotherapy- i n d u c e d neutropaenia i i . Infection and haemostasis c o n t r o l  18 19  in  1.3  TRANSPLANTATIONS  OF H A E M A T O P O I E T I C S T E M C E L L S :  CURRENT  STATUS  20  a. B o n e M a r r o w Transplantation ( B M T )  21  i. A l l o g e n e i c bone marrow transplantation ( a l B M T ) 1. H L A m a t c h i n g  21  2. Prevention and the management o f graft- versus- host disease ( G v H D )  22  3. Identification o f m i n o r histocompatibility antigens ( m H C )  23  4. N o v e l indications f o r a l B M T i n nonmalignant and malignant disorders  24  i i . A u t o l o g o u s bone m a r r o w transplantation ( a u B M T ) 1. a u B M T and chronic myelogenous l e u k a e m i a ( C M L ) b. Peripheral b l o o d stem c e l l transplantations c. U m b i l i c a l c o r d b l o o d transplantation  26 28 28  d. Future directions EXPLOITABLE  25 27  i. A u t o l o g o u s transplantation o f P B S C i n cancer patients  1.4  21  '  29  DIFFERENCES B E T W E E N N O R M A L AND L E U K A E M I C  CELLS  29  a. L e u k a e m o g e n e s i s  29  b. Genetic differences  30  c. Differences i n intracellular s i g n a l l i n g and c e l l death pathways  31  d. Response to extracellular regulatory signals  32  e. C e l l surface markers, adhesion properties and the haematopoietic m i c r o e n v i r o n m e n t 3 3 f. Susceptibility to c y t o t o x i c agents 1.5  PURGING  33  IN A U T O L O G O U S S T E M C E L L T R A N S P L A N T A T I O N S  34  a. Introduction  34  b. P u r g i n g strategies  35  c. C o m b i n a t i o n p u r g i n g  36  d. P u r g i n g outside the traditional p a r a d i g m  37  1.6 P H O T O D Y N A M I C T H E R A P Y (PDT) AND ITS  R O L E IN PURGING  37  a. Reactive o x y g e n intermediates ( R O I s ) i n b i o l o g y  37  b. P D T : c y t o t o x i c i t y and b i o l o g i c a l responses  38  iv  c. In v i v o uses o f P D T  40  d. T h e B e n z o p o r p h y r i n derivatives- second generation photosensitisers  40  e. E x v i v o uses o f P D T  41  f. B o n e m a r r o w p u r g i n g  42  g. C l i n i c a l trials  43  O B J E C T I V E S AND R A T I O N A L E  EXPERIMENTAL 2.1  43  PROCEDURES  46  EXPERIMENTAL REAGENTS  46  a. B e n z o p o r p h y r i n derivative m o n o a c i d r i n g A ( B P D , Verteporfin®)  46  b. C h e m i c a l reagents  46  c. T i s s u e culture reagents  46  d. Reagents f o r c o l o n y assays and long- term m a r r o w cultures  47  e. Fluorescence activated cell sorting ( F A C S ) reagents  47  f. Synthetic peptides  48  2.2 L I G H T S O U R C E 2.3  ...4 8  E X P E R I M E N T A L ANIMALS  49  a. M i c e 2.4  49  C E L L PREPARATION  50  a. M o u s e bone m a r r o w cells  50  b. M o u s e leukaemic cell lines  50  c. P r i m a r y human haematopoietic cells  51  d. H u m a n leukaemic cell lines  51  2.5 C Y T O T O X I C T R E A T M E N T O F C E L L S  51  a. P D T treatment o f murine bone m a r r o w cells and L I 2 1 0 cells f o r short term evaluations (combination experiments)  51  b. P D T treatment o f normal and leukaemic murine haematopoietic cells preincubated w i t h peptides (short- term c o l o n y assay)  v  52  c. P D T treatment o f murine bone m a r r o w cells for long- term bone m a r r o w cultures 53 d. P D T treatment o f human normal haematopoietic and l e u k a e m i c cells preincubated  2.6  w i t h peptides (short- term c o l o n y assay)  53  e. ara- C treatment o f D B A / 2 bone m a r r o w cells preincubated w i t h peptides  53  f. P D T o f D B A / 2 bone m a r r o w cells preincubated w i t h 5 0 u M ara- C  54  C Y T O T O X I C I T Y ASSAYS  54  a. A g a r c o l o n y assay o f treated m u r i n e bone m a r r o w cells  54  b. C o l o n y assay o f L 1 2 1 0 cells  55  c. Short-term c y t o t o x i c i t y assay ( M T T assay)  55  d. O n e step long- term bone marrow culture ( L T B M C ) o f m u r i n e haematopoietic cells56  2.7  e. C o l o n y assay o f treated normal and leukaemic h u m a n haematopoietic cells  57  f. C o l o n y assay o f the human l e u k a e m i c c e l l line K 5 6 2  57  P H O T O M E T R I C AND S P E C T R O S C O P I C  ANALYSES  58  a. Spectrophotometric analysis  58  b. Spectrofluorimetric analysis o f the interaction between 1 B P D and D o x  58  2.8 C E L L ANALYSIS  BY F L U O R E S C E N C E A C T I V A T E D C E L L S  SORTING  (FACS) 2.9  58  FLUORESCENT  3.0 ANALYSIS  MICROSCOPY  59  OF I N T R A C E L L U L A R G L U T A T H I O N E (GSH)  CONTENT  a. Tietze enzymatic assay 3.1 S T A T I S T I C A L  60 60  ANALYSIS  60  a. A n a l y s i s o f c o l o n y assay data f r o m P D T / D o x c o m b i n a t i o n experiments  60  b. A n a l y s i s o f c o l o n y assay data f r o m the inhibitory peptide pretreatment experiments61  C H A P T E R 3: C O M B I N E D HAEMATOPOIETIC AND  PDT:  T R E A T M E N T O F MURINE  AND LEUKAEMIC  CYTOTOXICITY  STUDY  vi  CELLS  WITH  NORMAL DOXORUBICIN 62  3.1  ABSTRACT  62  3.2  INTRODUCTION  63  3.3  RESULTS  65  Single agent cytotoxicity  65  C o m b i n a t i o n experiments w i t h B P D - mediated P D T and D o x : simultaneous a n d sequenced treatments o f D B A / 2 haematopoietic progenitor cells  72  C o m b i n a t i o n experiments with B P D - mediated P D T and D o x : simultaneous a n d sequenced treatments o f L 1 2 1 0 leukaemic cells  72  D i f f e r e n t i a l susceptibility o f L 1 2 1 0 cells and D B A / 2 haematopoietic progenitors to D o x - > P D T sequenced combination treatment  77  Statistical analysis o f combination treatments  77  DISCUSSION  CHAPTER  4: C O M B I N E D  HAEMATOPOIETIC AND  PDT:  84  AND  MECHANISTIC  T R E A T M E N T O F MURINE LEUKAEMIC  CELLS  WITH  NORMAL DOXORUBICIN  STUDY  87  4.1  ABSTRACT  87  4.2  INTRODUCTION  88  4.3  RESULTS  90  P h o t o p h y s i c a l properties o f B P D and D o x  90  Intracellular localisations o f B P D and D o x i n the human leukaemic c e l l line K 5 6 2 . . . 9 7 T h e role o f excitation wavelength (A, ) has o n the photobleaching o f B P D i n the ex  presence o f D o x  101  U p t a k e o f B P D b y L 1 2 1 0 cells as affected b y the presence o f D o x i n different c o m b i n a t i o n regimens  107  D e p l e t i o n o f cellular glutathione ( G S H ) i n L 1 2 1 0 cells b y preincubation w i t h D o x . 111 4.4  DISCUSSION  113  vii  CHAPTER 5: SELECTIVE PREPROTECTION OF HAEMATOPOIETIC COMMITTED PROGENITIOR WITH N-ACSDKP:  CYTOTOXICITY  NORMAL CELLS  FROM  PDT  STUDY  118  5.1  A B S T R A C T  118  5.2  INTRODUCTION  119  5.3  RESULTS  121  Photoprotective effect o f N - A c S D K P o n D B A / 2 haematopoietic progenitors  121  A b s e n c e o f N - A c S D K P mediated photoprotection o f L 1 2 1 0 cells  121  Statistical analyses o f progenitor assay data  128  N - A c S D K P protection o f D B A / 2 haematopoietic cells does not extend to earlier progenitors and stem cells  133  Selective photoprotection o f n o r m a l human bone m a r r o w cells but not l e u k a e m i c cells from P D T by N - A c S D K P  138  DISCUSSION  141  CHAPTER 6: PREPROTECTION OF NORMAL CELLS  WITH N-ACSDKP:  MECHANISTIC  HAEMATOPOIETIC  STUDY....  144  6.1  A B S T R A C T  144  6.2  INTRODUCTION  145  6.3  RESULTS  148  B P D uptake b y D B A / 2 bone m a r r o w cells preincubated w i t h 100 n M N - A c S D K P . 148 Inhibition o f the proliferative activity o f m u r i n e C F U - G M progenitors by 100 n M NAcSDKP  ..152  C e l l c y c l e inhibition o f D B A / 2 haematopoietic progenitor cells mediated by 100 n M N AcSDKP  152  Glutathione level i n D B A / 2 bone m a r r o w cells treated w i t h 100 n M N - A c S D K P . . .157  viii  6.4  DISCUSSION  CHAPTER PDT  7:  DISCUSSION  COMBINATION  N-ACSDKP-  FACTORS  159  164  T H E R A P Y  MEDIATED  AFFECTING  164  PHOTOPROTECTION  PDT  CYTOTOXICITY  F R O M  BPD  165  167  T h i o l s , a- t o c o p h e r o l , and haeme oxygenase  167  N F - K B and its role i n P D T c y t o t o x i c i t y  167  Summary  169  REFERENCES  171  ix  LIST OF  TABLES Title  Page 48  T a b l e 2.1  Reagents used f o r F A C S analysis  T a b l e 3.1a  T w o w a y analysis o f variance ( A N O V A ) o f c y t o t o x i c i t y data f r o m D B A / 2 haematopoietic progenitor cells treated w i t h the three different drug combinations i n v o l v i n g D o x and B P D - mediated P D T : significance o f treatment sequence and B P D dose  80  T a b l e 3.1b  Statistical analyses o f data f r o m the different D o x / P D T combinations f r o m D B A / 2 haematopoietic cells: B o n f e r r o n i (allpairwise) m u l t i p l e c o m p a r i s o n testings o f B P D concentrations and treatment sequences  81  T a b l e 3.2a  T w o w a y analysis o f variance ( A N O V A ) o f c y t o t o x i c i t y data f r o m L I 2 1 0 leukaemic cells treated w i t h the three different d r u g combinations i n v o l v i n g D o x and B P D mediated PDT: significance o f treatment sequence and B P D dose  82  T a b l e 3.2b  Statistical analyses o f data f r o m the different D o x / P D T combinations f r o m L I 2 1 0 leukaemic cells: B o n f e r r o n i multiplec o m p a r i s o n testings o f B P D concentrations and treatment sequences  83  T a b l e 5.1a  Bonferroni (all- pairwise) multiple c o m p a r i s o n test of clonogenicity data o f D B A / 2 haematopoietic progenitors: s i g n i f i c a n c e o f B P D doses  129  T a b l e 5.1b  Bonferroni (all- pairwise) multiple c o m p a r i s o n test of clonogenicity data o f D B A / 2 haematopoietic progenitors: significance o f peptides  130  T a b l e 5.2a  Bonferroni (all- pairwise) multiple c o m p a r i s o n test . o f clonogenicity data o f L 1 2 1 0 leukaemic cells: significance o f B P D doses  131  T a b l e 5.2b  Bonferroni (all- pairwise) multiple c o m p a r i s o n test of clonogenicity data o f L 1 2 1 0 leukaemic cells: significance o f peptides  132  T a b l e 5.3  S u s p e n s i o n cell numbers o f w e e k l y harvests o f l o n g term bone m a r r o w culture o f D B A / 2 haematopoietic cells preincubated i n the presence or absence o f 100 n M N - A c S D K P f o l l o w e d b y B P D mediated P D T  135  T a b l e 5.4  Numbers  138  of C F U -  GM  progenitor colonies generated f r o m  x  nonadherent cells harvested w e e k l y f r o m l o n g term bone m a r r o w culture o f D B A / 2 haematopoietic cells preincubated i n the presence or absence o f 100 n M N - A c S D K P f o l l o w e d b y B P D mediated P D T T a b l e 6.1  B P D uptake i n the C D 3 4 e x p r e s s i n g subpopulation o f D B A / 2 bone m a r r o w cells  xi  151  LIST OF FIGURES  F i g u r e 3.1  Title Page T h e effect o f B P D - mediated P D T o n the clonogenicity o f normal 66-7 D B A / 2 haematopoietic progenitors and the • leukaemic cell line L1210  F i g u r e 3.2  C y t o t o x i c i t y o f B P D in the absence or presence o f 15 J / c m red light irradiation on L 1 2 1 0 cells as determined by the M T T cytotoxicity assay  68  F i g u r e 3.3  T h e effect o f D o x and 15 J / c m red light o n the clonogenicity o f D B A / 2 haematopoietic progenitor cells and L 1 2 1 0 leukaemic cells  6970  F i g u r e 3.4  C y t o t o x i c i t y o f D o x i n the absence or presence o f 15 J / c m red light irradiation o n L 1 2 1 0 cells as determined by the M T T cytotoxicity assay  71  F i g u r e 3.5  S u r v i v a l fraction o f D B A / 2 haematopoietic progenitors and L 1 2 1 0 cells to combinations o f 5 ng/ml B P D and 2.5 u M D o x in c o n j u n c t i o n w i t h 15 J / c m o f red light exposure  73  S u r v i v a l fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 cells to combinations o f 5 ng/ml B P D and 1.25 u M D o x in c o n j u n c t i o n w i t h 15 J / c m o f red light exposure  74  S u r v i v a l fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 cells to combinations o f 2.5 ng/ml B P D and 2.5 u M D o x in c o n j u n c t i o n w i t h 15 J / c m o f red light exposure  75  S u r v i v a l fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 cells to combinations o f 2.5 ng/ml B P D and 1.25 u M D o x in c o n j u n c t i o n w i t h 15 J / c m o f red light exposure  76  F i g u r e 3.9  Differential k i l l i n g o f L 1 2 1 0 cells over D B A / 2 progenitors in the different P D T / D o x combinations  79  F i g u r e 4.1  C h e m i c a l structure o f the b e n z o p o r p h y r i n derivative ring- A ( B P D , Verteporfin®)  92  F i g u r e 4.2  C h e m i c a l structure o f D o x o r u b i c i n ( D o x )  93  F i g u r e 4.3  A b s o r p t i o n spectra o f 1 pg/ml B P D and 10 u M D o x in 10 % H I FCS/ PBS  94  F i g u r e 4.4a  Fluorescence e m i s s i o n spectra o f B P D (550- 7 0 0 nm)  95  2  2  2  2  F i g u r e 3.6  2  F i g u r e 3.7  2  F i g u r e 3.8  2  xn  monoacid  in the presence o f D o x  F i g u r e 4.4b  Fluorescence e m i s s i o n p r o f i l e o f B P D (650- 7 0 0 n m )  in the presence o f D o x  96  F i g u r e 4.5  Fluorescence m i c r o g r a p h o f K 5 6 2 leukaemic cells incubated w i t h BPD  98  F i g u r e 4.6  Fluorescence m i c r o g r a p h o f K 5 6 2 leukaemic cells incubated w i t h Dox  99  F i g u r e 4.7  Fluorescence m i c r o g r a p h o f K 5 6 2 leukaemic cells incubated w i t h B P D and D o x  100  F i g u r e 4.8  P h o t o b l e a c h i n g o f 10 ng/ml B P D  103  in the presence o f different  concentrations o f D o x (A, = 4 4 0 nm) ex  F i g u r e 4.9  S u m m a r y o f B P D photobleaching in the presence o f different concentrations o f D o x (440 n m excitation)  F i g u r e 4.10  -phe effect o f X on B P D absence of. 5 u M D o x  F i g u r e 4.11  L i g h t transmittance o f 1 m M D o x in 10 % HI- F C S / P B S  Figure 4.12a  Uptake of BPD by L1210 cells as spectrofluorimetric measurements o f c e l l lysates  determined  by  108  F i g u r e 4.12b  Uptake of Dox by L1210 cells as spectrofluorimetric measurement o f cell lysates  determined  by  109  F i g u r e 4.13  U p t a k e o f B P D b y L 1 2 1 0 cells w h e n incubated simultaneously ( B P D / D o x ) or after D o x (Dox-> B P D )  110  F i g u r e 4.14  C e l l u l a r glutathione levels i n L 1 2 1 0 cells after 1 h incubation w i t h different doses o f D o x  112  F i g u r e 5.1  Selective cytoprotective effects o f N - A c S D K P on D B A / 2 haematopoietic progenitor cells subjected to B P D - mediated P D T  1223  F i g u r e 5.2  Effects o f N - A c S D K P , control peptides N - A c S D K E or S D K P , and m e d i u m on the clonogenicity o f D B A / 2 haematopoietic progenitor cells  124  F i g u r e 5.3  A b s e n c e o f cytoprotective effects o f N - A c S D K P leukaemic cell line subjected to B P D - mediated P D T  1256  F i g u r e 5.4  Effects o f N - A c S D K P , control peptides N - A c S D K E or S D K P ,  e x  photobleaching in the presence and  xiii  104  ^  106  on L 1 2 1 0  127  and m e d i u m on the clonogenicity o f L 1 2 1 0 leukaemic cells F i g u r e 5.5  One step long- term bone m a r r o w culture ( L T B M C ) o f D B A / 2 bone m a r r o w cells  136  F i g u r e 5.6  C o m p a r i s o n o f w e e k 1 long- term bone m a r r o w culture ( L T B M C ) harvests o f nonadherent cells f r o m samples preincubated w i t h 100 n M N - A c S D K P or m e d i u m f o l l o w e d b y P D T  137  F i g u r e 5.7  Cytoprotection conferred b y 100 n M o f N - A c S D K P against subsequent B P D - mediated P D T was specific f o r n o r m a l h u m a n bone m a r r o w cells  140  F i g u r e 6. la-c  Representative F A C S histograms o f B P D uptake i n control and N - A c S D K P - treated murine bone m a r r o w mononuclear cells (BMMNC)  149150  F i g u r e 6.2  E f f e c t o f 1.5 h preincubation w i t h 100 n M N - A c S D K P had o n the c y c l i n g status o f C F U - G M f r o m D B A / 2 m i c e  154  F i g u r e 6.3  E f f e c t o f 5.0 h preincubation w i t h 100 n M N - A c S D K P h a d o n the c y c l i n g status o f C F U - G M f r o m D B A / 2 m i c e  155  F i g u r e 6.4  Correlation between cell c y c l i n g and susceptibility to B P D mediated P D T in D B A / 2 C F U - G M progenitors  156  F i g u r e 6.5  C e l l u l a r glutathione content i n D B A / 2 bone incubated w i t h 0, 10, or 100 n M N - A c S D K P  158  xiv  marrow  cells  LIST OF ABBREVIATIONS 4- H C  4- h y d r o p e r o x y c y c l o p h o s p h a m i d e  8- M O P  8- m e t h o x y p s o r a l e n  ACTH  A d r e n o - corticotrophic hormone  alBMT  A l l o g e n e i c one m a r r o w transplantation  ALDH  A l d e h y d e dehydrogenase  ALL  Acute lymphoid leukaemia  AlSPc  Sulphonated a l u m i n i u m phthalocyanine  AML  A c u t e m y e l o i d leukaemia  ANOVA  A n a l y s i s o f variance  APL  A c u t e p r o m y e l o c y t i c leukaemia  Ara-C  C y t o s i n e arabinoside  ATG  Antithymocyte globulin  ATRA  A l l - trans retinoic acid  auBMT  A u t o l o g o u s bone m a r r o w transplantation  BCG  B a c i l l u s Calmette- G u e r i n  BMMNCs  B o n e m a r r o w m o n o n u c l e a r cells  BMP  Bone marrow purging  BMT  B o n e m a r r o w transplantation  BPD  B e n z o p o r p h y r i n derivative m o n o a c i d ring- A  BSA  B o v i n e serum a l b u m i n  BSO  Buthionine sulphoximine  Bu  Busulphan  CFU- G M  C o l o n y f o r m i n g unit- granulocyte & macrophage  CFU-L  C o l o n y f o r m i n g unit- leukaemic cell  CML  Chronic myeloid leukaemia  CMV  Cytomegalovirus  CSF  C o l o n y stimulating factor  CTL  Cytotoxic T lymphocyte  CW  Continuous wavelength  CY  Cyclophosphamide  XV  CYA  Cyclosporin A  DHE  D i h a e m a t o p o r p h y r i n ether  DIC  D i s s e m i n a t e d intravascular coagulation  DMEM  Dulbecco' s modified Eagle' s medium  DTNB  5,5'- dithiobis- (2- n i t r o b e n z o i c acid)  Dox  D o x o r u b i c i n hydrochloride  EPO  Erythropoietin  EtNBS  5- ethylamino- 9- diethylaminobenzo[a] p h e n o t h i a z i n i u m chloride  FAB  French- A m e r i c a n - B r i t i s h classification  FACS  Fluorescence activated c e l l sorting  FCS  Foetal c a l f serum  FITC  F l u o r e s c e i n isothiocyanate  G- C S F  Granulocyte- c o l o n y stimulating factor  GM- CSF  G r a n u l o c y t e & macrophage- c o l o n y stimulating factor  GSH  Glutathione  GvHD  Graft- versus- host- disease  GvL  Graft- versus- l e u k a e m i a  HI- F C S  Heat- inactivated- foetal c a l f serum  HLA  H u m a n leukocyte antigen  HOC1  Hypochlorous acid  HPC  Haematopoietic progenitor c e l l  HPD  H a e m a t o p o r p h y r i n derivative  HSC  Haematopoietic stem cell  HUC  H u m a n u m b i l i c a l c o r d b l o o d cells  ICSBP  Interferon consensus b i n d i n g sequence protein  IFNa  Interferon a  M D M  Iscove's M o d i f i e d D u l b e c c o ' s M e d i u m  LAK  L y m p h o k i n e activated k i l l e r  LDL  L o w density lipoprotein  LDLr  L o w density lipoprotein receptor  LTBMC  L o n g - term bone m a r r o w culture  xvi  MC540  Merocyanine 540  MDR  M u l t i d r u g resistance  MESNA  S o d i u m 2- mercaptoethanesulfonate  MFI  M e a n fluorescence intensity  MHC  M a j o r histocompatibility c o m p l e x  mHC  m i n o r histocompatibility c o m p l e x  MIP- l a  M a c r o p h a g e inflammatory protein- l a  MLR  M i x e d lymphocyte reaction  MMC  Mitomycin C  MnSOD  M a n g a n e s e superoxide dismutase  MRD  M i n i m a l residual disease  MTT  3- [4,5- D i m e t h y l t h i a z o l - 2- yl]- 2,5- d i p h e n y l t e t r a z o l i u m bromide  MUD  M a t c h e d unrelated donor  N-AcSDKE  N - A c e t y l - Ser- A s p - L y s - A s p  N-AcSDKP  N - A c e t y l - Ser- A s p - L y s - P r o  NAC  N-Acetyl- cysteine  NCI  N a t i o n a l C a n c e r Institute  NEM  N- ethylmaleimide  NF- KP  N u c l e a r factor- K(3  ODN  Oligodeoxynucleotide  PBS  Phosphate buffered saline  PBSC  Peripheral b l o o d stem cell  PBSCT  Peripheral b l o o d stem c e l l transplantation  PCR  Polymerase chain reaction  PDT  P h o t o d y n a m i c therapy  PDTC  P y r r o l i d o n e derivative o f dithiocarbamate  pEEDCK  PyroGlu- Glu- Asp- Cys- L y s  PHA- L C M  phytohaemaggultinin stimulated- h u m a n leucocyte conditioned m e d i u m  PHSC  Pluripotent haematopoietic stem c e l l  PH'  Philadelphia chromosome  xvii  PMT  Photomultiplier tube  PTK  Protein tyrosine kinase  PWM- S C C M  P o k e w e e d mitogen- spleen cell conditioned m e d i u m  qRT- P C R  Quantitative reverse transcriptase- polymerase chain reaction  RAPA  Rapamycin  RB  Retinoblastoma protein  RBC  Red blood cell  RFLP  Restriction fragment length p o l y m o r p h i s m  Rhl23  R h o d a m i n e 123  rhIL- 2  R e c o m b i n a n t h u m a n interleukin- 2  ROIs  Reactive o x y g e n species  RT- P C R  Reverse transcriptase- polymerase chain reaction  SCA  S i c k l e c e l l anaemia  SCID  Severe c o m b i n e d i m m u n o d e f i c i e n c y  SDKP  Ser- A s p - L y s - P r o  SEM  Standard error o f the mean  SLT- 1  Shiga- l i k e t o x i n 1  SOD  S u p e r o x i d e dismutase  sODN  Phosphorothioate- m o d i f i e d o l i g o d e o x y n u c l e o t i d e  TBI  T o t a l b o d y irradiation  TCD  T cell depletion  TGF- P  T r a n s f o r m i n g g r o w t h factor- (3  TIL  T u m o u r infiltrating lymphocyte  TNFa  T u m o u r necrosis factor a  TNFr  T u m o u r necrosis factor receptor  TPO  Thrombopoietin  xviii  A C K N O W L E D G E M E N T S I w o u l d l i k e to thank the members o f m y thesis c o m m i t t e e : D r s . N o e l B u s k a r d , D a v i d D o l p h i n , and M i k e G o l d . I w o u l d also w i s h to a c k n o w l e d g e the advice and support p r o v i d e d b y D r . A n t h o n y C h o w , i n his capacity as the director o f the U B C M . D . - P h . D . p r o g r a m . I w o u l d l i k e to thank D r . J u l i a L e v y and L u c y . A n d lastly, P i g g y Y i p , i n his capacity as m y f a i t h f u l d o g f o r the past 10 years. B a B a R a . . .  xix  B l o o d is the o r i g i n a t i n g cause o f a l l m e n ' s diseases. The Talmud  Baba Nathra, III. 58a  INTRODUCTION  1.1 LEUKAEMIA  : HISTORY  a. C l i n i c a l observations T h e history o f l e u k a e m i a i n the past t w o centuries says as m u c h about the disease itself as about the scientific advancements and p a r a d i g m shifts i n the f i e l d o f m e d i c i n e . S i r W i l l i a m Osier, i n his seminal w o r k The Principles and Practice of Medicine, described the three components o f disease as manifestations, pathogenesis, and cause. T r u e appreciation o f an illness and therefore f o r m u l a t i o n o f rational treatments require understandings o f a l l three o f the above. U n f o r t u n a t e l y , the extent o f our m e d i c a l k n o w l e d g e does not a l l o w us to f u l l y comprehend their c o m b i n e d significance. W e are often left w i t h the most o b v i o u s facet o f the disease t r i l o g y , the manifestations, w h i c h w a s never more true than i n Osier's days. T h e manifestations o f a disease b e l o n g to the d o m a i n o f signs, s y m p t o m s , and the treatments designed to ameliorate them. S y m p t o m a t i c treatments were universal during Osier's time, some o f w h i c h are still i n use today. In 1893, w h e n the first e d i t i o n o f the textbook was published, p h y s i c i a n s already had an idea that l e u k a e m i a constituted a distinct entity o f the systemic disorders. T h e first documented case o f l e u k a e m i a was reported i n 1  1827 b y the F r e n c h p h y s i c i a n A l f r e d V e l p e a u . " T h e b l o o d was thick, l i k e gruel... r e s e m b l i n g i n consistency a n d c o l o r the yeast o f red wine... O n e m i g h t have asked i f it were not rather laudable pus, m i x e d w i t h b l a c k i s h c o l o r i n g matter, than b l o o d . "  1  T e n years later, gross observations were c o m p l e m e n t e d w i t h rudimentary m i c r o s c o p i c examination o f the b l o o d f r o m leukaemic patients. D o n n e c o n c l u d e d that the proliferation o f " m u c o u s c e l l s " w a s responsible f o r the patient's hepatosplenomegaly. A t that time, there was still great controversy concerning the aetiology o f l e u k a e m i a . M a n y prominent physicians b e l i e v e d that the disease was a severe f o r m o f general p y a e m i a and therefore was suppurative i n nature. V i r c h o w correctly defined l e u k a e m i a as a distinct disease w i t h " a n increase i n the number o f colorless b l o o d corpuscles to the extent that the red c o l o r o f b l o o d turns into r e d d i s h , y e l l o w i s h , or greenish w h i t e " and w a s associated w i t h the findings o f hepatosplenomegaly and lymphadenopathy. H e also attempted to classify l e u k a e m i a into t w o categories: the splenic and l y m p h a t i c f o r m . In 1854, V o g e l described 1  in detail the signs and symptoms o f l e u k a e m i a w h i c h i n c l u d e d lethargy, diarrhoea, pallor, hepatosplenomegaly, and lymphadenopathy i n some cases. S i g n i f i c a n t l y , V o g e l questioned whether the l e u k a e m i c b l o o d p r o f i l e was caused b y enlargements o f the spleen, liver, and l y m p h a t i c s or the other w a y around. In the late 1800's, W o o d w a r d introduced the aniline dyes into diagnostic haematology. E h r l i c h later e x p l o i t e d the differential staining ability o f these dyes to identify the different b l o o d cells i n both n o r m a l subjects and leukaemic patients; his most significant contribution to the f i e l d w a s the demonstration that leukaemia c o u l d be diagnosed b y simple b l o o d smears. T h u s entered S i r W i l l i a m Osier's textbook i n 1893. T h e manifestations o f l e u k a e m i a were f a i r l y w e l l understood and physicians were starting to focus their attention o n the pathogenesis a n d aetiology o f the disease. H o w e v e r , leukaemic treatments were still symptomatic i n nature. In fact, m a n y were palliative and i n c l u d e d " f r e s h air, g o o d diet, a n d abstention f r o m mental w o r r y and c a r e . . . " .  2  b. Diagnostics and Therapeutics T h e 20th century has brought significant progress i n both the diagnosis a n d treatment o f leukaemia. O n e important landmark was the discovery o f the P h i l a d e l p h i a c h r o m o s o m e ( P H ' ) b y N o w e l l a n d H u n g e r f o r d i n I 9 6 0 . P H ' is a product o f the t(9; 3  22)(q34; q21) rearrangement in c h r o n i c myelogenous l e u k a e m i a ( C M L ) . T h i s discovery not o n l y p r o v i d e d a unique and reliable marker f o r C M L , it also p a v e d the path f o r further discoveries i n the m o l e c u l a r pathogenesis and aetiology o f this f o r m o f leukaemia. Other  2  c h r o m o s o m a l abnormalities were soon l i n k e d to different f o r m s o f cancers; i r o n i c a l l y , the majority o f c h r o m o s o m a l translocations are f o u n d to be associated w i t h l e u k a e m i a s . Great 4  strides were made in elucidating the cause o f l e u k a e m i a f r o m studies in the viral transmission of l e u k a e m i a in animals and f r o m e p i d e m i o l o g i c a l studies o f the patients exposed to radiation and chemicals. A t the same time, development o f n o v e l chemotherapy was b e i n g conducted at the N a t i o n a l C a n c e r Institute ( N C I ) under C . G o r d o n Z u b r o d ' s d i r e c t i o n . T h e systemic nature o f l e u k a e m i a precluded the effective use (except during 1  o n c o l o g i c a l emergency or in l o c a l control) o f surgery and radiation treatments; therefore, chemotherapy by default became the therapy o f choice. E a r l y use o f c h e m i c a l c o m p o u n d s i n the control o f leukaemic growth was often e m p i r i c a l i n nature. In 1907, O s i e r mentioned the use o f potassium arsenic by his contemporaries in the management o f l e u k a e m i a ; the results, however, were predictably unpredictable. E h r l i c h , p e s s i m i s t i c i n regard to the future o f chemotherapy in cancer management, d i s p l a y e d a sign w i t h the line f r o m Dante's Inferno; " A b a n d o n all hope all ye w h o enter here," prominently at the entrance to his laboratory. P e s s i m i s m c o n c e r n i n g the success o f l e u k a e m i a chemotherapy also was shared b y W o g l o m half a century later. " T h o s e w h o have not been trained i n chemistry or medicine... m a y not realize how d i f f i c u l t the p r o b l e m o f cancer treatment really is. It is almostnot quite, but almost- as hard as f i n d i n g some agent that w i l l d i s s o l v e away the left ear, say, yet leave the right ear unharmed: S o slight is the difference between the cancer cell and its n o r m a l ancestor." 5  Research into a l k y l a t i n g agents such as busulphan and c y c l o p h o s p h a m i d e f o l l o w e d rapidly after the initial discovery o f the use o f nitrogen mustard i n l e u k a e m i a control. B u s u l p h a n , d e v e l o p e d by S i r A l e x a n d e r H a d d o w , was f o u n d to be extremely m y e l o s u p p r e s s i v e and was therefore useful in the control o f excessive neutrophils in c h r o n i c phase C M L .  6  Early  observations of the b i o l o g i c a l activities o f corticosteroids had lead to the subsequent use o f adreno- corticotrophic hormone ( A C T H ) and cortisone o n patients w i t h l y m p h o c y t i c l e u k a e m i a . A t the same time, development o f the different antimetabolites was underway. F o l i c acid plays an important role in haematopoiesis; its deficiency leads to retarded haematopoiesis and its abundance accelerates the g r o w t h o f l e u k a e m i c c e l l s . ' T h e 7  8  therapeutic potential o f antimetabolites was realised initially w i t h aminopterin and later w i t h  3  methotrexate, both f o l i c a c i d antagonists. In 1948, H i t c h i n g s and E l i o n , w h o later w o n the N o b e l p r i z e for m e d i c i n e and p h y s i o l o g y , developed the purine analogue 6- mercaptopurine w h i c h was subsequently used f o r the maintenance o f patients w i t h acute l y m p h o c y t i c leukaemia ( A L L ) . C l i n i c a l introduction o f the p y r i m i d i n e analogue cytosine arabinoside 9  (Ara- C ) i n 1963 s i g n i f i e d a milestone in acute myelogenous l e u k a e m i a ( A M L ) management.  10  C o m m o n l y used in conjunction w i t h anthracycline antibiotics such as  daunorubicin or more recently i d a r u b i c i n , A r a - C has been the mainstay o f i n d u c t i o n therapy f o r a variety o f A M L s . T h e next b i g breakthrough i n therapeutics was the discovery of the v i n c a a l k a l o i d vincristine and its extraordinary efficacy in paediatric A L L w h e n used in c o m b i n a t i o n w i t h p r e d n i s o n e . ' 1 1  1 2  D i s c o v e r y o f the anthracycline antibiotics and the  p o d o p h y l l o t o x i n s further c o m p l e m e n t e d an already potent arsenal o f drugs. Realisation o f the potential o f c o m b i n a t i o n chemotherapy i n the early 1950's was due p r i m a r i l y to understanding o f c e l l u l a r m e t a b o l i s m . 1 3  1 4  E n c o u r a g i n g results f r o m  experiments that used drugs to effect sequential and concurrent b l o c k i n g o f b i o c h e m i c a l pathways lead to the initiation o f more ambitious c l i n i c a l t r i a l s .  14  T h e rationale f o r  c o m b i n a t i o n therapy has changed somewhat since then and drugs w i t h different mechanisms o f action have been used together to increase the potency o f treatment, decrease the c o m b i n e d adverse effects as w e l l as the p o s s i b i l i t y o f d e v e l o p i n g m u l t i d r u g resistance. ' 15  16  Current treatment protocols often c a l l for c o m b i n a t i o n s o f different  chemotherapeutic agents at different stages o f the disease so as to tailor the p h a r m a c o l o g i c a l profiles o f the drugs to the unique growth d y n a m i c and peculiarity o f the l e u k a e m i a .  c. S u p p o r t i v e t h e r a p y Significant i m p r o v e m e n t in the prognosis of leukaemic patients in the p e r i o d between 1900- 1970 was also due to the introduction o f various ancillary therapies. A d v a n c e s i n transfusion science a l l o w e d c l i n i c i a n s to provide " c u s t o m " haematological supports i n the f o r m o f p a c k e d red b l o o d c e l l or platelet transfusions. E a r l y recognition of the relationship between thrombocytopaenia and bleeding diathesis i n patients had lead to the c l i n i c a l introduction o f platelet transfusions, i n i t i a l l y as w h o l e b l o o d and later as platelet concentrates.  17  Improvement i n both purification and storage techniques also contributed to  4  the universal use o f platelet transfusions i n l e u k a e m i c patients suffering f r o m thrombocytopaenia. H o w e v e r , early recognition o f the l i n k between t h r o m b o c y t o p a e n i a and b l e e d i n g diathesis d i d not lead to increased use o f platelet replacements. T h e delay was m a i n l y due to technical inexperience in the h a n d l i n g of platelets ex vivo. In a d d i t i o n , scepticism c o n c e r n i n g the merits o f the procedure also contributed to delays i n its implementation i n the clinics. P a r a d o x i c a l l y , increased alio- transfusions o f platelets lead to i m m u n o l o g i c a l l y mediated- "transfusion r e f r a c t o r i n e s s " .  18  T h i s p r o b l e m was later  ameliorated w i t h the use o f partially matched donors. Ironically, adequate control o f haemostasis by platelet transfusions accentuated the p r o b l e m o f infections related to the u n d e r l y i n g disease or chemotherapy- i n d u c e d neutropaenia. T h e role o f neutrophils i n nonspecific i m m u n i t y was clearly established i n the 1800's. C o r r e c t i o n o f neutropaenia not o n l y decreases the incidence o f life- threatening infections, it also permits the use o f more aggressive chemotherapy. S t r u m i a , in 1934, attempted to reinfuse leukocytes into a patient w i t h unmitigated infection. H e described the m i r a c u l o u s effects that " l e u k o c y t e c r e a m " , a dubious mixture o f w h o l e cell and c e l l debris, had o n the p a t i e n t .  19  Later, " s u r p l u s " neutrophils f r o m c h r o n i c phase C M L patients were  used as supplements i n neutropaenic patients, a rather adventurous f o r m o f replacement therapy.  20  Increased use o f neutrophil transfusions e x p o s e d the recipient to the p r o b l e m o f  cytomegalovirus ( C M V ) transmission, even f r o m asymptomatic donors w i t h latent C M V infections. 21  22  D e v e l o p m e n t o f C M V infections in l e u k a e m i a patients is extremely  problematic because of suppressed i m m u n i t y caused by the disease and the c y t o t o x i c treatments. T h e appropriate use o f a n t i m i c r o b i a l therapy later supplanted the use o f neutrophil transfusions i n patients. G e r a l d B o d e y was an early advocate of the appropriate yet aggressive use o f antibiotics in neutropaenic p a t i e n t s .  23  The emerging problem with  fatal fungal infections i n leukaemic patients m i r r o r e d that o f bacterial infection. A g a i n , the p r i n c i p a l culprit was persistence o f the neutropaenic state initiated by the l e u k a e m i a w h i c h was sustained by the ever more aggressive m y e l o s u p p r e s s i v e chemotherapy regimens. A retrospective r e v i e w conducted b y B a k e r i n 1962 demonstrated the increased incidence o f fatal f u n g a l infections and c o n c l u d e d that " s o m e factor o f modern therapy is responsible f o r the f a t a l i t i e s " .  24  L i b e r a l and invaluable use o f i n d w e l l i n g catheters such as the H i c k m a n l i n e  also contributed to the emergence o f systemic infections. A m p h o t e r i c i n B was the d r u g o f choice f r o m a s m a l l arsenal o f antifungal agents. U n f o r t u n a t e l y , the side effects were  5  horrendous and patients often c o m p l a i n e d o f the " b a k e and s h a k e " effects o f "amphoterrible".  25  d. Bone marrow transplantation A p p r e c i a t i o n o f the curative potential o f B M T in humans began not l o n g after the d a w n o f the atomic age. Fear o f the consequences o f radiation exposures; inadvertently f r o m c i v i l i a n accidents or deliberately and more sinisterly, as a result o f m i l i t a r y c o n f l i c t s , perpetuated early research i n h u m a n B M T .  2 6  In 1922 Fabricious- M o e l l e r demonstrated,  albeit o b l i q u e l y , the principles o f B M T . H e s h o w e d that shielding o f the legs d u r i n g lethal irradiation protected guinea pigs f r o m subsequent bone m a r r o w a p l a s i a .  27  Jacobson and  colleagues repeated similar experiments i n m i c e w i t h splenic s h i e l d i n g ; i n addition, they observed that intraperitoneal injections o f splenocytes also rescued the irradiated a n i m a l s .  28  M e a n w h i l e , M o r r i s o n and S a m w i c k successfully treated a patient afflicted w i t h idiopathic aplastic anaemia w i t h sternal injections o f donated bone m a r r o w . T h e y were, however, mistaken i n their interpretation o f the result in that they thought the donated m a r r o w contained h u m o r a l factors that p r o m o t e d the maturation o f the diseased host haematopoietic compartments.  29  R e c o g n i t i o n o f a stable population o f haematopoietic c e l l s o f donor o r i g i n  or c h i m e r i s m posttransplantation and its role in the protection o f the lethally irradiated host further strengthened the theoretical foundation o f B M T .  3 0  A d d i t i o n a l a n i m a l experiments  also helped to define the relatively n o v e l concepts o f graft- versus- host disease ( G v H D ) , also k n o w n as secondary or runt disease, and i m m u n e t o l e r a n c e .  3 1 - 3 3  H o w e v e r , it was not  until m u c h later that the human equivalents o f the mouse major histocompatibility antigens H- 2 were identified and the concept o f h u m a n leucocyte antigen ( H L A ) m a t c h i n g was introduced into c l i n i c a l m a r r o w t r a n s p l a n t a t i o n .  34-36  In a landmark paper p u b l i s h e d i n  1957, E. D o n a l d T h o m a s and colleagues demonstrated the feasibility o f c l i n i c a l m a r r o w transplantation i n leukaemia and more importantly, they s h o w e d evidence o f transient m a r r o w engraftment i n a p a t i e n t .  37  W i t h great prescience, the authors foresaw the use o f  B M T as a f o r m o f salvage r e g i m e n i n h i g h dose radiotherapy,  6  "In selected patients w i t h disseminated neoplasia it m a y be advantageous to use total- body radiation in large dosage and to c o v e r the resultant aplasia b y m a r r o w transplantation";  they also predicted the increasing demand for this n o v e l f o r m o f m e d i c a l technology r i v a l l i n g that o f k i d n e y transplantations. P r i o r to 1968, allogeneic B M T s were p e r f o r m e d without the benefits o f H L A m a t c h i n g and the results were d i s m a l . T h e rare cases o f success were later ascribed to fortuitous H L A m a t c h i n g between the host and the s i b l i n g d o n o r .  2 6  In 1968, a patient w i t h  congenital severe c o m b i n e d i m m u n o d e f i c i e n c y disease ( S C I D ) received m a r r o w f r o m a H L A - matched s i b l i n g , thus heralding in a new era in c l i n i c a l m a r r o w transplantation, one that is based o n the rational selection o f d o n o r s .  38  T o compensate f o r the paucity o f  genotypically H L A - matched s i b l i n g donors, partly as a result o f the l i m i t e d size o f the W e s t e r n f a m i l y and also a consequence o f the enormous p o l y m o r p h i s m o f the H L A haplotypes - different m a t c h i n g criteria were used. C o m p l e x algorithms were developed for the identification o f m a r r o w donors, starting f r o m the best to the least c o m p a t i b l e match i.e. f r o m a m o n o z y g o t i c t w i n donor to a H L A - matched unrelated donor ( M U D ) , respectively.  39  U t i l i s a t i o n o f less rigorously matched m a r r o w donors necessitated the use  o f more e f f i c a c i o u s i m m u n o s u p p r e s s i v e agents. Introduction o f c y c l o s p o r i n A and later, a more effective c y c l o s p o r i n A and methotrexate c o m b i n a t i o n significantly decreased the incidence o f G v H D and hence i m p r o v e d the outcomes o f allogeneic B M T s .  4 0  -  4 1  D e v e l o p m e n t o f new c o n d i t i o n i n g regimens not o n l y reflected the p a r a d i g m shifts i n B M T , but also the n e w l y acquired k n o w l e d g e in i m m u n o b i o l o g y and haematology. O r i g i n a l l y intended for the rescue o f v i c t i m s o f accidental radiation exposure, B M T was later adapted as a f o r m o f experimental therapy for endstage l e u k a e m i c patients. Subsequently, people w i t h congenital or acquired d e f i c i e n c y o f the haematopoietic system also benefited f r o m B M T . T h e purpose o f the p r e c o n d i t i o n i n g r e g i m e n is to achieve adequate i m m u n o s u p p r e s s i o n o f the host to effect graft acceptance, to " r e l i e v e " the m a r r o w m i c r o e n v i r o n m e n t o f diseased host haematopoietic cells so that the donor cells can "take up r e s i d e n c e " ; and, i n the case o f a host w i t h m a l i g n a n c y , to destroy a significant n u m b e r o f residual cancer c e l l s .  4 2  T h e need f o r unique p r e c o n d i t i o n i n g regimens was evident f r o m the  different types o f disease indicated above. F o r e x a m p l e , patients w i t h F a n c o n i ' s anaemia, a f o r m o f congenital aplastic aplasia, are exceptionally sensitive to a l k y l a t i n g agents therefore  7  necessitating a less severe r o u n d o f pretransplant c o n d i t i o n i n g . T h e f u n c t i o n a l status o f 43  the host's i m m u n e system, in addition to the degree o f donor- host disparity, also determines the intensity o f the r e g i m e n . O b v i o u s l y , a foetal host is different f r o m an adult host w i t h congenital i m m u n o d e f i c i e n c y and is quite different f r o m a host w i t h an intact i m m u n e system, most l i k e l y a l e u k a e m i c patient. E a r l y B M T recipients, w i t h their restricted range o f disease indications, were often preconditioned w i t h radiation (mostly unintentional) or h i g h doses o f chemotherapeutic agents. Later, w i t h allogeneic B M T assuming a more " p r o a c t i v e " role i n the overall management o f diseases such as congenital i m m u n o d e f i c i e n c y , n o v e l and often more effective c o n d i t i o n i n g regimens were developed. S i n g l e dose total b o d y irradiation ( T B I ) was o r i g i n a l l y designed to suppress the i m m u n e system and ablate the host m a r r o w ; later, fractionated T B I was used to reduce extramedullary t o x i c i t y . C y c l o p h o s p h a m i d e ( C Y ) , a nitrogen mustard derivative, and 4 4  antithymocyte g l o b u l i n ( A T G ) are both potent immunosuppressants that d o not appreciably ablate the m a r r o w ; therefore, regimens that utilise C Y or A T G were successful o n l y in patients w i t h intrinsic stem c e l l d y s f u n c t i o n i.e. severe m a r r o w aplasia. A transplant candidate suffering f r o m T cell- mediated aplastic anaemia w o u l d require a regimen that both suppresses host i m m u n i t y and ablates residual endogenous haematopoiesis. S i n c e busulphan (Bu) has the capability to damage cells that reside i n the G / G , phases o f the cell 0  c y c l e , i.e. the quiescent stem c e l l compartment, B u along w i t h T B I became the rational c h o i c e f o r m a r r o w a b l a t i o n . In 1968, Santos and Haghshenass o u t l i n e d a n e w r e g i m e n 42  busulphan- c y c l o p h o s p h a m i d e ( B u C y ) , w h i c h was subsequently used for the pretransplant c o n d i t i o n i n g o f various types o f d i s e a s e s . 45  46  In the same year, B a c h and colleagues  performed a successful allogeneic B M T on a paediatric patient suffering f r o m WiskottA l d r i c h s y n d r o m e ; donor m a r r o w was obtained f r o m a K L A - identical s i b l i n g .  4 7  Shortly  thereafter, the first detailed study o f allogeneic B M T in acute l e u k a e m i a was performed by the F r e d H u t c h i n s o n g r o u p ; H L A - matched marrows were infused into e n d stage l e u k a e m i c patients c o n d i t i o n e d w i t h C Y and T B I f o l l o w e d by a t w o w e e k anti- G v H D regimen o f methotrexate  4 8  T h e history o f leukaemia mirrors the progress o f m e d i c i n e in general; it e v o l v e d f r o m an area o f e m p i r i c a l observations and speculations to more substantive (and often more objective) investigations. L e u k a e m i c therapy also i m p r o v e d f r o m the early use o f  8  alkylating agents to the development and incorporation o f B M T into the overall management plan o f the disease.  9  1.2 LEUKAEMIA  a. C l i n i c a l  : THE STATE  OF  THE  ART  advances  Recent developments in the f i e l d o f l e u k a e m i c research have not only significantly contributed to the understanding o f the disease but also, more importantly, have l e d to improvements i n both the prolongation and the quality o f life o f the leukaemic patient.  b. D i a g n o s t i c s a n d  i. Epidemiology  &  therapeutics  aetiology  E p i d e m i o l o g i c a l studies o f c h i l d h o o d l e u k a e m i a in geographical regions exposed to h i g h levels o f radioactive fallout f r o m C h e r n o b y l further illustrated the correlation between foetal radiation exposure and an increased incidence o f l e u k a e m i a .  49  H o w e v e r , the  contribution o f previous paternal radiation exposure, w h i c h implicates mutations i n the male g e r m c e l l compartment, to the increased incidence of c h i l d h o o d l e u k a e m i a is still controversial.  50  F r o m retrospective studies o f the occurrence o f A L L in B r i t i s h c h i l d r e n  that were relocated during the S e c o n d W o r l d W a r , M e l G r e a v e s proposed that c o m m o n c h i l d h o o d infections promote leukaemogenesis o f haematopoietic precursor cells that have received mutations (first hit but silent ?) in utero. *' 5  BCR-  and PML-  ABL  RARa  52  T h e n o v e l f u s i o n gene products  are i m p l i c a t e d i n the initiation and maintenance o f c h r o n i c  myelogenous l e u k a e m i a ( C M L ) and acute p r o m y e l o c y t i c l e u k a e m i a ( A P L ) , respectively. In fact, countless c h r o m o s o m a l abnormalities have been attributed to the different types o f leukaemia  4  Unfortunately, the role o f m a n y o f these genomic events in the actual initiation  and progression of l e u k a e m i a is not certain. S i n c e l e u k a e m i a , and cancer i n general, results f r o m m u l t i p l e genetic and sometimes epigenetic events, f i n d i n g the proverbial " s m o k i n g g u n " has proven to be difficult. O n e must appreciate the c o m b i n e d role o f protooncogenes, t u m o u r suppressor genes, and the environment in l e u k a e m i a pathogenesis.  10  H o w e v e r , there is little doubt that definitive identification o f an aberrant genetic event i n the initiation and maintenance o f l e u k a e m i a provides a unique target for m o l e c u l a r therapy. T e c h n i c a l advances in molecular diagnostics permit the sensitive detection o f genetic changes i n leukaemic cells, w h i c h provide clinicians w i t h invaluable leukaemia- specific markers f o r the purpose o f detecting m i n i m a l residual d i s e a s e .  53  D i f f e r e n t mutagenic agents  leave b e h i n d unique basepair alterations in the p53 tumour suppressor gene w h i c h afford the investigator with clues as to the identity the aetiologic agent, thereby p r o v i d i n g an important tool i n the emerging f i e l d o f m o l e c u l a r e p i d e m i o l o g y .  54  O n e has to realise that  societal and p o l i c y changes often c o m e as a result o f the positive identification o f leukaemogenic agents, especially i n the face o f "irrefutable scientific p r o o f s " . Therefore, the i m p r o v e d understanding o f the m o l e c u l a r events that underlie the initiation and maintenance o f leukaemia w i l l not o n l y translate into i m p r o v e d diagnostics and therapeutics in the c l i n i c , but also the o v e r a l l w e l l n e s s of s o c i e t y .  //. Novel  55  chemotherapeutics T h e search for novel chemotherapeutic agents diverges into t w o paths: identification  o f naturally o c c u r r i n g antineoplastic agents and the c h e m i c a l synthesis o f potentially useful cytotoxic c o m p o u n d s . T h e majority o f the anticancer drugs i n current c l i n i c a l use are naturally derived and are usually c h e m i c a l l y m o d i f i e d to achieve reasonable pharmacokinetic and p h a r m a c o d y n a m i c characteristics. L a t e l y , significant developments in the f i e l d o f combinatorial libraries have created large databases o f potentially useful pharmacological agents.  56  >  57  A foreseeable use of this technology i n l e u k a e m i a therapy  w o u l d be the production o f a large number o f c o m p o u n d s w h i c h c o u l d potentially interact with the signal transduction machinery o f the leukaemic c e l l . M e y d a n and colleagues have s h o w n that the tyrphostin analogue A G - 4 9 0 , synthesised u s i n g traditional c h e m i c a l techniques, i n h i b i t e d the g r o w t h o f l y m p h o b l a s t i c l e u k a e m i c cells in vitro and in vivo. Importantly, the authors s h o w e d that the c o m p o u n d selectively inhibited the J A K 2 protein tyrosine kinase, a downstream component of the c y t o k i n e receptor superfamily s i g n a l l i n g cascade; J A K 2 is s h o w n to be elevated in relapsing l y m p h o b l a s t i c leukaemic cells, thus p r o v i d i n g a rational m o l e c u l a r target for future i n v e s t i g a t i o n s .  11  58  Several groups are  w o r k i n g on the development o f other c o m p o u n d s w h i c h c o u l d inhibit the tyrosine kinase activity o f the p a t h o g n o m o n i c P 2 1 0 BCR-  ABL  f u s i o n protein i n C M L . R e c e n t l y , D r u k e r  and colleagues s h o w e d that the 2- p h e n y l a m i n o p y r i m i d i n e - based c o m p o u n d C G P 57148 selectively inhibited the growth o f leukaemic cells f r o m C M L patients w i t h no deleterious effects on the c o l o n y formation o f normal haematopoietic c e l l s .  5 9  Research into the m y r i a d  signal transduction pathways o f b l o o d cells w i l l h o p e f u l l y identify more potential targets for p h a r m a c o l o g i c a l intervention. Screening o f naturally derived c o m p o u n d s still remains the backbone o f chemotherapeutic drug discovery. Treatment o f hairy cell l e u k a e m i a was significantly i m p r o v e d w i t h the d i s c o v e r y o f the new purine analogues such as 2'- d e o x y c o f o r m y c i n , c h l o r o d e o x y a d e n o s i n e , and f l u d a r a b i n e .  60  D e v e l o p m e n t o f chemotherapy protocols based  on all- trans retinoic a c i d ( A T R A ) , i n c o m b i n a t i o n w i t h traditional i n d u c t i o n therapy, remains one o f the major achievements o f m e d i c a l science i n the late 2 0 century and th  illustrates that m u t u a l l y beneficial cross talk exists between science and m e d i c i n e .  61  Leo  Sachs initially pioneered the theory that growth o f some l e u k a e m i c clones c o u l d be successfully c o n t r o l l e d w i t h differentiation- i n d u c i n g c h e m i c a l c o m p o u n d s or c y t o k i n e s . ' 62  6 3  Later, B r e i t m a n and colleagues demonstrated that retinoic a c i d ( R A ) c o u l d induce the  A M L c e l l line H L - 6 0 to differentiate in vitro.  64  Subsequently, A M L M 3 or acute  p r o m y e l o c y t i c leukaemia ( A P L ) became the o b v i o u s therapeutic candidate for R A - mediated differentiation therapy because o f the cumulative in vitro data and the therapeutic urgency in its management. T h e use o f oral A T R A successfully controlled life- threatening episodes o f haemorrhagic diathesis o f A P L by f o r c i n g the differentiation o f the l e u k a e m i c promyelocytes into endstage cells o f the granulocyte lineage; c l i n i c i a n s also f o u n d that a " 3 + 7 " regimen o f cytosine arabinoside /anthracycline f o l l o w i n g A T R A therapy was successful i n m a i n t a i n i n g the r e m i s s i o n . A p p a r e n t l y , l e u k a e m i c p r o m y e l o c y t e s contain vast amounts o f tissue factor whose release leads to v i c i o u s c y c l e s o f c l o t t i n g and t h r o m b o l y s i s , a condition k n o w n c l i n i c a l l y as disseminated intravascular coagulopathy or D I C . Subsequent m o l e c u l a r studies demonstrated that the canonical t(15; 17) translocation o f A P L results i n the f u s i o n o f the retinoic a c i d receptor a gene (rara) E c t o p i c c e l l u l a r expression o f the f u s i o n protein PML-  RARa  w i t h the pml gene.  and inappropriate  dimerisation w i t h the n o r m a l b i n d i n g partners o f PAR e x p l a i n e d partly the dysregulated growth o f A P L c e l l s ; however, the exact m e c h a n i s m s o f enforced differentiation o f the  12  l e u k a e m i c cells by h i g h dose A T R A is still not c l e a r .  61  Reports f r o m C h i n a suggested  arsenic trioxide ( A s 2 0 3 ) is effective i n treating A P L patients refractory to A T R A and conventional chemotherapeutic regimens; in addition, C h e n and colleagues recently demonstrated that A s 2 0 3 promotes apoptotic c e l l death in A P L cells v i a the downregulation o f the antiapoptotic protein BCL-  PML-  RARa and  2 as w e l l as m o d u l a t i o n o f the intracellular localisation o f  PML.  65  Recent developments in the area o f tumour angiogenesis have lead to the discovery o f positive and negative regulators o f angiogenesis, as w e l l as the appreciation o f angiogenesis in tumour growth and m e t a s t a s i s .  66  L e u k a e m i a has traditionally been v i e w e d  as " b l o o d t u m o u r " i n w h i c h angiogenesis is irrelevant; however, m a n y o f the haematopoietic growth factors are angiogenic factors and vice v e r s a .  67  F o l k m a n and  colleagues recently s h o w e d increased m i c r o v e s s e l density in l e u k a e m i c bone m a r r o w w i t h the capillaries surrounded, b y cords o f l e u k a e m i c cells, an observation c o m m o n l y made in s o l i d tumours; i n addition, the levels o f urinary basic fibroblastic g r o w t h factor, a potent inducer o f angiogenesis, were elevated i n all the l e u k a e m i c subjects studied ( A L L )  6 8  This  discovery has lead to c l i n i c a l trials o f the use o f angiostatin, a potent angiogenesis inhibitor derived f r o m proteolytic cleavage o f f i b r i n o g e n , on paediatric A L L patients w h o have failed initial therapy.  iii. Antibody- based diagnostics & therapeutics T h e concept o f eradication o f n e o p l a s m b y the specific targeting o f cancer cells first e v o l v e d f r o m E h r l i c h ' s " m a g i c b u l l e t " theor)' o f i m m u n e recognition. Initial o p t i m i s m in antibody- directed immunotherapy o f l e u k a e m i a was tempered by various scientific and technical p r o b l e m s . ' 6 9  7 0  T h e majority o f the p r o b l e m s associated w i t h i m m u n o t h e r a p y is  due to the paucity o f genuine leukaemia- specific antigens and subsequently the c o m p r o m i s e d choice o f the target antigens. In addition, therapeutically effective eradication of m i n i m a l residual disease requires the expression o f the target antigen on l e u k a e m i c stem cells. Fortunately, j u d i c i o u s selection o f the disease and target antigen can sometimes c i r c u m v e n t the above problems. N o r m a l expression o f the B c e l l developmental antigens C D 19 and C D 2 0 are tightly regulated; both are not expressed on haematopoietic stem cells  13  and p l a s m a cells. H o w e v e r , B c e l l l y m p h o m a and B c e l l precursor l e u k a e m i a express abundant levels o f the above t w o antigens, thus o f f e r i n g unique targets f o r i m m u n o t h e r a p y without significantly affecting haematopoiesis and antibody secretion. K a m i n s k i and colleagues demonstrated the e f f i c a c y o f I- a n t i B l ( C D 2 0 ) radioimmunoconjugates o n B 13I  c e l l l y m p h o m a patients refractory to chemotherapy i n a recent t r i a l .  71  A n o t h e r approach,  u s i n g immunoconjugates o f the tyrosine kinase inhibitor genistein- a n t i B 4 ( C D 19), was successfully used b y U c k u n ' s group i n abrogating the g r o w t h o f lethal doses o f h u m a n B c e l l precursor leukaemia, w h i c h constitutes 7 5 % o f all adult A L L s , i n a S C T D mouse model.  7 2  U s i n g radioimmunoconjugates against the panhaematopoietic antigen C D 4 5 , a  group at the F r e d H u t c h i n s o n C a n c e r Research Centre was able to deplete B M T recipients o f host lymphohaematopoietic cells f o r the purpose o f pretransplant c o n d i t i o n i n g w h i l e sparing the radiosensitive organs such as the l u n g s . 7 3  7 4  Identification o f therapeutically  useful m o n o c l o n a l antibodies and the p r o d u c t i o n o f h u m a n i s e d mouse antibody w i l l significantly i m p r o v e the utility o f in vivo immunotherapy  7 5  M o n o c l o n a l antibodies have  also been used therapeutically i n the ex vivo manipulation o f autologous or allogeneic bone m a r r o w grafts f o r the purpose o f l e u k a e m i c c e l l p u r g i n g or the r e m o v a l o f G v H D m e d i a t i n g donor T l y m p h o c y t e s , r e s p e c t i v e l y .  76  Regardless, w i d e s p r e a d use o f  immunoconjugates i n l e u k a e m i a treatment is far f r o m reality. D e v e l o p m e n t o f antibody- based diagnostics has made more o f an impact i n the overall c l i n i c a l management o f l e u k a e m i a , m u c h more so than the antibody- based therapeutics discussed above. Haematopoietic maturation is a c c o m p a n i e d by the appearance and disappearance o f developmentally- restricted c e l l surface antigens. Leukaemogenesis f r o m normal haematopoietic precursor cells means that l e u k a e m i c cells w o u l d share many o f the lineage antigens present o n n o r m a l cells, hence the.difficulty i n the specific targeting o f l e u k a e m i c cells i n therapeutics. I m m u n o p h e n o t y p i n g o f peripheral b l o o d and bone m a r r o w aspirates w i t h panels o f antibodies against lineage- specific antigens has dramatically i m p r o v e d the specificity o f traditional m o r p h o l o g i c and cytochemical diagnosis; f o r example, the definitive diagnosis o f F A B M 7 acute megakaryoblastic leukaemia using m o n o c l o n a l antibodies that recognise the platelet antigens glycoprotein Ib and l i b - I l i a .  7 7  '  7 8  In a d d i t i o n , antibody- based diagnostics also p r o v i d e i n f o r m a t i o n w i t h  significant prognostic implications such as the identification o f biphenotypic leukaemia, w h i c h coexpresses both m y e l o i d and l y m p h o i d markers a n d m a y require more aggressive  14  treatment.  79  I m p r o v e d detection o f l e u k a e m i c cells w i t h f l o w cytometry also permits the  sensitive detection o f m i n i m a l residual disease d u r i n g and posttherapy, w h i c h h o p e f u l l y s h o u l d translate into better c l i n i c a l management o f the p a t i e n t .  iv. Cell- mediated  80  immunotherapy  B a c i l l u s Calmette- G u e r i n ( B C G ) and other adjuvants such as Parvum  Corynebacterium  have been used experimentally as i m m u n o m o d u l a t o r y agents for l e u k a e m i a ,  unfortunately w i t h m i x e d results due to the nonspecific nature o f the elicited i m m u n e response.  8 1 - 8 3  Several groups were actively i n v o l v e d i n therapeutic trials i n w h i c h patients  were vaccinated w i t h allogeneic l e u k a e m i c cells (live or irradiated), often coadministered with adjuvants.  84  H o w e v e r , realisations o f the i m m e n s e potential o f c e l l u l a r i m m u n i t y i n  l e u k a e m i a therapy came o n l y after publication o f results f r o m c l i n i c a l trials conducted by Steven R o s e n b e r g ' s group at the N C I . L y m p h o c y t e s isolated f r o m the d r a i n i n g l y m p h nodes or b i o p s y samples o f patients w i t h s o l i d tumours were stimulated in vitro w i t h recombinant human interleukin- 2 (rhIL- 2). R e i n j e c t i o n o f these activated l y m p h o k i n e activated k i l l e r ( L A K ) cells or tumour- infiltrating l y m p h o c y t e s ( T I L s ) , often f o l l o w e d w i t h in vivo administration o f rhIL- 2, was f o u n d to have tremendous therapeutic effects on selected patient p o p u l a t i o n s .  8 5 , 8 6  D e v e l o p m e n t o f c l i n i c a l protocols that take advantage o f  the above discoveries i n leukaemia patients first came about in trials studying the efficacy o f in vivo administration o f rhIL- 2 . " 8 7  8 9  Surface M H C I presentation o f antigenic peptides  consisting o f the f u s i o n region o f the translocation product on l e u k a e m i c cells, f o r e x a m p l e BCR-  ABL  i n C M L , presents the p o s s i b i l i t y o f c e l l mediated a n t i l e u k a e m i a response i n the  patient. Presence o f f u s i o n peptide- specific c y t o t o x i c T l y m p h o c y t e s ( C T L s ) has been demonstrated; h o w e v e r , the c l i n i c a l s i g n i f i c a n c e o f these cells remains a m b i g u o u s . 9 0  9 1  Investigators soon realised that active and adoptive immunotherapy c o u l d be incorporated into B M T s f o r various malignancies. H a n d l i n g o f the autologous or allogeneic graft ex vivo offers unique opportunities for the generation o f a leukaemia- specific i m m u n e response in vitro as a f o r m o f bone m a r r o w p u r g i n g in a u B M T and in v i v o to eradicate m i n i m a l residual  15  disease in both a l B M T and a u B M T patients postgraft reinfusion. These f o r m s o f i m m u n e manipulation w i l l be discussed in a later section.  v. Murine  models of human  leukaemia  B a s i c i m m u n o l o g i c a l research p r i n c i p a l l y focuses on the development and effector function o f members o f the l y m p h o i d compartment. H o w e v e r , one can easily appreciate that information garnered f r o m the study o f the i m m u n e system can be applied to leukaemia research (and not just restricted to l y m p h o i d leukaemia). O n e e x a m p l e is the development o f the S O D - hu m o d e l o f h u m a n l y m p h o p o i e s i s by M c C u n e ' s t e a m . ' 9 2  9 3  T h i s concept  was later extended to the study o f human haematopoietic development i n a murine host. T h e S C I D - hu system is an invaluable tool f o r the study o f human haematopoiesis because the available in vitro assays can o n l y detect progenitor cells w i t h l i m i t e d proliferating and replating potentials; the murine system also offers an opportunity to test the in vivo effects of various cytokines and therapeutic protocols o n h u m a n b l o o d c e l l d e v e l o p m e n t .  94  The  same system later a l l o w e d f o r the identification o f a rare population o f h u m a n m y e l o i d leukaemic precursor cells that behave s i m i l a r l y in the i m m u n o c o m p r o m i s e d murine hosts as i n the o r i g i n a l h u m a n p a t i e n t s .  95  T h e above examples illustrate the importance o f the  murine system for the study o f h u m a n haematopoiesis; i n addition, phylogenetic closeness between the t w o species means the sharing o f genes, developmental pathways, and signalling pathways. Genetic alterations i n transgenic m i c e and gene k n o c k o u t m i c e provide numerous clues to the normal and abnormal f u n c t i o n i n g o f the h u m a n haematopoietic system. E x p r e s s i o n (increased or ectopic) or deletion o f genes i m p l i c a t e d i n h u m a n leukaemias in the mouse w i l l often recapitulate the human phenotype. N e o p l a s t i c development in ber- abl transgenic m i c e and i n m i c e reconstituted w i t h haematopoietic stem cells infected with ber- abl carrying retroviruses partially recapitulated the human phenotype.  9 6 - 9 8  H o w e v e r , the occasional unexpected results serve to r e m i n d the  investigators of the c o m p l e x i t y o f the b i o l o g i c a l system and the fact that cancer often results f r o m polygenetic cooperativity. Serendipitous discovery by T a k M a k ' s group o f natural l e u k a e m i a resistance in the T c R V l . U y 4 C Y 4 transgenic m i c e w i l l certainly spur additional y  16  research into the role o f the y8 T c e l l population i n i m m u n e s u r v e i l l a n c e . " In a separate study, T a k M a k ' s group deleted the mouse equivalent o f the gene c o d i n g f o r the h u m a n H o d g k i n ' s Disease antigen C D 3 0 ; the resulting impairment i n l y m p h o c y t e c e l l death signalling and t h y m i c deletion offers an intriguing g l i m p s e into the n o r m a l regulatory role o f this protein i n the o r g a n i s m , and p o s s i b l y its role i n H o d g k i n ' s D i s e a s e .  1 0 0  Recently,  H o l t s c h k e and colleagues engineered a k n o c k o u t mouse strain that faithfully recapitulates the h u m a n equivalent o f C M L .  1 0 1  M i c e without functional interferon consensus b i n d i n g  sequence protein (ICSBP"'") develop acute l e u k a e m i a after an initial p r o d r o m a l period characterised b y benign neutrophilia. N u m e r o u s other examples also serve to illustrate the importance o f the mouse m o d e l f o r the study o f h u m a n l e u k a e m i a , either i n its pathogenesis or i n the search f o r different treatment modalities - in vivo  vi. Gene- based  Veritas.  therapeutics  A s mentioned i n an earlier section, identification o f leukaemia- specific genetic changes i n patients offers unique targets f o r m o l e c u l a r therapy. E n c o u r a g i n g results i n the development o f antisense technology w i t h o l i g o d e o x y n u c l e o t i d e s ( O D N s ) , retroviral vectors, or r i b o z y m e s serve to p r o p e l the d i s c i p l i n e f o r w a r d . E x a m p l e s i n c l u d e the specific eradication o f ber- abt m R N A and the growth o f ber- abt cells w i t h junction- region specific antisense O D N s .  1 0 2  S i m i l a r results were obtained w i t h retrovirus expressing  antisense ber- abl c D N A and r i b o z y m e s .  1 0 3  '  1 0 4  C l e a r l y , one can appreciate the potential  applications o f this technology i n l e u k a e m i a therapy, either i n bone m a r r o w p u r g i n g in vitro or the eradication o f m i n i m a l residual disease in vivo. Recent technical advances i n the design o f nuclease- resistant nucleotides and o f cationic l i p o s o m e delivery systems enable greater latitude i n antisense- based t h e r a p y .  105  T h e current system is restricted to the  i n h i b i t i o n o f m R N A transcription without affecting the source o f the corrupted genetic code; however, the future o f gene- specific therapeutics appears to lie w i t h catalytic O D N s or r i b o z y m e s w i t h the capability to effect site- specific D N A hence permanent corrections.  1 0 6  17  c. S u p p o r t i v e t h e r a p y  i. G- CSF and GM-  CSF support for chemotherapy-  induced  neutropaenia  Haematotrophic cytokines, particularly the c o l o n y stimulating factors ( C S F s ) in their current capacity are not considered to be true therapeutic agents. A notable exception is the successful application o f a- interferon ( I F N a ) f o r the treatment o f c h r o n i c phase C M L and hairy c e l l l e u k a e m i a .  1 0 7  '  1 0 8  C S F s essentially play an ancillary role in l e u k a e m i a  management; principally in accelerating normal haematopoietic recovery postradiochemotherapy to reduce the incidence o f opportunistic infections, thereby f u l f i l l i n g the same function as S t r u m i a ' s " l e u k o c y t e c r e a m " more than fifty years ago. U t i l i s a t i o n o f C S F s in leukaemia management poses the theoretical d i l e m m a o f the unintentional stimulation o f l e u k a e m i c g r o w t h . In vitro studies have s h o w n that many l e u k a e m i c cells upregulate the expression o f both C S F s and their c o r r e s p o n d i n g receptors.  109  C S F s are mitogenic for both p r i m a r y l e u k a e m i c cells and c e l l lines in vitro  and in vivo:  In a d d i t i o n , p r o l o n g e d use o f granulocyte- C S F (G- C S F ) in patients w i t h  95  severe congenital neutropaenia ( K o s t m a n n ' s syndrome) m a y augment an already increased risk o f A M L .  A recent study revealed mutations in the d o m a i n responsible for the  1 1 0  transmission o f maturation signals in the G- C S F receptor gene and is i m p l i c a t e d in the pathogenesis o f congenital neutropaenia and A M L , as w e l l as the possible contributory role o f therapeutic G- C S F in l e u k a e m o g e n e s i s .  111  Nevertheless, at the present time the c l i n i c a l  benefits o f exogenous G- C S F in congenital neutropaenia o u t w e i g h the elevated risk o f •subsequent-AML d e v e l o p m e n t ,  112  ,  Results f r o m two recent multicentre, double- b l i n d , r a n d o m i s e d , placeboc o n t r o l l e d c l i n i c a l trials questioned the efficacy o f G- C S F and G M - C S F in elderly A M L patients.  1 1 3  '  1 1 4  B o t h studies s h o w e d that use o f either c y t o k i n e d i d not lead to stimulation  o f l e u k a e m i c relapse; however, despite improvements in objective parameters such as shortening o f the neutropaenic p e r i o d , overall s u r v i v a l was not enhanced. Treatmentrelated failures (i.e. intolerance to chemotherapy) and possibly the presence o f a b i o l o g i c a l l y distinct f o r m o f A M L remain the major problems that plague A M L management i n the e l d e r l y .  1 1 5  U n f o r t u n a t e l y , 6 0 % o f A M L s o c c u r in patients 6 0 years and older. A g e -  18  related intolerance to chemotherapy also extends to the intense preparative regimen for a l B M T and the subsequent episodes o f G v H D ; therefore, most centres refuse to p e r f o r m a l B M T o n patients 55 years and older. O n e can clearly appreciate the urgency in identifying new agents that c o u l d protect the patient f r o m the severity o f l e u k a e m i a treatments.  ii. Infection and haemostasis control A n t i b i o t i c s are often used p r o p h y l a c t i c a l l y or e m p i r i c a l l y to manage episodes o f p y r e x i a o f u n k n o w n o r i g i n in neutropaenic p a t i e n t s .  1 1 6  -  1 1 7  F u n g a l infections have proven  to be more recalcitrant to therapy; however, introduction o f the less t o x i c f l u c o n a z o l e and l i p o s o m a l a m p h o t e r i c i n B s h o u l d i m p r o v e somewhat the c l i n i c a l e x i g e n c y .  1 1 8  -  1 1 9  Itraconazole, another antifungal agent, was d i s c o v e r e d to have the ability to reverse the m u l t i d r u g resistance phenotype, p o s s i b l y v i a steric interference o f the membrane glycoprotein p u m p .  1 2 0  C a r e f u l selection o f b l o o d product donors contribute to the  reduction o f v i r a l transmission; in addition, application o f f l o w cytometry i m p r o v e d the rapidity o f viral diagnosis and therefore t r e a t m e n t .  121  Nevertheless, i n f e c t i o n still remains  one o f the thorniest problems in the management o f l e u k a e m i c p a t i e n t s .  122  Limited  availability o f effective antifungal and antiviral agents has created significant interest in the use o f intravenous g a m m a g l o b u l i n as p r o p h y l a x i s against infections, especially i n the B M T setting.  1 2 3  -  1 2 4  Recent advances i n platelet storage techniques s h o u l d enable the  p r o l o n g e d and safe storage o f p l a t e l e t s .  125  H o w e v e r , the most important development in  haemostatic research was the successful identification and c l o n i n g o f the h u m a n platelet growth factor or thrombopoietin ( T P O ) .  1 2 6  P r e c l i n i c a l research has demonstrated that T P O ,  often i n c o m b i n a t i o n w i t h other cytokines, has significant m i t o g e n i c effects on megakaryocytes and the resultant platelet counts; clearly desirable i n patients receiving myelosuppressive chemotherapy.  127  U n d e r s t a n d i n g o f leukaemia and the c l i n i c a l management o f the disease has come a l o n g w a y since O s i e r ' s era. H o w e v e r , except f o r selected diseases such as paediatric A L L or A P L , overall survival o f the leukaemic patient is still fairly d i s m a l . T e c h n o l o g i c a l advances i n m o l e c u l a r diagnosis does not necessarily translate into better outcome.  19  N o w a d a y s , c l i n i c i a n s have k n o w l e d g e o f the exact location o f the c h r o m o s o m a l translocation or the type o f basepair alteration. Unfortunately, the majority o f treatments are still archaic i n v o l v i n g various c y t o t o x i c substances w i t h little selectivity; not m u c h different f r o m O s i e r ' s prescription o f " c l e a n air and absence o f w o r r y " f o r l e u k a e m i c patients. T h e next hurdle i n disease management w i l l be the translation o f k n o w l e d g e and s k i l l gained i n m o l e c u l a r diagnosis to m o l e c u l a r therapeutics - i.e. targeting and the corrections o f the genetic lesions.  1.3 TRANSPLANTATIONS  OF HAEMATOPOIETIC  STEM CELLS:  CURRENT  STATUS Intravenous infusion o f haematopoietic progenitor and stem cells ( H P C s & H S C s ) into a patient w i t h damaged or defective bone m a r r o w is already an established f o r m o f treatment for several benign and malignant haematological conditions. C e l l s can be harvested f r o m the m a r r o w cavities o f the donor, hence the term bone m a r r o w transplantation ( B M T ) ; i n peripheral b l o o d stem c e l l transplantation ( P B S C T ) , circulating stem and progenitor cells are harvested f r o m the patient (autologous) or an allogeneic donor through apheresis, an extracorporeal process w h i c h p h y s i c a l l y separates b l o o d into its cellular components. Placental and u m b i l i c a l c o r d b l o o d represents a third source o f stem cells w i t h unique properties w h i c h appear to permit greater margins i n donor: recipient H L A disparity. T h e f o l l o w i n g section w i l f f o c u s o n recent;advances-in stem c e l l transplantation.  20  a. Bone Marrow Transplantation  i. Allogeneic  bone marrow transplantation  (BMT)  (alBMT)  In brief, successful engraftment o f allogeneic haematopoietic cells i n a foreign host requires a modus vivendi between the graft and the host. T h i s is aided b y the careful selection o f donors based on the m a t c h i n g o f major and even m i n o r h i s t o c o m p a t i b i l i t y antigen l o c i as w e l l as the effective posttransplantation management o f the patient in the f o r m o f adequate i m m u n o s u p p r e s s i o n w i t h vigilant infection c o n t r o l and c l i n i c a l precautions.  1. H L A m a t c h i n g L i m i t a t i o n in the size o f the o p t i m a l m a r r o w donor p o o l i.e. h u m a n leukocyte 'antigen or H L A matched siblings drives the search f o r other sources o f stem cells. L e s s desirable donors include H L A matched unrelated donors ( M U D s ) and related donors w i t h partial H L A matches. T h e science o f H L A matching has progressed f r o m p h e n o t y p i n g through the use o f the m i x e d l y m p h o c y t e reaction ( M L R ) and serology to the more precise method o f genotyping u s i n g the polymerase chain reaction ( P C R ) . A c c u r a t e m a t c h i n g o f donors and recipients s h o u l d decrease the incidence o f graft rejection and o f graft- versushost disease ( G v H D ) ; h o w e v e r , the paucity o f potential w e l l - m a t c h e d donors often necessitates the use o f m a r r o w s f r o m the suboptimal donor p o o l s m e n t i o n e d above. D N A t y p i n g o f - H L A genes u s i n g analytical techniques such as D N A restriction fragment length p o l y m o r p h i s m ( R F L P ) , P C R - sequence- s p e c i f i c o l i g o n u c l e o t i d e probe t y p i n g , and P C R 'fingerprinting' p l a y e d significant roles i n i m p r o v i n g the outcome o f M U D transplantations b y o p t i m i s i n g the H L A m a t c h i n g between unrelated donors and r e c i p i e n t s .  39  Creation of  the national and global registries o f volunteer bone m a r r o w donors facilitates the search for potential M U D donors. Nonetheless, G v H D and graft rejection w i l l r e m a i n a fact o f life in a l B M T regardless o f the improvements made in H L A m a t c h i n g technology s i m p l y because of the enormous p o l y m o r p h i s m o f the H L A l o c i and consequently the scarcity o f perfectly matched donors. Therefore, i m p r o v e m e n t o f the outcome o f a l B M T requires developments  21  o f n o v e l immunosuppressive regimens and patient management techniques such as the effective control o f infections, in addition to better H L A matching technology.  2. Prevention and the management o f graft- versus- host disease ( G v H D ) C y c l o s p o r i n A ( C Y A ) , the venerable antirejection drug, revolutionised the management o f graft/ host H L A discordance in B M T . H o w e v e r , C Y A is nephrotoxic and requires frequent drug level m o n i t o r i n g . T h e n o v e l m i c r o e m u l s i o n f o r m u l a t i o n o f C Y A appears to i m p r o v e the pharmacokinetics o f the drug in patients, therefore a l o w e r d r u g dose is required to maintain the same level o f i m m u n o s u p p r e s s i o n .  128  Several ongoing  a l B M T trials are being conducted to compare the efficacy o f C Y A w i t h the macrolide antibiotic F K 5 0 6 , an i m m u n o s u p p r e s s i v e agent w i t h a relatively successful history in l i v e r transplantations.  129  -  130  R a p a m y c i n ( R A P A ) , another naturally d e r i v e d macrolide- based  immunosuppressant, binds to the same m o l e c u l a r target as F K 5 0 6 yet mediates different d o w n s t r e a m signalling pathways. P r e l i m i n a r y m u r i n e studies indicated the potential o f R A P A in M H C - mismatched B M T ; h o w e v e r , this agent has several unique i m m u n o l o g i c a l effects w h i c h warrant additional studies p r i o r to any c l i n i c a l testing on p a t i e n t s .  131  Ex vivo  T c e l l depletion ( T C D ) using a - C D 3 ( O K T 3 ) or a - C D 5 2 ( C a m p a t h l ) m o n o c l o n a l antibodies is an effective method o f preventing acute G v H D by r e m o v i n g donor T l y m p h o c y t e s . H o w e v e r , this often comes w i t h the price o f increased incidences o f l e u k a e m i a relapse and graft f a i l u r e .  1 3 2  -  1 3 3  T h e expression o f other T cell- specific antigens  are also e x p l o i t e d in antibody-, mediated ex vivo and in vivo T C D ; i n fact, several trials have c o m b i n e d both modalities of T C D i n the hope o f i m p r o v i n g the c l i n i c a l outcome, especially o f M U D transplantations. M o n o c l o n a l antibodies specific f o r the T c R afj c h a i n , L F A 1 , C D 2 , and C D 5 have all been used s i n g l y or i n different c o m b i n a t i o n s in T C D . 1 3 6  1 3 4 -  Ex vivo T C D is t y p i c a l l y a c c o m p l i s h e d by antibody- c o m p l e m e n t mediated lysis o f the  target cells w h i l e in vivo T C D is often carried out by antibody- t o x i n conjugates. Nonetheless, the c l i n i c i a n must realise that T cells mediate proper engraftment o f the donor stem cells, i m m u n e surveillance o f leukaemic relapses ( G v L ) as w e l l as viral- associated l y m p h o p r o l i f e r a t i v e disorders, and unfortunately also G v H D .  22  1 3 7  T h e graft- versus-  l e u k a e m i a effect ( G v L ) is mediated by donor T cells w h i c h recognise residual leukaemic cells in the patient. Studies have demonstrated that G v L is responsible f o r the l o w e r rates o f l e u k a e m i c relapses in patients r e c e i v i n g a l B M T ; in addition, reinfusions o f donor- derived "buffy- c o a t " containing l y m p h o c y t e s have been s h o w n to suppress l e u k a e m i c relapse posta l B M T in C M L patients.  138  . Therefore, overzealous and i n j u d i c i o u s use o f T C D w i l l affect  not only the f i n a l outcome o f the graft but ultimately the welfare o f the p a t i e n t .  139  Better  definition o f the T c e l l subset(s) responsible f o r G v H D s h o u l d help in the selective r e m o v a l o f this p o p u l a t i o n without affecting the o t h e r s .  140  C h r o n i c G v H D , a different f o r m o f graft/host c o n f l i c t than acute G v H D , can still occur despite p h a r m a c o l o g i c a l control o f the latter; c h r o n i c G v H D manifests itself as a m u l t i s y s t e m disorder w h i c h resembles the c o l l a g e n vascular d i s e a s e s .  141  Prolongation of  the s u r v i v a l o f the transplantation recipient highlights the problems associated w i t h this disorder. M a n a g e m e n t o f chronic G v H D w i t h t h a l i d o m i d e offers an attractive alternative to the traditional regimen o f prednisolone and azathiaprine because o f the f o r m e r ' s effectiveness in treating s k i n c h r o n i c G v H D without m y e l o t o x i c i t y .  1 4 2  3. Identification o f m i n o r histocompatibility antigens ( m H C ) T h e m i n o r histocompatibility antigens ( m H C ) denote transplantation antigens w h i c h are not encoded by the major histocompatibility antigen l o c i ( H L A in the h u m a n and H- 2 in the mouse). P o l y m o r p h i s m s o f these genes in the p o p u l a t i o n contribute to their roles as allorgactive, antigens in organ transplantations. T h e most-obvious m H C is the sex- l i n k e d male antigen H- Y , w h i c h elicits alloreactive T c e l l responses in male patients receiving M H C - matched female g r a f t s .  143  S i n c e o n l y H L A m a t c h i n g is p e r f o r m e d i n c l i n i c a l  a l B M T , G v H D often can result f r o m mismatches in the m H C even in transplantations between H L A - identical i n d i v i d u a l s . R e c e n t l y , t w o reports h i g h l i g h t e d the importance o f the non- sex- l i n k e d m H C i n c l i n i c a l a l B M T s . G o u l m y and colleagues demonstrated the importance o f H A - 1 m a t c h i n g in H L A - matched a l B M T .  1 4 4  Interestingly, the authors  s h o w e d that mismatches i n H A - 1 contributes to G v H D o n l y i n donors and recipients positive f o r H L A - A 2 ; therefore, a c o m p l e x interplay exists between m H C (simple "protein  23  antigens" presented b y the M H C ) and the M H C (alloantigens, protein antigens, and b i n d i n g partners o f potential m H C ) , therefore not a l l m H C s contribute to G v H D . In another study, direct sequencing o f the c o m p l e m e n t a r y D N A  o f the C D 3 1 ( P E C A M - 1)  adhesion m o l e c u l e revealed a single p o l y m o r p h i s m i n n o r m a l subjects; further investigation i n a l B M T patients w h o received bone m a r r o w cells f r o m H L A - identical s i b l i n g donors revealed a positive correlation between donor: recipient C D 3 1 p o l y m o r p h i s m and the incidence of G v H D .  1 4 5  U n f o r t u n a t e l y , the i n s u f f i c i e n c y o f H L A - matched donors precludes the routine matching o f m H C in actual c l i n i c a l practice. H o w e v e r , as indicated in an a c c o m p a n y i n g editorial, m H C m a t c h i n g m a y be beneficial in situations where more than one H L A identical donor is available or w h e n the degree o f m H C disparity can be used to tailor the extent o f the i m m u n o p r o p h y l a x i s for the p a t i e n t .  146  4. N o v e l indications for a l B M T in nonmalignant and malignant disorders A recent multicentre trial demonstrated the curative potential o f a l B M T in paediatric patients s y m p t o m a t i c f o r s i c k l e cells anaemia ( S C A ) .  1 4 7  T h e authors s h o w e d that a l B M T  was able to halt the various s y m p t o m s o f S C A and to abolish the need f o r R B C transfusions. S i m i l a r l y , patients suffering f r o m another b e n i g n h a e m a t o l o g i c a l disorder, |3thalassaemia, also benefited f r o m a l B M T .  1 4 8  A l B M T , w h e n used r e s p o n s i b l y , is able to  i m p r o v e the quality o f l i f e in patients suffering f r o m various f o r m s o f b e n i g n haematological'disorders.  149  T h i s however w i l l increase the d e m a n d f o r an already scarce  resource and w i l l p r o b a b l y p r o v i d e an impetus for u t i l i s i n g n o v e l sources o f stem cells such as u m b i l i c a l c o r d b l o o d and m o b i l i s e d peripheral b l o o d stem cells. F i n a l l y , several groups are actively pursuing the use o f a l B M T as a f o r m o f adoptive i m m u n o t h e r a p y ; i n f u s i o n o f donor leukocytes f o r l e u k a e m i c relapse control post- a l B M T and the transfer o f tumour antigen specific i m m u n i t y f r o m an i m m u n i s e d allogeneic d o n o r are t w o such e x a m p l e s .  24  1 5 0  ii. Autologous  bone marrow transplantation  (auBMT)  " w a i t i n g for a M U D is l i k e w a i t i n g f o r G o d o t " - Sometimes, a patient becomes an u n w i t t i n g p l a y e r i n B e c k e t t ' s p l a y . [ G o l s t o n e , 1993 #610] A u t o l o g o u s B M T ( a u B M T ) offers the o n l y hope for elderly patients or without H L A - compatible donors; elderly patients are especially susceptible to the deleterious effects o f G v H D associated w i t h a l B M T and therefore most centres do not p e r f o r m the procedure i n patients m o r e than 5 0 years o l d .  1 5 1  -  1 5 2  T h e o r e t i c a l l y , bone m a r r o w harvest  should be performed w h e n the p r o b a b i l i t y o f neoplastic c e l l contamination is at its lowest i.e. d u r i n g r e m i s s i o n . T h i s p e r i o d u s u a l l y f o l l o w s successful intensive c h e m o t h e r a p y , i.e. at first complete r e m i s s i o n postinduction therapy i n A M L and d u r i n g c h r o n i c phase i n C M L . T h e patient then receives high- dose chemotherapy and radiation therapy f o l l o w e d by reinfusion o f the harvested m a r r o w cells. T h e l a c k o f G v H D i n a u B M T unfortunately means the absence o f the G v L effect; therefore, the higher rate o f l e u k a e m i c relapses essentially balances out the l o w e r incidence o f B M T procedure- related mortality (i.e. G v H D ) in a u B M T patients.  152  In a d d i t i o n , r e i n f u s i o n o f contaminating t u m o u r cells also  increase the chance o f relapse. T w o independent studies, indirectly and directly, demonstrated the contributory role o f u n p u r g e d autografts i n subsequent relapses. T h e tumour- specific translocation t(14; 18), w h i c h j o i n s together the bcl- 2 gene w i t h the i m m u n o g l o b u l i n heavy chain promoter, is c o m m o n to a majority o f f o l l i c u l a r nonH o d g k i n ' s l y m p h o m a ( N H L ) and 3 0 % o f B- c e l l N H L and a l l o w s the use o f P C R to detect rare l y m p h o m a cells. P C R p o s i t i v i t y o f the reinfused purged autograft correlated w i t h the incidence o f subsequent l y m p h o m a r e l a p s e .  153  B r e n n e r and colleagues u n a m b i g u o u s l y and  directly demonstrated, v i a ex vivo retroviral m a r k i n g o f the harvested product, that neoplastic cells i n the reinfused graft contribute to subsequent relapses i n A M L and neuroblastoma p a t i e n t s .  1 5 4  '  1 5 5  H o w e v e r , this does not discount the presence o f  endogenous neoplastic cells that have s u r v i v e d the c o n d i t i o n i n g r e g i m e n . S e v e r a l groups have tried to i m p r o v e the odds o f a u B M T b y the administration o f l o w dose c y c l o s p o r i n A , w h i c h appears to disrupt i m m u n e tolerance and thereby provokes a f o r m o f G v L but also G v H D i n the p a t i e n t .  156  25  1. a u B M T and chronic myelogenous l e u k a e m i a ( C M L ) A l B M T is currently the sole curative regimen f o r chronic myelogenous l e u k a e m i a ( C M L ) p o s s i b l y because o f the role o f G v L .  1 4 0  C M L can strike at all ages yet the m e d i a n  age o f incidence is 5 0 years, w h i c h renders most patients unsuitable f o r a l B M T f o r reasons discussed a b o v e .  1 5 7  T h e r e f o r e , intensive chemotherapy c o u p l e d w i t h autologous bone  m a r r o w rescue becomes an alternative regimen f o r those patients i n w h i c h a l B M T is not indicated.  1 5 8  '  1 5 9  C o n t a m i n a t i o n o f the harvested m a r r o w b y C M L c e l l s and the absence o f  G v L effects c o m b i n e to increase the incidence o f relapse i n patients r e c e i v i n g unpurged autologous m a r r o w g r a f t s .  160  Presence o f the unique m o l e c u l a r lesion ber- abl i n C M L  cells a l l o w s c l i n i c i a n s to assess the extent o f m i n i m a l residual disease ( M R D ) i n the patient's m a r r o w and c i r c u l a t i o n u s i n g sensitive reverse transcriptase- polymerase c h a i n reaction (RT- P C R ) , w h i c h can detect a single ber- abl  +  c e l l i n 1 0 to 1 0 n o r m a l c e l l s . 5  6  1 6 1  N e w e r quantitative R T - P C R ( q R T - P C R ) m e t h o d o l o g y based o n the c o m p e t i t i v e a m p l i f i c a t i o n o f exogenous competitor templates permits the quantitation o f in vivo M R D or the degree o f autologous graft contamination as w e l l as the efficacy o f the graft p u r g i n g procedure. Serial m o n i t o r i n g o f the patient posttransplantation u s i n g q R T - P C R facilitates the early detection o f r e l a p s e .  162  H o w e v e r , M i y a m u r a and colleagues have s h o w n that bcr-  abl P C R positivity post- a l B M T does not necessary correlate w i t h c l i n i c a l r e l a p s e s .  163  Therefore, c l i n i c a l r e m i s s i o n m a y exist as a state o f e q u i l i b r i u m between the residual l e u k a e m i c cells and the i m m u n e s y s t e m .  1 6 4  Regardless, P C R technology a l l o w s f o r the  rapid evaluation o f ex vivo p u r g i n g e f f i c a c y and is i nval uabl e f o r the c o m p a r i s o n o f the different p u r g i n g systems. W i t h o u t significant improvements in the management o f G v H D and'the development o f other forms o f therapies, a u B M T w i l l remain a feasible alternative i n l e u k a e m i a treatment. D e v e l o p m e n t o f novel ex vivo techniques such as l e u k a e m i c c e l l p u r g i n g and i m m u n e m o d u l a t i o n w i l l make a u B M T a more viable option than a l B M T . 166  26  1 6 5  '  b. Peripheral blood stem cell transplantations Peripheral b l o o d stem c e l l ( P B S C ) transplantation can be performed i n either autologous or allogeneic setting. Advantages and problems associated w i t h a l B M T and a u B M T are also applicable respectively to allogeneic and autologous P B S C grafts. H o w e v e r , transplantation o f P B S C s i n general has several advantages over B M T - the f o r m e r procedure does not require general anaesthesia and P B S C grafts mediate faster haematological recovery than m a r r o w grafts, p o s s i b l y because o f an abundance o f c o m m i t t e d progenitor cells i n the c i r c u l a t i o n .  1 6 7  '  1 6 8  T h e procedure is especially useful i n  paediatric patients because o f the risks associated w i t h bone m a r r o w harvests i n children.  1 6 9  T h e harvesting o f P B S C can be a c c o m p l i s h e d b y leukapheresis, an  extracorporeal procedure i n w h i c h the p e r s o n ' s b l o o d is circulated through a b l o o d c e l l separator such as the F e n w a l l 3 0 0 0 C S and cells w i t h i n a certain range o f specific density (mainly mononuclear cells) are isolated v i a centrifugal elutriation. L y m p h o c y t e s and monocytes are collected along w i t h the c o m m i t t e d progenitor and stem cells, w h i c h can be selected positively using m o n o c l o n a l antibody specific f o r the haematopoietic stem c e l l antigen C D 3 4 . M o s t o f the experience i n P B S C transplantation has been w i t h autologous grafts; h o w e v e r , allogeneic P B S C transplantation is g a i n i n g more acceptance s i m p l y because o f the relative ease o f the harvesting procedure. " M o b i l i s a t i o n " is necessary to increase the number o f stem and progenitor cells in the circulation prior to the actual harvests. R e c e n t advances i n the functional characterisation o f P B S C and the development o f n o v e l m o b i l i s a t i o n and stem cell expansion protocols have established P B S C transplantation as an integral part o f the standard treatment regimen i n o n c o l o g y . F o r e x a m p l e , f l o w cytometric analysis , 0 f i C D 3 4 cells i n the P B S C harvests enables direct .and +  rapid enumeration o f progenitor and stem cells w h i c h provides an index o f the reconstitutive potential o f the harvested product without resorting to the labour- intensive and time- c o n s u m i n g b i o l o g i c a l assays ( C F U - G M assays, delta assays, and long- term culture- initiating c e l l a s s a y s ) .  170  Furthermore, B r u g g e r and colleagues recently s h o w e d  that ex vivo e x p a n s i o n o f p o s i t i v e l y selected C D 3 4 cells f r o m a single session o f +  leukapheresis was sufficient to effect rapid and sustained haematopoietic e n g r a f t m e n t . T h e above findings c o u p l e d w i t h enhanced design o f the c e l l separator and better  27  171  h a e m o d y n a m i c support d u r i n g leukapheresis significantly increased the universality o f P B S C transplantation.  /. Autologous transplantation of PBSC in cancer patients P B S C transplantation in this setting bypasses problems associated w i t h the autologous m a r r o w such as inadequate s p e c i m e n size due to m a r r o w f i b r o s i s (from p e l v i c irradiation or disease process) and tumour contamination o f the m a r r o w space. T o obtain enough P B S C , m o b i l i s a t i o n o f the o n c o l o g y patient can be a c c o m p l i s h e d b y myeloablative chemotherapy in conjunction w i t h the administration o f recombinant haematopoietic growth factors such as G- C S F . F o r o b v i o u s reasons, o n l y g r o w t h factors are u s e d f o r al l og eneic donor m o b i l i s a t i o n . T h i s procedure increases the n u m b e r o f P B S C to a l e v e l w h i c h is practical for harvesting; usually three leukapheresis sessions are required to obtain the 2 X 1 0 C D 3 4 P B S C / k g o f recipient needed f o r adequate h a e m a t o l o g i c a l e n g r a f t m e n t . 6  +  172  C l i n i c i a n s , however, must be vigilant in dealing w i t h m o b i l i s e d autologous harvests f r o m these patients because the operation also increases the c i r c u l a t o r y t u m o u r l o a d .  1 7 3  -  1 7 4  Therefore, additional steps such as C D 3 4 positive selection c o u p l e d w i t h t u m o u r c e l l +  p u r g i n g are necessary i n autologous P B S C transplantations i n v o l v i n g l e u k a e m i c patients; whereas C D 3 4 positive selection alone w o u l d suffice for patients w i t h s o l i d tumours +  because o f the absence o f C D 3 4 expression on these t u m o u r c e l l s .  1 7 5  "  1 7 7  In allogeneic  P B S C transplantation, positive selection o f C D 3 4 c e l l s also reduces the p o s s i b i l i t y o f +  r e i n f u s i o n o f donor l y m p h o c y t e s , w h i c h can contribute to G v H D .  1 7 8  c. Umbilical cord blood transplantation H u m a n u m b i l i c a l c o r d b l o o d ( H U C ) , n o r m a l l y discarded after d e l i v e r y o f the foetus, is an unique source o f haematopoietic stem and progenitor c e l l s f o r transplantation. M o s t studies have f o c u s e d on H U C transplantations i n the paediatric setting because o f the fear o f insufficient cells f o r adult recipients, even though H U C contains approximately the same concentration o f stem cells as adult bone m a r r o w but o n l y at a t y p i c a l harvest v o l u m e  28  of 100 m l .  1 7 9  T h e unique developmental state or i m m u n o l o g i c a l naivete o f the H U C s  endows them w i t h the capability to engraft across disparate H L A barriers without resulting in s i g n i f i c a n t G v H D .  1 8 0  H U C s also appear to be u n u s u a l l y robust, p o s s i b l y due to their  developmental status, i n their ability to effect haematopoietic engraftment; therefore adult patients m a y benefit f r o m H U C t r a n s p l a n t a t i o n s .  181  Standardisation o f H U C c o l l e c t i o n ,  storage, and characterisation s h o u l d increase the use o f this f o r m o f stem c e l l transplantation.  182  d. Future directions T h e raison d ' etre o f stem cell rescue i n l e u k a e m i a therapy is the reconstitution o f the haematopoietic system damaged b y the h i g h dose chemotherapeutic agents and radiation treatment needed to eradicate leukaemic cells f r o m the host. T h e relative nonselectivity o f the current generation o f treatments results i n " c o l l a t e r a l " damage to n o r m a l tissues such as cells o f the haematopoietic system and other rapidly d i v i d i n g cells. D e v e l o p m e n t o f more specific antineoplastic agents such as gene- based or s i g n a l l i n g pathway specific antagonists w i l l m a k e this particular i n d i c a t i o n r e d u n d a n t .  152  In the future, stem c e l l  transplantation probably w i l l be used f o r the correction o f intrinsic genetic defects or i n customised m o d i f i c a t i o n s to the host i m m u n e system.  >>.k  1.4 EXPLOITABLE  a.  DIFFERENCES  -  BETWEEN NORMAL AND LEUKAEMIC  CELLS  Leukaemogenesis Oncogenesis i n cells o f haematopoietic o r i g i n is often a multistep event and involves  the acquisition o f oncogenic changes as w e l l as the loss or inactivation o f n o r m a l antiproliferative control m e c h a n i s m s .  1 8 3  '  1 8 4  A n i m a l viruses, especially those b e l o n g i n g to  the f a m i l y o f retroviridae, can cause l e u k a e m i a i n a variety o f m a m m a l i a n hosts. Recently,  29  G r o s s speculated that dormant o n c o g e n i c viruses may play a even bigger role i n a n i m a l leukaemogenesis than p r e v i o u s l y t h o u g h t .  185  S o m e oncogenic events are so potent that n o  other genetic changes are necessary f o r l e u k a e m i c transformation; h o w e v e r , successful evolution o f the leukaemic clone in the host often needs more than one genetic " h i t " i n the guise o f additional oncogenic changes or abrogation o f n o r m a l g r o w t h c o n t r o l .  1 8 6  B i o c h e m i c a l and p h y s i o l o g i c a l differences between normal and leukaemic cells f o r m the basis o f antileukaemic therapy; h o w e v e r , the differences can be quite subtle since leukaemogenesis arises f r o m n o r m a l haematopoietic precursors. T h e therapeutic w i n d o w o f opportunity between normal and malignant cells relies on the differential sensitivity to treatment w h i c h results f r o m a range o f d i s s i m i l a r i t i e s .  187  T h e f o l l o w i n g is a b r i e f  description of selected leukaemia- specific changes resulting f r o m the ber- abl translocation in C M L , and their implications in therapy.  b. Genetic differences Gene- based differences f o r m the foundation o f subsequent deviations i n signal transduction, adhesion properties, and response to extracellular s t i m u l i i n l e u k a e m i c cells  5 5  A c q u i s i t i o n o f transforming oncogenes for e x a m p l e , through point mutations and  c h r o m o s o m a l translocations, in c o n j u n c t i o n w i t h the loss o f tumour suppressor genes conceive the script for the ensuing l e u k a e m i c transformation. These changes, w h i c h are absent in normal cells, are responsible f o r the initiation as w e l l as maintenance o f the leukaemic phenotype. M e n t i o n e d p r e v i o u s l y , ber- abl and pml- raraaxe  respectively unique  to C M L and A P L ; both translocation events result in a plethora o f changes w h i c h e n d o w the cells w i t h growth autonomy. A b s e n c e o f oncogenic changes i n n o r m a l haematopoietic cells therefore allows specific targeting o f the leukaemic clone. C o m p l e m e n t a r y b i n d i n g o f specific antisense oligodeoxynucleotides ( O D N s ) or r i b o z y m e s can target leukaemic- specific sequences at the m R N A l e v e l .  1 0 5  S z c z y l i k and  colleagues have demonstrated the specificity o f the ber- abl junction- region antisense O D N in the i n h i b i t i o n o f ber- abl  +  cells by both R T - P C R as w e l l as c o l o n y a s s a y s .  102  Therefore,  identification o f genetic aberrations w h i c h are pathognomonic for l e u k a e m i a remains important i n not only the diagnosis, but also the treatment o f the disease.  30  c. Differences in intracellular signalling and cell death pathways S i g n a l transduction is paramount in the conveyance o f extracellular i n f o r m a t i o n to the nucleus or executive centre o f the c e l l ; it is also important i n the regulation o f cellular development. Aberrant signal transduction events can lead to dysregulated g r o w t h or oncogenesis, therefore such pathways have been deemed appropriate targets in cancer therapy.  188  T y r o s i n e p h o s p h o r y l a t i o n , w h i c h consists o f less than 2 % of total c e l l u l a r  protein phosphorylation, plays an extremely important role i n the mediation o f cellular s i g n a l l i n g . D y s r e g u l a t i o n of the expression and regulation o f protein tyrosine kinases ( P T K ) can result i n catastrophic changes i n cells and the o r g a n i s m .  1 8 9  F o r e x a m p l e , genetic  k n o c k o u t o f the CSK tyrosine kinase i n m i c e , w h i c h negatively regulates the SRC P T K , results i n substantial upregulation o f SRC kinase activity. A phenotype o f e m b r y o n i c lethality w i t h significant neural tube defects was e l i c i t e d f r o m the CSK k n o c k o u t m i c e .  1 9 0  T h e BCR- ABL f u s i o n protein i n C M L has s i g n i f i c a n t l y upregulated tyrosine kinase activity in c o m p a r i s o n to the w i l d - type ABL protein in n o r m a l haematopoietic c e l l s .  1 9 1  In  a d d i t i o n , BCR- ABL localises to the c y t o p l a s m whereas w i l d - type ABL e x p r e s s i o n i s restricted to the nucleus where it interacts w i t h growth regulatory proteins such as the retinoblastoma p r o t e i n .  1 9 2  '  1 9 3  . S i n c e tyrosine k i n a s e activation o f BCR- ABL appears to  be crucial i n the initiation and maintenance o f the leukaemic phenotype in C M L , signal transduction events mediated b y BCR- ABL therefore b e c o m e l o g i c a l targets for therapeutic interventions.  194  Several examples o f the successful use o f tyrosine kinase inhibitors have  been m e n t i o n e d p r e v i o u s l y . 5 8  5 9  T r a n s f o r m e d cells, in addition to changes w h i c h promote proliferation and growth autonomy, also are deficient in regulatory pathways resulting i n resistance to n o r m a l homeostatic controls. F o r e x a m p l e , loss o f the w i l d - type p53 phenotype results i n the abrogation o f intrinsic senescence control and sensitivity to apoptosis- i n d u c i n g anticancer agents.  1 9 5  -  1 9 6  Restoration o f the w i l d - type p 5 3 p h e n o t y p e , v i a retrovirus- or adenovirus-  mediated gene transfer, can therefore increase sensitivity to chemotherapeutic agents or effect cellular differentiation.  31  d. Response to extracellular regulatory signals L e u k a e m i c cells exhibit heightened responses to c y t o k i n e stimulation o r m a y even b e c o m e independent o f s u r v i v a l o r m i t o g e n i c factors. BCR- ABL e n d o w s c y t o k i n e independence to the interleukin- 3 (IL- 3) dependent c e l l line F D C P - 1 b y substituting f o r n o r m a l signal transduction events mediated b y IL- 3 b i n d i n g to its receptor; i n addition, BCR-  ABL, v i a its Src- h o m o l o g y 2 d o m a i n ( S H 2 ) , induces the secretion o f IL- 3 to effect  paracrine s t i m u l a t i o n .  1 9 7  '  1 9 8  Furthermore, S h u a i and colleagues have demonstrated that  S T A T 5 , a c y t o p l a s m i c protein w h i c h mediates G M - C S F s i g n a l l i n g , is constitutively activated b y BCR- ABL i n the absence o f G M - C S F .  1 9 9  T h e r e f o r e , BCR- A B L , through  its exaggerated P T K activity and the presence o f various adapter o r b i n d i n g regions such as the S H 2 and S H 3 d o m a i n s , can substitute f o r m i t o g e n i c and s u r v i v a l c y t o k i n e s w h i c h n o r m a l l y maintain homeostasis. A t the other extreme, evidence has s h o w n that l e u k a e m i c cells d o not respond to the growth inhibitory effects o f a f a m i l y o f cytokines loosely termed haematopoietic o r stem cell i n h i b i t o r s .  2 0 0  O n e such m o l e c u l e , the macrophage i n f l a m m a t o r y protein- l a ( M I P -  l a ) , is a m e m b e r o f the c h e m o k i n e f a m i l y but was o r i g i n a l l y characterised as an inhibitor o f haematopoietic stem c e l l p r o l i f e r a t i o n .  2 0 1  -  2 0 2  E a v e s and colleagues reported that C M L  cells are unresponsive to the regulatory effects o f M I P - l a .  2 0 3  A n o t h e r e x a m p l e is the lack  o f response to the tetrapeptide N - A c e t y l - Ser- A s p - L y s - P r o ( N - A c S D K P ) b y C M L but not b y n o r m a l haematopoietic c e l l s .  2 0 4  N - A c S D K P and p y r o G l u - G l u - A s p - C y s - L y s  ( p E E D C K ) b e l o n g to a new class o f l o w m o l e c u l a r weight haematopoietic inhibitors w h i c h b l o c k c e l l c y c l e progression f r o m the quiescent o r G phase; n o r m a l l y , quiescent cells are 0  not susceptible to the c y t o t o x i c effects o f cycle- specific chemotherapeutic agents and to ionising radiation.  205  D i f f e r e n t i a l sensitivity to the above m o l e c u l e s can be e x p l o i t e d i n  therapeutics. Selective c y c l e i n h i b i t i o n o f n o r m a l cells b y A c S D K P protects them f r o m chemotherapy, hyperthermia, radiation, as w e l l as P D T t r e a t m e n t s .  206  "  209  Selective  inhibition o f n o r m a l haematopoietic c e l l c y c l i n g b y N - A c S D K P , p E E D C K , and M I P - l a should permit dose intensification i n the in vivo treatment o f l e u k a e m i a patients or during ex vivo p u r g i n g .  2 1 0  32  e. Cell surface markers, adhesion properties and the haematopoietic microenvironment Haematopoiesis is a tightly regulated process and depends a great deal on the physical interactions between the haematopoietic cells and the stromal element i n the bone marrow. T h i s is c o n t r o l l e d b y the expression and activity o f m u l t i p l e adhesion m o l e c u l e s and their counter receptors. Engagement o f selected receptors o n the surface o f progenitor cells and stromal ligands results i n the transduction o f g r o w t h i n h i b i t o r y s i g n a l s .  2 1 1  G o r d o n and colleagues first demonstrated that C M L progenitor cells exhibit attenuated adhesion to the m a r r o w stroma, even though C M L progenitors express s i m i l a r numbers o f integrin adhesion receptors as than n o r m a l c o u n t e r p a r t s .  2 1 2  '  2 1 3  Subsequent studies  s h o w e d that treatment w i t h interferon- a (IFN- a) restores n o r m a l (31 integrin- dependent adhesion i n C M L progenitor c e l l s , p o s s i b l y v i a m e d i a t i o n b y M I P -  la.  2 1 4  '  2 1 5  Reestablishment o f stromal contact and transduction o f g r o w t h i n h i b i t o r y signals b y I F N a may explain its c l i n i c a l effects i n C M L m a n a g e m e n t .  108  S i n c e P T K s are i m p l i c a t e d in the  mediation o f integrin s i g n a l l i n g , abnormality i n P T K activation c o u l d therefore affect normal cellular responses to integrin- mediated c e l l a d h e s i o n .  2 1 6  In a recent review article,  V e r f a i l l i e and colleagues speculated the BCR- ABL fusion protein i n C M L interfered w i t h integrin- mediated signal transduction events and brings about a b n o r m a l adhesion, t r a f f i c k i n g and as w e l l as proliferation observed in C M L .  2 1 3  f. Susceptibility to cytotoxic agents In theory, the sum o f the above differences should lead to a comfortable therapeutic w i n d o w o f opportunity i n l e u k a e m i a treatment. Unfortunately, most practical experience i n therapy does not give credence to the scientific evidence. In a d d i t i o n , most neoplastic cells have e v o l v e d sophisticated protective mechanisms that confer resistance to various radiochemotherapeutic regimens. F o r e x a m p l e , expression o f the P I 7 0 A T P - dependent membrane glycoprotein p u m p , encoded b y the mdrl gene, is s i g n i f i c a n t l y increased i n cells w h i c h express the m u l t i d r u g resistance ( M D R ) p h e n o t y p e .  217  In one study o f adult acute  leukaemias, heightened mdrl expression was documented i n 5 0 % o f l e u k a e m i c relapses but o n l y 1 9 % o f n e w l y diagnosed l e u k a e m i a ; i n addition, an inverse correlation was made  33  between the complete r e m i s s i o n rate and the amount o f mdrl e x p r e s s i o n .  2 1 8  Successful  treatment o f leukaemia w i l l therefore depend on the application o f n e w l y acquired k n o w l e d g e in the m o l e c u l a r b i o l o g y o f l e u k a e m i a to the c l i n i c .  7.5 PURGING  IN AUTOLOGOUS  STEM  CELL  2 1 9  TRANSPLANTATIONS  a. I n t r o d u c t i o n B o n e m a r r o w p u r g i n g ( B M P ) is deemed beneficial in certain situations i n v o l v i n g the reinfusion o f autologous m a r r o w cells because o f contamination o f the harvested product b y neoplastic c e l l s .  2 2 0  P u r g i n g assumes that exploitable differences exist between  n o r m a l and leukaemic cells such that selectivity can be ensured in the removal or destruction o f the undesirable population. C l i n i c a l l y effective B M P presents a c h a l l e n g i n g d i l e m m a because the c l i n i c i a n must achieve a balance between sufficient eradication o f the l e u k a e m i c cells with preservation o f enough o f the n o r m a l cells for haematopoietic reconstitution. Overzealous p u r g i n g w i l l lead to eventual graft failure or a delay i n engraftment; on the other hand, inadequate p u r g i n g may contribute to leukaemic relapses i n the patient. B M P has been studied extensively i n C M L autografts and provides an excellent m o d e l system for the study o f the different p u r g i n g strategies because o f the availability o f  ber- abT  c e l l lines and the sensitivity o f the P C R detection a s s a y .  2 2 1  -  2 2 2  Accumulating  evidence also points to the effectiveness o f B M P i n A M L patients receiving autografts. A report f r o m a recent conference s h o w e d significant survival advantage in patients r e c e i v i n g purged c o m p a r e d to unpurged autografts and l e d the coauthor o f the report, E l i z a b e t h j . S h p a l l to declare " T h e s e results suggest that autotransplantation o f acute myelogenous l e u k a e m i a s h o u l d use purged bone m a r r o w f o r restoration o f b l o o d p r o d u c t i o n " .  2 2 3  Lastly,  p u r g i n g is also b e i n g considered f o r autologous P B S C transplantations i n order to reduce the level o f tumour cell contamination o f the m o b i l i s e d leukapheresed p r o d u c t .  34  2 2 4  b. Purging strategies T h e four c h i e f modalities o f p u r g i n g ; p h y s i c a l , i m m u n o l o g i c a l , b i o l o g i c a l , and p h a r m a c o l o g i c a l , are used singly or i n combinations to effect c l i n i c a l l y beneficial results.  2 2 2  '  2 2 5  D i f f e r e n c e s in b i o p h y s i c a l characteristics are e x p l o i t e d in p h y s i c a l p u r g i n g .  E x a m p l e s include counterflow elutriation, density gradient separation, filtration, and heating. F o r e x a m p l e , clusters o f neuroblastoma cells can be r e m o v e d f r o m the autograft v i a filtration through a 4 0 p m m u l t i l a y e r m e s h filter w h i c h results in the e l i m i n a t i o n of 0.5 l o g o f the cancer cells. Several groups have discovered that leukaemic c e l l lines are m u c h m o r e susceptible to hyperthermia than n o r m a l haematopoietic c e l l s .  2 2 6  -  2 2 7  Surface expression o f differentiation markers permit recognition o f cells by antibodies. H o w e v e r , as mentioned earlier, l e u k a e m i c and n o r m a l haematopoietic cells share m a n y o f the same markers since the former population derives f r o m neoplastic transformation o f the l a t t e r .  228  F o r e x a m p l e , m a n y A M L cells and essentially all A M L stem  cells express the haematopoietic stem c e l l antigen C D 3 4 ; therefore, procedures w h i c h i n v o l v e o n l y the positive selection o f C D 3 4 cells are applicable solely for s o l i d +  nonhaematopoietic tumours, w h i c h do not express C D 3 4 . Nevertheless, several c o m m e r c i a l ventures have been started to take advantage o f C D 3 4 positive selection technology in the ex vivo m a n i p u l a t i o n o f stem cells. C D 3 4 - specific m o n o c l o n a l antibodies conjugated to a solid- phase support, either i n the f o r m o f ferromagnetic beads or the surface o f tissue culture flasks, are i n various stages o f c l i n i c a l trials f o r stem c e l l p u r i f i c a t i o n and such approaches afford p r o m i s e f o r conditions l i k e neuroblastoma and breast c a n c e r .  229  Positive selection o f C D 3 4 cells f r o m a l e u k a e m i c sample w i l l therefore +  require additional steps such as negative selection or p h a r m a c o l o g i c a l p u r g i n g to eradicate C D 3 4 - expressing leukaemic cells. N e g a t i v e selection based on the expression of mature  .  lineage markers provides greater latitude i n p u r g i n g since n o r m a l c o m m i t t e d progenitor cells and haematopoietic stem cells do not express such markers. F o r e x a m p l e , presence o f C D 2 , 3, 5, 7, and C D 1 l a on l e u k a e m i c cells targets them f o r destruction by i m m u n o t o x i n s and radioisotope immunoconjugates, without significant reduction in the v i a b i l i t y and c l o n o g e n i c i t y o f n o r m a l haematopoietic c e l l s . 6 9  7 0  B i o l o g i c a l p u r g i n g operates on the basis  that normal and leukaemic cells behave differently i n culture due to unique expression profiles o f haematopoietic g r o w t h factors, their receptors, as w e l l as adhesion m o l e c u l e s . S c h e f f o l d and associates s h o w e d that C M L - specific cytokine- i n d u c e d k i l l e r cells can be  35  generated f r o m the peripheral circulation o f C M L patients; furthermore, they s h o w e d that these C D 5 6 cells can be used as a f o r m o f bone m a r r o w p u r g i n g i n C M L . +  2 3 0  Growth  kinetics i n response to cytokines also differentiate n o r m a l cells f r o m their malignant counterpart, as m e n t i o n e d i n a previous section. In vitro culture o f bone m a r r o w cells harvested f r o m selected C M L patients resulted i n the gradual disappearance o f Philadelphiachromosome positive leukaemic cells w i t h a concomitant expansion o f n o r m a l haematopoietic c e l l s .  2 3 1  '  2 3 2  Established chemotherapeutic agents such as 4-  h y d r o p e r o x y c y c l o p h o s p h a m i d e (4- H C ) and its congenor m a f o s f a m i d e (or A S T A - Z®) are both analogues o f the c o m m o n l y used a l k y l a t i n g agent c y c l o p h o s p h a m i d e ; the t w o former drugs have been m o d i f i e d such that in vivo hepatic activation is not required. A S T A - Z ® and 4- H C r e m a i n the t w o most c o m m o n l y used p h a r m a c o l o g i c a l p u r g i n g a g e n t s .  In  2 3 3 - 2 3 5  addition, other chemotherapeutic agents are also b e i n g c o n s i d e r e d f o r ex vivo p u r g i n g .  2 3 6  A l k y l - l y s o p h o s p h o l i p i d s , analogoues o f the platelet- activating factors, also appear to be p r o m i s i n g candidates i n various p u r g i n g a p p l i c a t i o n s .  237  c. Combination purging A s s h o w n i n c l i n i c a l pharmacology, c o m b i n a t i o n therapy i n cancer management is often superior to the single drug approach. Therefore, research i n p u r g i n g w h i c h c o m b i n e s the different m o d a l i t i e s , such as i m m u n o l o g i c a l and p h a r m a c o l o g i c a l p u r g i n g , or different drugs, such as m a f o s f a m i d e w i t h etoposide, are b e i n g a c t i v e l y p u r s u e d .  2 2 5  T h e ex vivo  nature o f the treatment affords the c l i n i c i a n with greater latitude o f graft manipulation without m u c h fear o f systemic t o x i c i t y ; the singular goal o f p u r g i n g is to. effect maximal-log reduction o f the contaminating neoplastic cells w h i l e preserving an adequate number o f n o r m a l haematopoietic cells f o r rapid yet durable engraftment. Other examples o f c o m b i n a t i o n p u r g i n g include combinations o f 4- H C or A S T A - Z ® w i t h antibodymediated- c o m p l e m e n t lysis, i m m u n o t o x i n s , and ber- abl j u n c t i o n specific antisense oligodeoxynucleotide ( O D N ) .  2 3 8  "  2 4 0  36  d. Purging outside the traditional paradigm N o v e l purging modalities are constantly being developed to exploit the preferential k i l l i n g o f leukaemic cells. Gene- based strategies are used to target leukaemia- specific oncogenes such as the ber- abl translocation sequence i n C M L . S o m e examples include the ber- abl j u n c t i o n region- specific antisense O D N and retroviral expression v e c t o r .  1 0 3  -  2 4 1  E n f o r c e d reexpression o f the w i l d - type t u m o u r suppressor gene p53 c a n lead to.a reversion o f the tumour phenotype or even apoptosis i n cells w i t h mutated or deleted p53 sequence. Seth and colleagues used an adenoviral vector i n a h u m a n breast cancer p u r g i n g m o d e l to effect the transfer o f the w i l d - type p 5 3 tumour suppressor gene, w h i c h i n d u c e d apoptosis preferentially i n the breast cancer c e l l s .  2 4 2  O f t e n , aberrant signal transduction  events are implicated i n the initiation and maintenance o f the leukaemic phenotype; therefore, specific targeting o f s i g n a l l i n g pathways can lead to the selective destruction o f the cells dependent o n their s u r v i v a l . N o v e l c o m p o u n d s w h i c h disrupt the s i g n a l l i n g pathways o f neoplastic cells or promote apoptosis w i l l increasingly f i n d their w a y into purging. - 5 8  5 9  7 2  L a C a s s e and colleagues d e s c r i b e d the successful p u r g i n g o f the h u m a n  B- c e l l l y m p h o m a c e l l line D a u d i w i t h the protein synthesis inhibitor Shiga- l i k e t o x i n 1 ( S L T - 1), w h i c h b i n d s to C D 7 7 , a c e l l surface g l y c o l i p i d  2 4 3  Restricted e x p r e s s i o n o f  C D 7 7 to a subset o f activated B cells and l y m p h o m a cells means that n o r m a l haematopoietic stem and precursor cells are spared f r o m S L T - 1- mediated k i l l i n g . A s i m i l a r strategy utilises a fusion G M - C S F / diphtheria t o x i n w h i c h selectively targets and destroys G M C S F dependent leukaemic cells (as w e l l as n o r m a l granulocyte- macrophage progenitors) in vitro.  244  T h e above examples illustrate some o f the n o v e l methods o f p u r g i n g w h i c h s h o w  p r o m i s e f o r future c l i n i c a l success.  1.6 PHOTODYNAMIC  THERAPY  (PDT) AND ITS ROLE IN  PURGING  a. Reactive oxygen intermediates (ROIs) in biology M o l e c u l a r o x y g e n is essential f o r the survival o f the vast majority o f terrestrial b i o l o g i c a l organisms. It, however, c a n behave deleteriously i f not f o r the elaborate protective mechanisms that are evolutionarily conserved in the different species. R e a c t i v e o x y g e n intermediates ( R O I s ) such as superoxide anions (0 ~) and h y d r o g e n p e r o x i d e 2  37  ( H 0 ) are generated during oxidative respiration and are n o r m a l l y deactivated b y 2  2  superoxide dismutase ( S O D ) and catalase, respectively. T h e f a m i l y o f glutathione ( G S H ) dependent enzymes are extremely important in the detoxification o f a variety o f R O I s and lipid peroxides.  2 4 5  In addition to their role as byproducts o f m i t o c h o n d r i a l respiration, R O I s are also i m p l i c a t e d in a m y r i a d o f intracellular and intercellular signal transduction pathways.  2 4 6  A c t i v a t e d phagocytic cells produce 0 " that is bactericidal f o r intracellular pathogens. 2  Phagocytes f r o m patients suffering f r o m chronic granulomatous disease are defective i n the generation o f 0 , w h i c h predisposes these patients to persistent and severe infections. 2  N e u t r o p h i l s contain the e n z y m e myeloperoxidase, w h i c h catalyses the o x i d a t i o n o f chloride i o n b y H 0 i n t o the h i g h l y reactive h y p o c h l o r o u s acid ( H O C 1 ) . Therefore, the b o d y 2  2  utilises R O I s for various aspects o f n o n s p e c i f i c i m m u n e d e f e n c e .  247  O x i d a t i v e injury to  cells can occur at the different levels o f cellular constituencies. L i p i d p e r o x i d a t i o n , oxidative damage to the thiol groups o f structural proteins as w e l l as e n z y m e s , and direct damage to D N A are observed in cells e x p o s e d to o x i d a t i v e s t r e s s .  2 4 8  '  2 4 9  Increasing  evidence f r o m different experimental systems also demonstrate the role o f R O I s i n the i n d u c t i o n o f apoptotic c e l l d e a t h . antiapoptotic protein BCLand actions o f R O I s .  2 5 1  -  2 5 0  In addition, some studies have s h o w n that the  2 has antioxidative capability w h i c h can inhibit the formation 2 5 2  H o w e v e r , contradictory f i n d i n g s also suggest that R O I s are  not necessary f o r the induction of apoptosis and that BCLeven i n a n o x i c c o n d i t i o n s .  2 retains its antiapoptotic ability  2 5 3  In summary, R O I s are generated d u r i n g n o r m a l cellular m e t a b o l i s m and are also i m p l i c a t e d in the various signal transduction pathways. Phagocytes use R O I s as part o f the nonspecific defence against pathogens.-All cellular components are potential•targets^of oxidative damage; in addition, oxidative stress can effect its c y t o t o x i c i t y v i a apoptosis.  b. P D T : cytotoxicity and biological responses P h o t o d y n a m i c therapy ( P D T ) exploits the b i o p h y s i c a l characteristics o f a group o f photoreactive c o m p o u n d s that, w h e n activated by light of the appropriate wavelengths, enter into a higher energy state and w i l l subsequently excite either cellular b i o m o l e c u l e s (type I photosensitised reaction) or ground state o x y g e n (type II photosensitised reaction);  38  singlet o x y g e n ( ' 0 ) , w h i c h is extremely reactive, is generated as a result o f the type II 2  reaction.  2 5 4  -  2 5 5  T h e relatively short l i f e span o f ' 0 (4 |is) in aqueous solution means that 2  the sites o f oxygen- dependent photodamage are dictated by the initial localisation o f the photosensitiser.  256  B o t h apoptosis and necrosis have been observed after P D T and this  appears to be dependent on the type and the dose o f the photosensitiser. S i n c e these agents are inert until activated b y light, the specificity o f the P D T c y t o t o x i c response can be finetuned b y m a n i p u l a t i n g the time and the site o f photoirradiation. N e o p l a s t i c tissues and other rapidly proliferating tissues such as a n g i o g e n i c a l l y stimulated endothelial cells readily take up the photosensitising c o m p o u n d , one o f the m e c h a n i s m s is mediated by the upregulation o f l o w density lipoprotein ( L D L ) r e c e p t o r s .  257  V a r i o u s p o r p h y r i n - based photosensitisers associate w i t h p l a s m a lipoproteins; therefore, upregulation o f the L D L receptors ( L D L r ) w i l l increase the accumulation o f the photosensitiser- L D L c o m p l e x , and hence subsequent P D T c y t o t o x i c i t y .  2 5 8  Other factors  such as the l o c a l m i l i e u o f the c e l l and the c h e m i c a l characteristics o f the c o m p o u n d also determine the uptake profile and susceptibility to P D T .  2 5 9  O x i d a t i v e damage to cellular  components results i n various changes to the c e l l s , tissues, and the o r g a n i s m  2 6 0  . The  degree o f P D T cytotoxicity is dependent on the amount and activity o f the various cellular protective e n z y m e s mentioned p r e v i o u s l y .  261  R e c e n t l y , m u c h research has been devoted to  the role o f apoptosis i n P D T c y t o t o x i c i t y , w h i c h appears to be mediated by phospholipaseC in L 5 1 7 8 Y l y m p h o m a c e l l s . antiapoptotic protein BCL-  2 6 2  H e and colleagues s h o w e d that overexpression o f  2 in the Chinese hamster ovary c e l l l i n e effected protection f r o m  P D T - mediated c y t o t o x i c i t y .  263  Several established stress signal transduction pathways are  also initiated b y P D T ; increased expression o f specific heat shock proteins and various early response:genes'as w e l l as the activation o f the N F - K{3, J N K / S A P K pathways as w e l l as increased p r o d u c t i o n o f ceramide are observed post- P D T .  2 6 4 - 2 6 8  These pathways m a y  be i n v o l v e d either as a n o r m a l protective response to P D T - mediated stress or as part o f the c e l l death pathways initiated by P D T . N u m e r o u s photosensitising c o m p o u n d s w i t h different b i o p h y s i c a l and b i o c h e m i c a l characteristics have been proposed f o r P D T . T w o members o f the p o r p h y r i n f a m i l y , Photofrin® (porfimer sodium) and the b e n z o p o r p h y r i n - derivatives, are in current c l i n i c a l use.  2 6 9  Other photosensitisers such as sulphonated a l u m i n i u m phthalocyanine ( A l S P c ) and  M e r o c y a n i n 5 4 0 ( M C 540) b e i n g considered as w e l l . T h e " p e r s o n a l i t y " o f each  39  photosensitiser, w h i c h includes h y d r o p h o b i c i t y , e x t i n c t i o n coefficient, absorption spectra, and singlet o x y g e n y i e l d , is determined b y the unique c h e m i c a l structure o f the c o m p o u n d . These factors, i n conjunction w i t h the b i o l o g i c a l attributes o f the c e l l (in vitro and in vivo P D T ) and the o r g a n i s m (in vivo P D T ) determine the overall effectiveness o f P D T .  2 7 0  c. In vivo uses of PDT A c c e s s i b i l i t y o f the s k i n to photoirradiation and the h i g h occurrence o f cutaneous tumours c o m b i n e d to p r o v i d e an ideal i n d i c a t i o n f o r P D T . I n a d d i t i o n , P D T has s h o w n p r o m i s e i n the treatment o f tumours o f the b r a i n , l u n g , oesophagus, and o f the b l a d d e r . 271,272  249  -  D a m a g e to the tumour- associated vasculature by P D T also contributes to the  destruction o f the t u m o u r .  2 7 3  H o w e v e r , l i m i t e d photopenetration remains one o f the major  impediments i n the application o f in vivo P D T f o r the treatment o f large tumours; this limitation was evident i n the first generation haematoporphyrin derivative- based photosensitiser Photofrin® (porfimer sodium) w i t h an activation w a v e l e n g t h at 6 3 0 n m , w h i c h c o i n c i d e s w i t h tissue absorption.  d. The Benzoporphyrin derivatives- second generation photosensitisers T o further i m p r o v e the efficacy o f P D T , various second generation photosensitising c o m p o u n d s w i t h absorption peaks between 650- and 7 0 0 n m are under investigation. S o m e of these include the b e n z o p o r p h y r i n - derivatives, phthalocyanines, p u r p u r i n s , and c h l o r i n C f  . 274-276  T h e b e n z o p o r p h y r i n derivatives absorb m a x i m a l l y at a p p r o x i m a t e l y 6 9 0 n m , a  wavelength w i t h at least t w i c e the •tissue penetration o f Photofrin® .  2 7 7  >  2 7 8  O n e analogue i n 1  current c l i n i c a l testing, benzoporphyrin- derivative m o n o a c i d r i n g A ( B P D - M A or B P D ) , possesses rather h i g h singlet and triplet o x y g e n quantum yields w h i c h contribute to its potential as an efficient p h o t o s e n s i t i s e r .  279  In a d d i t i o n , B P D has a p h a r m a c o k i n e t i c p r o f i l e  w h i c h favours tumour accumulation and therefore optimal tumour- to- tissue ratios i n D B A / 2 m i c e w i t h the i m p l a n t e d r h a b d o m y o s a r c o m a tumour M l .  2 8 0  -  2 8 1  E v e n though B P D  persists f o r up to 72 h i n the mouse s k i n , photosensitivity o n l y lasts f o r 2 4 h postinjection possibly as a result o f inactivation rather than clearance o f the p h o t o s e n s i t i s e r .  282  Furthermore, L o g a n a n d colleagues have s h o w n in vivo P D T mediated b y B P D increased  40  the i m m u n o g e n i c i t y o f M l tumour i n the m u r i n e h o s t .  2 8 3  In s u m m a r y , the advantages o f  B P D make it an effective and ideal photosensitiser f o r in vivo a p p l i c a t i o n s .  284  e. Ex vivo uses of PDT T h e ex vivo use o f P D T , p r i n c i p a l l y i n the m a n i p u l a t i o n o f b l o o d products, bypasses the m a n y constraints o f in vivo P D T ; n a m e l y , interference o f photoactivation b y the tissues and b l o o d surrounding the target site, and concerns w i t h a c h i e v i n g a therapeutic concentration o f the photosensitiser i n the target without e l i c i t i n g significant s k i n photosensitivity and other side effects. M a n y o f the techniques d e v e l o p e d f o r p r e c l i n i c a l in vitro studies o f P D T can be adapted into c l i n i c a l ex vivo applications. In a d d i t i o n , w h o l e b l o o d can be separated into its different components prior to P D T . T h e concentration and purification o f leukocytes through apheresis eliminates most o f the red b l o o d cells, w h i c h may interfere w i t h both the uptake o f the photosensitiser and subsequent light activation. E s s e n t i a l l y , ex vivo P D T s i m p l i f i e s the pharmacokinetics and p h a r m a c o d y n a m i c s b y restricting the potential target populations. F o r e x a m p l e , leukapheresis o f a patient w i t h autoimmune disease w i l l y i e l d both activated (proliferating) and nonactivated (quiescent) l y m p h o c y t e s ; the f o r m e r population has been s h o w n to be m o r e susceptible to P D T because o f its proliferative s t a t e .  285  G a s p a r r o ' s group has c o m p l e t e d several p r o m i s i n g c l i n i c a l  trials o n the extracorporeal U V A irradiation o f b l o o d cells pretreated w i t h 8methoxypsoralen (8- M O P ) , f o r the management o f cutaneous T- c e l l l y m p h o m a and several other a u t o i m m u n e d i s e a s e s .  286  Photophoresis u s u a l l y takes place t w o hours after  the ingestion o f 8- M O P w i t h m i n i m u m p l a s m a drug level at 5 0 ng/ml. A specially designed photophoresis unit is used to separate the patient's b l o o d into different components f o l l o w e d b y photoactivation o f the leukocytes i n recirculating clear plastic tubings sandwiched between banks o f U V A l a m p s ; the treated cells are then returned to the patient's systemic c i r c u l a t i o n . A n o t h e r use o f P D T is the extracorporeal inactivation o f enveloped viruses i n b l o o d products; B P D has s h o w n p r o m i s e i n the inactivation o f F e L V and HIV1.287.288  A n o t h e r emerging use o f the technology is i n the i m m u n o m o d u l a t i o n o f organ grafts prior to transplantation. O b o c h i and colleagues have observed that sublethal doses o f P D T u s i n g B P D was sufficient to p r o l o n g the s u r v i v a l o f allogeneic s k i n grafts i n m i c e ,  41  w h i c h was attributed to the reduction o f the expression o f i m m u n o s t i m u l a t o r y c e l l surface m o l e c u l e s i n the Langerhan cells, professional antigen presenting cells w h i c h reside in the skin.  2 8 9  f. Bone marrow purging P D T - mediated bone m a r r o w p u r g i n g ( B M P ) , w h i c h requires the extracorporeal photoirradiation o f bone m a r r o w cells preincubated w i t h photosensitiser, naturally progressed f r o m the other ex vivo P D T applications discussed a b o v e .  2 9 0  -  2 9 1  Preferential  uptake o f the photosensitiser b y l e u k a e m i c cells in c o m p a r i s o n to n o r m a l nonproliferating cells provides a w i n d o w o f opportunity f o r this m o d a l i t y in B M P . T h e f e a s i b i l i t y o f u s i n g B P D - mediated P D T i n p u r g i n g has been investigated exhaustively in both c y t o t o x i c i t y assays and ex vivo p u r g i n g s y s t e m s .  2 9 2  F l u o r e s c e n t m i c r o s c o p y and f l o w c y t o m e t r y have  demonstrated that leukaemic cell lines and primary leukaemic cells preferentially take up m o r e B P D than n o r m a l b l o o d cells. S p e c i f i c a l l y , Jamieson et a l . reported that the h u m a n l e u k a e m i c cell line K 5 6 2 took up 10- f o l d and primary C M L c l i n i c a l samples took up 7f o l d more o f B P D than n o r m a l h u m a n bone m a r r o w c e l l s .  2 9 3  F u r t h e r m o r e , short- term  c o l o n y assays and long- term bone m a r r o w cultures as w e l l as m u r i n e bone m a r r o w p u r g i n g experiments all demonstrated the utility o f B P D in B M P .  2 9 4  -  2 9 5  L e m o l i and  colleagues f o u n d that m u l t i d r u g resistant ( M D R ) leukaemic c e l l lines that express the P I 7 0 membrane glycoprotein are refractory to B P D - mediated but not to dihaematoporphyrin ether ( D H E ) - mediated P D T ; in addition, other cellular protection mechanisms m a y also affect the effectiveness o f p u r g i n g .  2 6 1  '  2 9 6  Therefore, the heterogeneity o f the c y t o t o x i c  response to . P D T depends on. the photosensitiser, its f o r m u l a t i o n (eg,-liposomeencapsulated), and the type o f cells targeted. A c t i v e drug e f f l u x as w e l l as defence mechanisms against oxidative stress can be attenuated p h a r m a c o l o g i c a l l y in order to enhance P D T cytotoxicity. R e c e n t l y , G l u c k and colleagues demonstrated the effectiveness o f B P D - mediated P D T p u r g i n g o f m u l t i p l e m y e l o m a cells ex vivo.  291  Photosensitisers  other than B P D are also being considered for p u r g i n g , some notable examples i n c l u d e the phthalocyanines and m e r o c y a n i n e 5 4 0 ( M C 5 4 0 ) .  2 9 8  '  3 0 0  P D T p u r g i n g has also been  s h o w n to be effective i n the eradication o f cancers other than l e u k a e m i a ; Sieber and colleagues demonstrated the effectiveness o f M C 5 4 0 in p u r g i n g contaminating  42  neuroblastoma c e l l s .  3 0 1  Therefore, P D T p u r g i n g can be considered f o r a u B M T s i n  neoplasms other than the leukaemias.  g. Clinical trials C l i n i c a l bone m a r r o w p u r g i n g u s i n g P D T is still relatively u n c o m m o n c o m p a r e d to the other methodologies such as m o n o c l o n a l antibody- mediated negative selection. Sieber and associates have conducted phase I c l i n i c a l testing o f M C 5 4 0 - mediated P D T - p u r g i n g o f autologous bone m a r r o w cells f r o m l y m p h o m a and l e u k a e m i a p a t i e n t s .  302  A n ongoing  c l i n i c a l trial o f B M P in acute leukaemia undergoing a u B M T u s i n g the photosensitiser B P D is b e i n g conducted i n M o n t r e a l . Initial f i n d i n g s f r o m the s m a l l group o f patients are extremely encouraging. P u r g i n g at 10 and 2 0 ng/ml o f B P D w i t h 15J/cm photoactivation 2  resulted in the p r o l o n g e d survival in f i v e o f seven patients w i t h the longest disease- frees u r v i v a l interval at 349 days posttransplantation. O n e patient relapsed on day 84 and another patient required i n f u s i o n o f the unmanipulated b a c k u p m a r r o w and subsequently relapsed on day 122. B P D dose escalation w i l l be incorporated into future trials in order to increase the efficacy o f the purging. A phase I trial studying the feasibility o f u s i n g B P D to purge C D 3 4 cells isolated f r o m the autologous P B S C harvests o f patients w i t h N o n +  H o d g k i n ' s L y m p h o m a is i n its f i n a l p l a n n i n g stage.  OBJECTIVES  AND  RATIONALE  '  In this thesis, we attempted to i m p r o v e the efficacy o f B P D - mediated P D T i n an in vitro setting. T h i s w o r k is based on previous f i n d i n g s b y J a m i e s o n that B P D - mediated P D T was selective i n the destruction o f l e u k a e m i c c e l l s .  2 9 1  -  2 9 2  W e decided to approach the  p r o b l e m v i a two independent angles. There exists a considerable amount o f literature on the various forms o f P D T combination t h e r a p y .  3 0 3 - 3 0 6  Several groups have already p e r f o r m e d studies o n the  c o m b i n a t i o n o f P D T and D o x , albeit w i t h different r e s u l t s .  43  3 0 7  -  3 0 8  W e therefore decided to  study the c o m b i n e d effects o f B P D - mediated P D T and D o x f o r the purpose o f in vitro P D T , i.e. for stem c e l l p u r g i n g . Furthermore, we were interested on the effects o f treatment sequence on the cytotoxic outcome and selectivity o f the regimen. Treatment sequence is an important parameter in P D T combination therapy w h i c h c o u l d potentially affect the efficacy o f the t h e r a p y .  3 0 3  -  3 0 9  . B P D , a recently d e v e l o p e d s e c o n d generation p o r p h y r i n - based  photosensitiser, is m u c h more potent than Photofrin® and is considered for various c l i n i c a l applications.  2 6 9  -  2 8 4  B P D , u n l i k e its predecessor Photofrin®, is a relatively pure  photosensitiser w i t h characterised active c o m p o n e n t s .  278  Therefore, P D T c o m b i n a t i o n  therapy u s i n g B P D is a n o v e l area o f research w i t h relevant c l i n i c a l potentials especially f o r stem c e l l p u r g i n g because o f its in vitro nature. C i n c o t t a et al. recently demonstrated the advantage o f a P D T r e g i m e n c o m b i n i n g B P D w i t h another p h o t o s e n s i t i s e r .  305  W e were  also interested in the possible interactions, p h y s i c a l and b i o c h e m i c a l , between B P D and D o x . M u r i n e haematopoietic cells, n o r m a l and l e u k a e m i c , were used throughout the experiment. T h e standard agar c o l o n y assay a l l o w s the convenient and reproducible measurement o f c l o n o g e n i c i t y o f cells as a result o f the various permutations o f treatment parameters. W e therefore e m p l o y e d the agar c o l o n y assay as a primary assessment tool o f the effectiveness o f the different combinations. W e f o u n d that the murine l e u k a e m i c cell line L 1 2 1 0 was m u c h more susceptible to the c o m b i n a t i o n o f D o x - > P D T than n o r m a l D B A / 2 haematopoietic progenitors (Chapter 3). Therefore, we decided to further explore some o f the possible m e c h a n i s m s b e h i n d the f i n d i n g s (Chapter 4). T h e second approach i n v o l v e s the incorporation o f the haematopoietic inhibitory peptide N - A c S D K P into B P D - mediated P D T o f n o r m a l and l e u k a e m i c cells. N - A c S D K P has been demonstrated to effect the selective protection o f normal haematopoietic cells f r o m various forms o f c y t o t o x i c treatments and therefore was considered to be an ideal protector o f n o r m a l bone m a r r o w cells d u r i n g r a d i o c h e m o t h e r a p y .  2 0 5  -  2 1 0  C o u t t o n and colleagues  showed that N - A c S D K P also selectively protected n o r m a l h u m a n haematopoietic cells f r o m Photofrin®- mediated P D T i n a p u r g i n g s e t t i n g .  209  W e therefore wanted to extend their  f i n d i n g s to the second generation c o m p o u n d B P D . W e also w i s h e d to extend the experimental system to the murine setting such that, i f needed, bone m a r r o w p u r g i n g and transplantation experiments c o u l d be p e r f o r m e d to assess the protective effect o f N A c S D K P . A g a i n , w e were able to determine the effectiveness o f the scheme b y u s i n g the standard agar c o l o n y assay. W e f o u n d that a p r e i n c u b a t i o n p e r i o d o f 1.5 h w i t h 100 n M N-  44  A c S D K P significantly and selectively protected D B A / 2 late haematopoietic progenitors but not the l e u k a e m i c c e l l line L 1 2 1 0 f r o m B P D - mediated P D T (chapter 5). Furthermore, w e attempted to study some o f the potential mechanisms responsible f o r N - A c S D K P photoprotection (chapter 6). O u r f i n d i n g s suggested that c e l l c y c l e i n h i b i t i o n mediated by the peptide was partly responsible f o r the observed protective effect. Therefore, the overall theme o f this thesis is to i m p r o v e the e f f i c a c y o f B P D mediated P D T . In addition, w e hope that in the process we w i l l reveal f i n d i n g s beneficial to the c l i n i c a l applications o f P D T as w e l l as c o n d u c i v e to further understanding o f the m o l e c u l a r mechanisms o f P D T .  45  EXPERIMENTAL  2.1  EXPERIMENTAL  PROCEDURES  REAGENTS  a. B e n z o p o r p h y r i n d e r i v a t i v e m o n o a c i d r i n g A ( B P D ,  Verteporfin®)  T h e m o n o a c i d ring- A analogue o f b e n z o p o r p h y r i n derivative i n a l i p o s o m a l f o r m u l a t i o n ( B P D , Verteporfin®) was obtained f r o m Q L T PhotoTherapeutics Inc. ( Q L T , V a n c o u v e r , B C , Canada). L y o p h i l i s e d B P D was reconstituted to a concentration o f 1.5 mg/ml w i t h sterile double- d i s t i l l e d water ( d d H 0 ) every two weeks and stored at 4 ° C u n t i l 2  use. Further dilutions were carried out d u r i n g the experiment w i t h tissue culture m e d i u m i n a reduced light environment.  b. C h e m i c a l reagents D o x o r u b i c i n h y d r o c h l o r i d e ( D o x ) and cytosine arabinoside (Ara- C ) were purchased f r o m the S i g m a C h e m i c a l C o . (St. L o u i s , M O ) and were s u p p l i e d i n p o w d e r f o r m . D o x was reconstituted i n sterile d d H 0 to a concentration o f 10 m M and stored as 5 0 2  p i aliquots at -20°C. A r a - C was reconstituted i n sterile d d H 0 to a concentration o f 5 0 0 2  m M and stored as 50 u M aliquots at - 2 0 ° C . A l l other c h e m i c a l reagents, except where s p e c i f i c a l l y noted, were purchased f r o m S i g m a C h e m i c a l C o . (St. L o u i s , M O ) .  c. Tissue c u l t u r e reagents Tissue culture m e d i a D u l b e c c o ' s M o d i f i e d Eagles M e d i u m ( D M E M ) and I s c o v e ' s M o d i f i e d D u l b e c c o ' s M e d i u m ( I M D M ) were purchased f r o m G i b c o / B R L L i f e T e c h . Inc. ( G r a n d Island, N . Y . ) and were prepared a c c o r d i n g to the manufacturer's instructions. F e t a l c a l f serum ( F C S ) was purchased f r o m the S i g m a C h e m i c a l C o . (St. L o u i s , M O ) and was heat- inactivated at 5 7 ° C for 15 m i n . D M E M was further supplemented w i t h streptomycin  46  (final 100 pg/ml) and p e n i c i l l i n (final 100 U / m l ) ( G i b c o / B R L ) , 1 m M s o d i u m pyruvate ( G i b c o / B R L ) , and 25 m M o f (N- [2- H y d r o x y e t h y l ] piperazine- N ' - [2- ethanesulfonic acid]) ( H E P E S , S i g m a ) . S u p p l e m e n t a l glutamine i n the f o r m o f G l u t a M A X I ( G i b c o / B R L ) , a L- A l a n y l - L- G l u t a m i n e dipeptide, was added to a f i n a l concentration o f 2 m M to D M E M that had been in storage for more than one month. A l l tissue culture reagents were stored at 4 ° C .  d. Reagents for colony assays and long- term marrow cultures Reagents f o r p e r f o r m i n g p r i m a r y haematopoietic c o l o n y assays and long- term bone m a r r o w m e d i u m were purchased f r o m S t e m C e l l T e c h n o l o g i e s ( S C T , V a n c o u v e r , B . C . , Canada). T h e y were p o k e w e e d mitogen- spleen c e l l c o n d i t i o n e d m e d i u m ( P W M - S C C M , H e m o S t i m M 2 1 0 0 ) w h i c h contained interleukin- 3 (fL- 3) and granulocyte- macrophage c o l o n y stimulating factor ( G M - C S F ) , and m u r i n e m y e l o i d long- term culture m e d i u m ( M y e l o C u l t M 5 3 0 0 ) . Tissue culture grade b o v i n e serum a l b u m i n ( B S A ) in I M D M was purchased f r o m B o e h r i n g e r M a n n h e i m C a n a d a . ( L a v a l , Quebec, Canada).  e. Fluorescence activated cell sorting (FACS) reagents A n t i b o d i e s and f l u o r o c h r o m e s used f o r F A C S analysis were purchased f r o m P h a r m i n g e n , Inc. (San D i e g o , C A ) and are listed b e l o w (Table 2.1). F A C S w a s h i n g buffer c o n s i s t e d o f 1 % heat- inactivated F C S (HI- F C S , S i g m a C h e m i c a l C o . , St. L u o i s , M O ) and 0 . 1 % s o d i u m azide ( S i g m a ) in phosphate buffered saline ( P B S ) . A 4 % paraformaldehyde (Sigma) double- strength F A C S f i x a t i o n buffer was prepared i n P B S .  47  Table 2.1  Reagents used for F A C S analysis  Specificity  Antibody  Clone  Isotvpe  Form  Reference Number  mouse  CD34  haematopoietic  Rat I g G , K  Biotin  09432D  R 3 5 - 95  Rat I g G , K  Biotin  11022C  -  -  Steptavidin-  13024D  RAM34  2 a  (49E8)  progenitor cells isotypic control Biotin  -  2 a  FITC F I T C , fluorescein isothiocyanate  f. Synthetic peptides T h e tetrapeptides acetylated- serine- aspartate- lysine- proline ( N - A c S D K P ) , acetylated- serine- aspartate- lysine- glutamate ( N - A c S D K E ) , and serine- aspartate- lysineproline ( S D K P ) were synthesised at the M i c r o s e q u e n c i n g Centre o f the U n i v e r s i t y o f V i c t o r i a ( V i c t o r i a , B.C., Canada). T h e peptides were further p u r i f i e d to homogeneity by h i g h performance l i q u i d chromatography ( H P L C ) and characterised by a m i n o acid analysis, c a p i l l a r y electrophoresis, and fast atom b o m b a r d m e n t mass spectroscopy. Peptides were supplied as l y o p h i l i s e d powders and were stored at -80°C in a desiccated environment. Peptides were reconstituted to 5 m M w i t h 1 0 % HI- F C S / I M D M and stored as single-use aliquots, a t - 8 0 ° C  2.2 LIGHT  SOURCE  A s p e c i f i c a l l y constructed light b o x was used f o r photoactivation o f cells. T h e unit consisted o f upper and l o w e r banks each o f eight G E F 1 5 T 8 - R red fluorescent tubes ( S y l v a n n i a , D r u m m o n d v i l l e , Q u e b e c , Canada) w h i c h e m i t between 600- 9 0 0 n m . In addition, t w o electric fans were incorporated into the unit to ensure adequate c o o l i n g o f the samples during exposure. C e l l samples were placed on a clear plexiglass p l a t f o r m at  48  equidistant between the t w o banks o f light. T h e unit was w a r m e d u p f o r thirty minutes before irradiation and photometric measurements i n three axes (x, y , z ) were made w i t h an EL 1350 Radiometer/Photometer p o w e r meter (International L i g h t , Inc., M A ) i m m e d i a t e l y before exposure. E x p o s u r e time ( Y ) w a s calibrated to provide 15 J / c m o f light energy 2  u s i n g the f o l l o w i n g f o r m u l a : Y (min)=  15 J / c m X mW/cm  2  2  x 0.06 ( W s/mW m i n )  T h e average o f the six photometric measurements o f the three axes constituted the variable X.  2.3 EXPERIMENTAL  ANIMALS  a. Mice PJ3A/2 (H- 2 ) m i c e (6- 8 weeks o f age) were purchased f r o m C h a r l e s R i v e r d  B r e e d i n g Laboratories C a n a d a ( M o n t r e a l , Quebec, Canada) and maintained under pathogen- free conditions i n the animal f a c i l i t y . o f the Department o f M i c r o b i o l o g y & I m m u n o l o g y at the U n i v e r s i t y o f B r i t i s h C o l u m b i a ( V a n c o u v e r , B . C . , Canada). A l l animals were housed i n m i c r o i s o l a t o r units and were given standard laboratory rodent diet (Ralston Purina) and H C 1 - a c i d i f i e d water ( p H 2.5) ad libitum.  N e w l y arrived animals were  kept i n quarantine for t w o weeks p r i o r to introduction into the core f a c i l i t y . E x p e r i m e n t s were conducted on animals that had been i n the facility for t w o weeks o r more to ensure acclimatisation. T h e protocol for animal experimentation was approved b y the animal care committee o f U B C .  49  2.4 CELL  PREPARATION  a. Mouse bone marrow cells B o n e m a r r o w m o n o n u c l e a r cells ( B M M N C s ) were obtained f r o m the femurs o f CO2- euthanised 8-12 w e e k o l d D B A / 2 m i c e . B r i e f l y , the mouse was rinsed w i t h 7 0 % ethanol and a straight line vertical i n c i s i o n was made on the ventral surface f o l l o w e d by m a n u a l separation o f the s k i n e x p o s i n g the a b d o m e n and the legs o f the mouse. T h e muscles surrounding the f e m u r were r e m o v e d and the f e m u r was cut superior to the patella and inferior to the acetabulum. T h e m a r r o w cavity o f the f e m u r was then f l u s h e d w i t h I M D M supplemented w i t h 1 0 % HI- F C S d e l i v e r e d by a 22 gauge needle connected to a 5 c c syringe (Becton- D i c k i n s o n , R u t h e r f o r d , N J ) . T h e m a r r o w was then dispersed into a single c e l l suspension v i a gentle m a n i p u l a t i o n w i t h the syringe without the needle. T h e process was repeated f o r the other femur. O n e mouse n o r m a l l y y i e l d e d 1 x 10  7  BMMNCs  f r o m its two rear femurs. T h e harvested cells were then w a s h e d twice and resuspended i n I M D M . C e l l s intended f o r F A C S analysis underwent an additional step o f erythrocyte lysis i n w h i c h 5 x 10 bone m a r r o w c e l l s were m i x e d w i t h 5 m l o f T r i s / N H C 1 b u f f e r ( p H 7.3) 7  4  and incubated at 3 7 ° C f o r 10 m i n f o l l o w e d b y t w o washes w i t h I M D M .  b. Mouse leukaemic cell lines T h e L 1 2 1 0 murine l y m p h o c y t i c l e u k a e m i a c e l l line ( A T C C C C L 219) was purchased f r o m the A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C , R o c k y v i l l e , M a r y l a n d ) and maintained in D M E M supplemented w i t h 1 0 % heat- inactivated F C S (HI- F C S ) . L 1 2 1 0 was o r i g i n a l l y derived f r o m the D B A / 2 mouse strain f o l l o w i n g s k i n paintings w i t h 0 . 2 % methylcholanthrene in ethyl ether and has been used extensively in the screening o f new chemotherapeutic a g e n t s .  310  T h e cells were g r o w n at 3 7 ° C and maintained in a 5 % carbon  d i o x i d e ( C 0 , Praxair, M i s s i s s a u g a , O N , C a n a d a ) - r o o m air aerated, f u l l y h u m i d i f i e d 2  incubator ( F o r m a S c i e n t i f i c , M a r i e t t a , O h i o ) . O n g o i n g cultures o f L 1 2 1 0 cells were routinely replaced at two m o n t h intervals w i t h f r o z e n stocks f r o m l i q u i d nitrogen storage to ensure that the cells d i d not acquire any mutations in vitro w h i c h c o u l d affect their b eha v i o u r.  50  c. Primary human haematopoietic cells N o r m a l h u m a n bone m a r r o w cells were obtained d u r i n g sternotomy and were k i n d l y s u p p l i e d b y D r . L a w r e n c e B u r r at the V a n c o u v e r G e n e r a l H o s p i t a l ( V G H , V a n c o u v e r , B . C . , Canada). C e l l samples were m a i n t a i n e d i n I M D M supplemented w i t h 2 0 0 units o f sterile s o d i u m heparin (Fisher S c i e n t i f i c , F a i r L a w n , N J ) at r o o m temperature and processed o n the same day o f extraction. C h r o n i c m y e l o g e n o u s leukaemic ( C M L ) cells were obtained f r o m venupuntures o f n e w l y d i a g n o s e d , untreated C M L patients a n d were k i n d l y s u p p l i e d b y D r . N o e l B u s k a r d at the M o n r o e outpatient c l i n i c o f V G H . T h e l e u k a e m i c b l o o d samples were stored i n 10 m l Vacutainers containing 143 U S P o f l i t h i u m heparin ( B e c t o n D i c k i n s o n ) and were processed o n the day o f extraction. T o isolate the fraction containing mononuclear cells, the b l o o d samples were m i x e d w i t h I M D M i n a 1: 1 ratio and 7 m l o f the resultant mixture was then layered o n top o f 3 m l o f r o o m temperature F i c o l l - Paque P l u s (Pharmacia B i o t e c h A B , U p p s a l a , S w e d e n ) and centrifuged at 3 0 0 R C F for 15 m i n . T h e interface was harvested w i t h a sterile Pasteur pipette and the cells were washed t w i c e w i t h I M D M .  d. Human leukaemic cell lines T h e human chronic myelogenous l e u k a e m i c c e l l line K 5 6 2 ( A T C C C C L 243), o r i g i n a l l y derived f r o m the pleural effusion o f a C M L patient i n blast crisis, was obtained f r o m A T C C a n d was maintained in 1 0 % HI- F C S / R P M I 1640. T h e cells were g r o w n at 3 7 ° C and maintained in a 5 % carbon d i o x i d e ( C 0 , P r a x a i r , M i s s i s s a u g a , O N , C a n a d a ) 2  r o o m air aerated, f u l l y h u m i d i f i e d incubator ( F o r m a S c i e n t i f i c , M a r i e t t a , O h i o ) .  2.5 CYTOTOXIC  TREATMENT  OF  CELLS  a. PDT treatment of murine bone marrow cells and L1210 cells for short term evaluations (combination experiments) E a c h mouse p r o v i d e d approximately 1 x 1 0 B M M N C s . Freshly extracted 7  B M M N C s were washed twice w i t h w a r m I M D M and resuspended i n the same m e d i u m . A  51  s m a l l aliquot o f the cells was then stained w i t h the vital stain E o s i n Y ( 0 . 3 % in P B S )  and  the c e l l concentration and viability were determined f o l l o w e d by dilution w i t h I M D M into 2.2 x 1 0 cells/ml. A t the same time, B P D and D o x were diluted into the appropriate 6  w o r k i n g concentrations in I M D M and 100 ul o f the 10 x drug was dispensed into the sterile 5 m l polystyrene test tubes (Falcon brand, B e c t o n D i c k i n s o n ) along w i t h 9 0 0 ul o f the B M M N C s . T h e tubes were then incubated at 3 7 ° C for 6 0 m i n after gentle m i x i n g . T h e cells were washed once w i t h I M D M and resuspended in 1.0 m l o f I M D M supplemented w i t h 1 0 % HI- F C S , dispensed into a 24 w e l l tissue culture plate ( L i n b r o b r a n d , F l o w L a b o r a t o r i e s , Inc., H a m d e s n , Connecticut) and were photoirradiation w i t h red light at 15J/cm . C l o n o g e n i c i t y o f the treated cells was then assessed (section 2.6a). T h e L 1 2 1 0 2  cells were processed in the same manner (section 2.6b). Experiments o f c o m b i n e d simultaneous B P D and D o x treatments were conducted w i t h both n o r m a l and leukaemic cells. Samples were incubated c o n c o m i t a n t l y w i t h both B P D and D o x in different combinations for one hour at 3 7 ° C . Subsequent manipulations f o l l o w e d the same p r o t o c o l as f o r the single agent- treated cells. E x p e r i m e n t s w h i c h i n v o l v e d sequential B P D / D o x or D o x / B P D treatments i n v o l v e d pretreatment of cells w i t h P D T f o l l o w e d by a one hour incubation p e r i o d w i t h D o x , or preincubation o f cells w i t h D o x f o l l o w e d by B P D incubation and photoirradiation. C e l l s were w a s h e d after the first treatment and handling protocols were identical to those f o r single agent treatment.  b. P D T treatment of normal and leukaemic murine haematopoietic cells preincubated with peptides (short- term colony assay) M u r i n e B M M N C s were obtained f r o m D B A / 2 m i c e as described previously (section 2.4a). B M M N C s or L 1 2 1 0 cells at 2.2 x 10 cells/ml in 9 0 0 pi o f I M D M were 6  m i x e d w i t h 10 pi o f N - A c S D K P or the control peptides N - A c S D K E and S D K P to a f i n a l concentration o f 100 n M . T h e cells were incubated at 3 7 ° C f o r 1.5 h f o l l o w e d by the addition o f 100 pi of B P D at 10 x the f i n a l concentrations. T h e cells were then incubated for an additional 1 h at 3 7 ° C and were washed 1 x w i t h I M D M . C e l l pellets were resuspended gently w i t h 1 m l o f 10 % HI- F C S / I M D M and dispensed into wells o f a 24  52  .  w e l l tissue culture plate and photoirradiated at 15 J / c m . C l o n o g e n i c i t y o f the treated cells 2  was then assessed (section 2.6a and 2.6b).  c. P D T treatment of murine bone marrow cells for long- term bone marrow cultures M u r i n e B M M N C s were obtained f r o m D B A / 2 m i c e as described previously (section 2.4a). B M M N C s at concentration o f 2.2 x 1 0 cells/ml i n 9 0 0 pi o f I M D M were 6  m i x e d w i t h 10 pi of N - A c S D K P to a f i n a l concentration o f 100 n M . T h e cells were incubated at 3 7 ° C for 1.5 h f o l l o w e d by the addition o f 100 pi o f B P D at 10 x the f i n a l concentrations. T h e cells were then incubated for an additional 1 h at 3 7 ° C and were washed 1 x w i t h I M D M . C e l l pellets were resuspended gently w i t h 1 m l o f 10 % HIF C S / T M D M and dispensed into wells o f a 24 w e l l tissue culture plate and photoirradiated at 15 J / c m . T h e treated cells were then assessed u s i n g the L T B M C assay (section 2.6d). 2  d. P D T treatment of human normal haematopoietic and leukaemic cells preincubated with peptides (short- term colony assay) N o r m a l or leukaemic cells at 2.2 x 10 cells/ml in 9 0 0 pi o f I M D M were m i x e d w i t h 6  10 pi o f N - A c S D K P or the control peptides N - A c S D K E and S D K P to a f i n a l concentration o f 100 n M . T h e cells were incubated at 3 7 ° C f o r 1.5 h f o l l o w e d by the addition o f 100 pi o f B P D at 10 x the final concentrations. T h e cells were then incubated f o r an additional 1 h at 3 7 ° C and were washed 1 x w i t h I M D M . C e l l pellets were resuspended gently w i t h 1 m l o f 10 % HI- F C S / I M D M and dispensed into wells o f a 24 w e l l tissue culture plate and photoirradiated at 15 J / c m . C l o n o g e n i c i t y o f the treated cells was then assessed (section 2  2.6e and 2.6f).  e. ara- C treatment of DBA/2 bone marrow cells preincubated with peptides M u r i n e B M M N C s were obtained f r o m D B A / 2 m i c e as described previously (section 2.4a). B M M N C s at concentration o f 2.2 x 1 0 cells/ml i n 9 0 0 p i o f I M D M were 6  53  m i x e d w i t h 10 ul o f N - A c S D K P to a f i n a l peptide concentration o f 100 n M . T h e cells were incubated at 3 7 ° C for 1.5 h f o l l o w e d by the addition o f 100 pi o f ara- C at 10 x the f i n a l concentrations. T h e cells were then incubated for an additional 1 h at 3 7 ° C and were w a s h e d 1 x w i t h I M D M . C e l l pellets were resuspended gently w i t h 1 m l o f 10 % HIF C S / I M D M and 140 pi (2.8 x 1 0 cells) was m i x e d w i t h the other ingredients o f the 5  standard agar c o l o n y assay for c l o n o g e n i c i t y determination (section 2.6a).  f. P D T of DBA/2 bone marrow cells preincubated with 50 u M ara- C M u r i n e B M M N C s were obtained f r o m D B A / 2 m i c e as described p r e v i o u s l y (section 2.4a). B M M N C s at concentration o f 2.2 x 1 0 cells/ml i n 9 0 0 pi o f I M D M were 6  m i x e d w i t h 100 p i o f ara- C to a f i n a l concentration o f 5 0 u M . T h e cells w e r e incubated at 3 7 ° C f o r 1 h, washed once w i t h I M D M , and resuspended in 9 0 0 pi o f I M D M . N e x t , 10 x solutions o f B P D (100 pi) were then added to the appropriate sample tubes to a f i n a l v o l u m e o f 1.0 m l . T h e cells were then incubated f o r an additional 1 h at 3 7 ° C and were w a s h e d 1 x w i t h I M D M . C e l l pellets were resuspended gently w i t h 1 m l o f 10 % HIF C S / I M D M and dispensed into w e l l s o f a 24 w e l l tissue culture plate and photoirradiated at 15 J / c m . C l o n o g e n i c i t y o f the treated cells was then assessed (section 2.6a). 2  2.6 CYTOTOXICITY  ASSAYS  a. Agar colony assay of treated murine bone marrow cells Standard agar- based c o l o n y assays were used to assess c y t o t o x i c i t y o n n o r m a l m u r i n e bone m a r r o w mononuclear cells ( B M M N C s ) . T h e protocol for the m u r i n e haematopoietic granulocyte- macrophage progenitor assay has been described elsewhere 294,311  u  j f treated D B A / 2 bone m a r r o w cells was added to a p l a t i n g m i x t u r e w h i c h 0  consisted o f 2 2 3 5 p i I M D M , 7 5 0 p i o f pretested HI- F C S , 375 p i o f 100 mg/ml (w/v) b o v i n e serum a l b u m i n ( B S A ) in I M D M (Boehringer M a n n h e i m , L a v a l , Q u e b e c ) , and 100 p i o f H e m o s t i m M 2 1 0 0 (see section 2. Id) as a source o f c y t o k i n e s . F i n a l l y , 4 0 0 pi o f p r e w a r m e d 3 % agar N o b l e solution (w/v) in sterile d d H 0 ( D i f c o Laboratories, D e t r o i t , 2  54  M i c h i g a n ) w a s dispensed into the c e l l m i x t u r e to achieve a f i n a l agar concentration o f 0 . 3 % and the resulting mixture was dispensed i m m e d i a t e l y i n 1.0 m l volumes to triplicate 35 m m tissue culture dishes (Sarstedt, N e w t o n , N C ) w i t h a p p r o x i m a t e l y 7 0 0 0 0 cells dispensed p e r tissue culture dish. T w o o f the agar c o n t a i n i n g plates were then p l a c e d inside a 100 m m petri dish (Fisher S c i e n t i f i c , E d m o n t o n , A l b e r t a , Canada) a l o n g w i t h a 35 m m dish c o n t a i n i n g 5 m l d d H 0 . T h e plates were then placed inside a 3 7 ° C incubator f o r seven days 2  prior to c o l o n y enumeration. A Z e i s s A x i o v e r t 35 inverted m i c r o s c o p e ( C a r l Z e i s s C a n a d a , , D o n M i l l s , O n t a r i o , C a n a d a ) was used f o r c o l o n y c o u n t i n g and a cluster consisting o f forty or m o r e cells w a s counted as a granulocyte- macrophage c o l o n y f o r m i n g unit ( C F U - G M ) . C l o n i n g e f f i c i e n c y f o r D B A / 2 C F U - G M w a s a p p r o x i m a t e l y 0.126 % .  b. Colony assay of L1210  cells  A g a r c o l o n y assay o f the leukaemic c e l l line L 1 2 1 0 w a s slightly different f r o m the p r e v i o u s l y described assay. A 334 p i aliquot o f diluted c e l l suspension was m i x e d w i t h 2 2 4 7 p i o f I M D M , 8 0 0 p i o f pretested H I - F C S , 4 0 0 p i o f B S A , and f i n a l l y 4 0 0 p i o f 3.0 % Agar- N o b l e . T o f u l l y appreciate to range o f L 1 2 1 0 l o g reduction, seeding numbers o f cells was c u s t o m i s e d f o r i n d i v i d u a l d r u g concentrations. A g a i n , 1.0 m l was dispensed into triplicate 35 m m tissue culture dishes. C o l o n i e s consisting o f forty or more cells were enumerated o n day 6 u s i n g the inverted m i c r o s c o p e . C l o n i n g e f f i c i e n c y o f L 1 2 1 0 C F U - L i n this system w a s a p p r o x i m a t e l y 36 % .  c. Short-term cytotoxicity assay ( M T T assay) C e l l s i n l o g phase growth, were washed twice w i t h D M E M to remove residual serum and 9 0 0 p i o f cells at 1.4 x 1 0 cells/ml were dispensed to 5 m l polystyrene test 6  tubes (Falcon). B P D and other chemotherapeutic agents at 10 x f i n a l concentrations were then added to each tube i n volumes o f 100 p i . T h e tubes were then gently agitated a n d a l l o w e d to incubate f o r 1 hour. T h e treated cells were then washed once and resuspended i n 9 0 0 p i o f D M E M . T h e content o f a single test tube was then distributed into eight w e l l s o f a 96 w e l l tissue culture plate (Falcon) i n 100 p i aliquots f o l l o w e d b y 15 J / c m o f red light 2  photoirradiation as described i n section 2.2. T h e plate w a s then incubated overnight at 3 7 ° C i n a 5 % CO2 incubator. T h e next day, 10 p i o f 5 mg/ml 3-[4,5-Dimethylthiazol-2-yl]-2,5-  55  d i p h e n y l t e t r a z o l i u m b r o m i d e ( M T T , S i g m a ) i n P B S was then dispensed into the w e l l s w i t h an E p p e n d o r f Repeator Pipette dispenser ( B r i n k m a n n Instruments, M i s s i s s a u g a , O n t a r i o , Canada) and a l l o w e d to incubate for 6 0 m i n at 37°C before f i x a t i o n w i t h 150 pi o f 0.05 N H C 1 / i. M i t o c h o n d r i a l dehydrogenase i n viable cells convert the y e l l o w M T T tetrazolium salt to purple f o r m a z o n crystals, w h i c h c o u l d be quantified spectrophotometrically at 595 nm (OD595).  3 1 2  '  3 1 3  T h e plate was read on a S p e c t r a M a x 2 5 0 scanning m u l t i w e l l  spectrophotometer ( M o l e c u l a r D e v i c e s , S u n n y v a l e , C A ) . O D 5 9 5 measurements o f control cells were averaged and arbitrarily set as 1 0 0 % survival and v i a b i l i t y o f the treated cells were calculated based on their relative values.  d. One step long- term bone marrow culture ( L T B M C ) of murine haematopoietic  cells  L o n g - term bone m a r r o w culture ( L T B M C ) was initiated to determine the fitness o f earlier haematopoietic progenitors and stem cells. M u r i n e L T B M C culture m e d i u m ( M y e l o C u l t M 5 3 0 0 , S t e m C e l l T e c h n o l o g i e s Inc.) was used to initiate and maintain the g r o w t h o f P D T - treated D B A / 2 B M M N C s f o r six weeks. A one step L T B M C p r o t o c o l was adapted i n w h i c h the i n o c u l u m was used to start the stromal layer as w e l l as to initiate haematopoiesis.  314  H y d r o c o r t i s o n e ( S i g m a C h e m c i a l C o . ) d i s s o l v e d in 100 % ethanol was  added to the L T B M C m e d i u m to a f i n a l concentration o f 1 0 " M before use. T h e P D T 6  treated c e l l s were d i v i d e d into two equal portions o f 1 x 1 0 cells and dispensed into w e l l s 6  o f a 2 4 w e l l tissue culture plate ( C o r n i n g C o s t a r C o r p . , C o r n i n g , N Y ) i n v o l u m e o f 1 m l . .The plates were i n c u b a t e d at 3 3 ° C during the first week and m o v e d to a 3 7 ° C 5 % C 0  2  incubator for the subsequent duration o f the experiment. C e l l s were harvested at w e e k l y intervals d u r i n g w h i c h the entire content o f the appropriate w e l l was used f o r c e l l number determination f o l l o w e d b y incorporation o f the cells into the short- term agar c o l o n y assay (section 2.6a). T h e rest o f the cells were d e m i d e p o p u l a t e d , i.e. gently agitated f o l l o w e d b y aspiration o f 5 0 0 pi o f the cells (50 % ) , and replenished w i t h 5 0 0 pi o f m u r i n e L T B M C culture m e d i u m containing 1 0 " M . T h e cells were then returned to the incubator ( 3 7 ° C , 5 % 6  C 0 , f u l l y h u m i d i f i e d ) until the next harvest seven days later. 2  56  e. C o l o n y assay of treated n o r m a l a n d l e u k a e m i c h u m a n h a e m a t o p o i e t i c cells Short- term c o l o n y assays o f h u m a n haematopoietic cells were i n i t i a l l y described b y M e s s n e r et a / .  3 1 5  P D T - treated or untreated cells at 4 0 0 pi v o l u m e were m i x e d w i t h 4 0 0 p i  phytohaemagglutinin- stimulated leucocyte conditioned m e d i u m ( P H A - L C M ) , 1200 pi pretested HI- F C S , 1600 pi I M D M , and 4 0 0 p i 10 % m e t h y l c e l l u l o s e d i s s o l v e d i n I M D M ( M e t h o c e l M C , F l u k a B i o c h e m i k a , Switzerland) to a total assay v o l u m e o f 4.0 m l . P H A L C M was harvested f r o m the peripheral b l o o d o f a healthy volunteer. B r i e f l y , b u f f y coat (mononuclear cells and plasma), w h i c h settled to the upper layer o f " r e s t e d " w h o l e b l o o d c o n t a i n i n g 5 0 U s o d i u m heparin (Fisher) per m l , was r e m o v e d b y aspiration. T h e m o n o n u c l e a r cells were then separated f r o m the p l a s m a b y centrifugation f o r 10 m i n at 4 0 0 x R C F . T h e cells were washed 2 x w i t h I M D M and the resuspended at 1 x 1 0 cells/ml i n 1 6  % P H A (v/v)/ I M D M and incubated at 3 7 ° C for 7 days. T h e c o n d i t i o n e d m e d i u m ( P H A L C M ) , separated f r o m the c e l l s , was dispensed and stored at -80°C. P H A - L C M contains a variety o f haematopoietic g r o w t h factors and supports the growth o f different c o l o n i e s . T h e mixture was then c o m b i n e d and 1 m l each was dispensed into triplicate 35 m m dishes (Sarstedt) u s i n g a 16 gauge needle connected to a 3 cc syringe (Becton- D i c k e n s o n ) . N o r m a l h u m a n B M M N C s and p r i m a r y leukaemic cells f r o m C M L patients were plated at 1 x 1 0 cells/ d i s h . C o l o n i e s c o n s i s t i n g o f forty or more cells were c o u n t e d after 14 days o f 5  culture at 3 7 ° C u s i n g an inverted m i c r o s c o p e ( C a r l Zeiss Canada).  f. C o l o n y assay of the h u m a n l e u k a e m i c cell line K 5 6 2 * P D T - treated or untreated K 5 6 2 cells were assayed s i m i l a r l y as the p r i m a r y h u m a n haematopoietic cells described above (section 2.6e) w i t h the f o l l o w i n g m o d i f i c a t i o n s . K 5 6 2 cells were plated at a f i n a l concentration o f 1 x 1 0 cells/ d i s h and P H A - L C M was replaced 4  w i t h I M D M i n the plating mixture.  57  2.7  PHOTOMETRIC  AND  SPECTROSCOPIC  ANALYSES  a. S p e c t r o p h o t o m e t r i c a n a l y s i s S t o c k B P D and D o x were d i s s o l v e d i n phosphate buffered saline ( P B S )  to  concentrations o f 1 pg/ml and 10 u M , respectively. E q u a l amounts o f B P D and D o x were dispensed into a spectrophotometric cuvette and then analysed w i t h the H P 8 4 5 2 A spectrophotometer (Palo A l t o , C A ) .  b . S p e c t r o f l u o r i m e t r i c analysis of the i n t e r a c t i o n between 1BPD T h e p r o t o c o l f o r B P D uptake analysis has been described elsewhere  and 2 8 5  Dox  . Briefly,  L 1 2 1 0 cells were incubated w i t h B P D alone, B P D in the presence o f D o x , and B P D after one hour preincubation w i t h D o x . C e l l s were w a s h e d twice w i t h P B S and then l y s e d i n the presence o f 5 0 0 pi o f 2 % T r i t o n X - 100 ( S i g m a ) / P B S . S a m p l e s were snap f r o z e n i n dry ice/ methanol bath and stored at -80°C i f readings were not planned on the same day o f the experiment. P r i o r to spectrofluorimetric readings, the lysates were put through three rounds o f freeze/ t h a w i n g . A n additional v o l u m e o f 5 0 0 p i P B S was added to the rest o f the c e l l lysate and the resultant mixture was used for analysis w i t h the A m i n c o S L M 5 0 0 C sprectrofluorimeter ( S L M instruments Inc., U r b a n a , IL). A l l measurements were performed u s i n g the continuous w a v e ( C W ) setting and at excitation and e m i s s i o n bandpass o f 0.5 n m . D e t e r m i n a t i o n o f B P D concentration was achieved u s i n g excitation reference at 4 4 0 n m and e m i s s i o n reference at 7 0 0 n m . M e a n fluorescence intensity  (MFI)  was measured at 6 9 4 n m for each sample. E m i s s i o n reference was adjusted to 6 0 0 n m f o r D o x concentration determination and M F I was measured at 5 9 0 n m . A l l experiments .were done i n duplicates.  2.8  CELL  ANALYSIS  BY FLUORESCENCE  ACTIVATED  CELLS  SORTING  (FACS)  T h e protocol f o r the determination o f cellular content o f B P D in murine splenocytes has been described e l s e w h e r e .  285  E s s e n t i a l l y the same p r o t o c o l has been adopted f o r  analysis o f D B A / 2 B M M N C s . O n e m i l l i o n o f freshly isolated B M M N C s i n 1.0 m l I M D M  58  were preincubated w i t h N - A c S D K P or S D K P (negative control) to a f i n a l concentration o f 100 n M . T i s s u e culture m e d i u m was used i n place o f peptide i n a second negative control. T h e cells were incubated at 3 7 ° C f o r 1.5 h f o l l o w e d b y 1 x w a s h w i t h I M D M . C e l l pellets were resuspended in 9 0 0 p i I M D M and m i x e d w i t h 100 p i o f 10 x B P D (100 ng/ml). T h e cells were incubated w i t h an additional 0.5 h f o l l o w e d by 2 x washes w i t h F A C S buffer and transferred to a round b o t t o m 96 w e l l plate (Costar, C a m b r i d g e , M A ) . T h e cells were labelled w i t h 10 pi o f biotin- C D 3 4 antibody (clone R A M 3 4 ) or 2 pi o f biotin- isotypic control f o r 0.5 h at 4 ° C . T h e secondary F I T C - strepavidin reagent was added after 2 washes w i t h F A C S buffer. A n additional i n c u b a t i o n p e r i o d o f 0.5 h was f o l l o w e d by 2 f i n a l washes w i t h F A C S buffer. T h e c e l l pellets were then transferred to 5 m l polystyrene tubes (Falcon) i n 5 0 0 p i o f F A C S buffer and 5 0 0 pi o f 4 % p a r a f o r m a l d e h y d e / P B S  and  stored at 4 ° C until analysis. A C o u l t e r E P I C S X L ® f l o w cytometry s y s t e m (Coulter C o r p . , M i a m i , F L )  was  u s e d f o r dual c o l o u r F A C S analysis o f B P D uptake and surface antigen e x p r e s s i o n . T h e excitation wavelength e m p l o y e d f o r F A C S analysis i n v o l v i n g B P D and F I T C was 488 n m w h i l e a 6 9 0 n m e m i s s i o n (longpass) filter was utilised to detect B P D (red) fluorescence and a 525 n m e m i s s i o n (longpass) filter was used to detect F I T C (green) fluorescence. O n e hundred thousand cells were analysed for each sample. D a t a f r o m three independent experiments are presented and source o f error is d e r i v e d f r o m the standard error o f the mean.  2.9 FLUORESCENT  MICROSCOPY  K 5 6 2 C e l l s were resuspended at a concentration o f 1 x 1 0 cells/ml i n D M E M and 6  incubated at 3 7 ° C w i t h 10 pg/ml of B P D f o r 6 0 m i n . C e l l s were then washed once and resuspended i n 1 m l o f D M E M . A 5 0 pi drop (5 x 10 cells) was placed on a glass slide 4  (Fisher S c i e n t i f i c , E d m o n t o n , A l b e r t a , Canada) and subsequently c o v e r e d w i t h a c o v e r slip (Fisher). T h e cells were observed under an O l y m p u s N e w V a n o x m i c r o s c o p e w i t h the A H 2 - F L transmitted light fluorescence attachment o n " B l u e " setting (excitation @ 3804 9 0 n m , observation @ 515 n m +). Pictures were taken w i t h the attached c a m e r a u s i n g F u j i c h r o m e 4 0 0 I S O f i l m at automatic settings.  59  3.0 ANALYSIS  OF  INTRACELLULAR  GLUTATHIONE  (GSH)  CONTENT  a. Tietze enzymatic assay C e l l u l a r glutathione ( G S H ) was determined using e n z y m a t i c c y c l i n g and E l l m a n ' s reagent (5,5'- dithiobis- (2- nitrobenzoic acid), D T N B ) as described by E y e r and Podhradsky.  3 1 6  B r i e f l y , cells were l y s e d i n 0.6 % s u l p h o s a l i c y c l i c acid/ 0.5 m M  EDTA  (Sigma) at a f i n a l concentration of 100 ul lysis buffer per 1 x 1 0 cells f o r 1 h at 4 ° C . T h e 6  supernatant was then separated after centrifugation at 16000 x R C F for 10 m i n ( m a x i m u m speed setting on the B e c k m a n n microcentrifuge) at 4 ° C . T h e c l a r i f i e d lysates were dispensed into a 96 w e l l microtitre plate (Falcon) and the v o l u m e made up to 180 ul w i t h P B S . A m i x t u r e c o n t a i n i n g 4 ug/ul N A D P H ( C a l b i o c h e m , L a J o l l a , C A ) and 1.2 ug/ul D T N B ( S i g m a ) was dispensed into each w e l l using a E p p e n d o r f repeating pipetter dispensing at 10 ul ( B r i n k m a n n ) . L a s t l y , 10 ul of yeast- d e r i v e d glutathione reductase at 0.012 U/ul ( C a l b i o c h e m ) in P B S was dispensed into each w e l l . T h e plate was then read at OD  4 1 2 n m  f o r 6 m i n i n 1 m i n intervals u s i n g a S p e c t r a M a x 2 5 0 scanning m u l t i w e l l  spectrophotometer ( M o l e c u l a r D e v i c e s , S u n n y v a l e , C A ) . T h e difference between the 1 m i n and 6 m i n readings was calculated and the concentration o f G S H was determined i n c o m p a r i s o n to k n o w n G S H standards ( S i g m a ) .  3.1  STATISTICAL  ANALYSIS  a. Analysis of colony assay data from PDT/Dox combination experiments D a t a were inputted into the N C S S 6.0.21 statistical analysis package ( N C S S Statistical software, K a y s v i l l e , Utah). T h e various treatment and control group means were c o m p a r e d u s i n g the Student's t- test w i t h paired sample f o r means. T h e data were further analysed u s i n g 2- w a y analysis o f variance (2- way A N O V A ) w i t h the t w o B P D concentrations (2.5 and 5.0 ng/ml) and the three different c o m b i n a t i o n s ( D o x / P D T , P D T - > D o x , D o x - > P D T ) set as the two parameters. A d d i t i o n a l statistical analyses were carried out w i t h the term significant at alpha (p value) = 0.05 and intragroup differences were e x a m i n e d using the B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test.  60  b. Analysis of colony assay data from the inhibitory peptide pretreatment experiments D a t a were inputted into the N C S S 6.0.21 statistical analysis package ( N C S S Statistical software, K a y s v i l l e , Utah). T h e various treatment and c o n t r o l group means were c o m p a r e d u s i n g the Student's t- test w i t h paired sample for means. T h e data were further analysed u s i n g two- w a y analysis o f variance (two- way A N O V A ) w i t h the parameters assigned to the three peptides ( N - A c S D K P , S D K P , N - A c S D K E ) as w e l l as the no peptide control groups. A d d i t i o n a l statistical analyses were carried out w i t h the term significant at alpha (p value) = 0.05 and intragroup differences were e x a m i n e d u s i n g the B o n f e r r o n i (allpairwise) m u l t i p l e c o m p a r i s o n test.  61  CHAPTER 3: COMBINED T R E A T M E N T OF MURINE N O R M A L HAEMATOPOIETIC AND L E U K A E M I C C E L L S WITH DOXORUBICIN AND PDT: CYTOTOXICITY  3.1  STUDY  ABSTRACT B e n z o p o r p h y r i n derivative m o n o a c i d r i n g A ( B P D ) , a p o r p h y r i n based  photosensitiser, shows ideal properties f o r the purpose o f p h o t o d y n a m i c therapy ( P D T ) based bone m a r r o w p u r g i n g and is currently undergoing c l i n i c a l testing f o r stem c e l l p u r g i n g in Canada. T o enhance the efficacy o f B P D - mediated P D T , a c o m b i n a t i o n approach w i t h D o x o r u b i c i n ( D o x ) was pursued. Short term c o l o n y assays were used to measure the frequency o f c o l o n y f o r m i n g unit- granulocyte/macrophage progenitors ( C F U G M assay) o f n o r m a l D B A / 2 bone m a r r o w cells and c o l o n y f o r m i n g unit- l e u k a e m i a ( C F U - L ) o f the murine leukaemic c e l l line L I 2 1 0 after drug treatment. B P D alone in the absence o f light resulted in no reduction o f C F U - G M ; however, 10 ng/ml o f B P D f o l l o w e d by 15 J / c m of red light activation resulted in the e l i m i n a t i o n o f 0.5 logs o f the 2  C F U - G M and 4 logs of C F U - L. D o x at 5.0 u M resulted i n one l o g reduction o f both n o r m a l as w e l l as leukaemic L 1 2 1 0 c o l o n y f o r m i n g units; i n addition, the c y t o t o x i c i t y o f D o x was neither enhanced nor d i m i n i s h e d in the presence o f 15 J / c m o f red light 2  exposure. Simultaneous treatment o f n o r m a l D B A / 2 bone m a r r o w cells or the L 1 2 1 0 c e l l line w i t h P D T and D o x (PDT/Dox).resulted i n the predicted l o g reductions. H o w e v e r , w h e n cells were preincubated w i t h D o x for one hour prior to P D T (Dox-> P D T ) , the resultant c y t o t o x i c i t y was 1.5- 2.5 f o l d more than that observed i n the simultaneous treatment ( P D T / D o x ) and in the reverse sequence ( P D T - > D o x ) . S i g n i f i c a n t l y , L I 2 1 0 cells were more susceptible to the D o x - > P D T treatment than n o r m a l C F U - G M resulting i n enlargement o f the therapeutic w i n d o w . T h i s augmentation is treatment sequence dependent as w e l l as drug dose dependent. T h e above findings suggest that care must be taken w h e n P D T is used in conjunction w i t h other chemotherapeutic agents i n the c l i n i c a l setting and that j u d i c i o u s use  62  of P D T c o m b i n a t i o n therapy can lead to the augmentation o f the therapeutic w i n d o w between n o r m a l and neoplastic cells.  3.2  INTRODUCTION  H i g h dose radiochemotherapy c o u p l e d w i t h haematopoietic stem c e l l rescue is an effective f o r m o f treatment f o r various k i n d s o f h u m a n malignancies. B o n e m a r r o w f r o m allogeneic h u m a n leukocyte antigen ( H L A ) - matched donors is an ideal source o f haematopoietic stem cells for leukaemic patients because o f the associated yet beneficial graft- versus- l e u k a e m i a ( G v L ) effect. U n f o r t u n a t e l y , the dearth o f H L A - matched donors restricts the use o f allogeneic bone m a r r o w transplantation ( a l B M T ) . In addition, many older patients exhibit a h i g h incidence o f age- related intolerance to graft- versus- hostdisease ( G v H D ) . Therefore, autologous bone m a r r o w s are used in older cancer patients or those without H L A - matched donors. C a n c e r patients u n d e r g o i n g autologous bone m a r r o w transplantations ( a u B M T ) have higher relapse rates than those receiving allogeneic bone marrow.  1 5 2  T h r o u g h retroviral m a r k i n g studies, B r e n n e r and colleagues have  u n e q u i v o c a l l y demonstrated the presence o f contaminating neoplastic cells i n autologous bone m a r r o w harvests and the contribution o f these cells to relapses p o s t t r a n s p l a n t a t i o n .  154  B o n e m a r r o w p u r g i n g therefore attempts to address this p r o b l e m b y the selective r e m o v a l or destruction o f the contaminating cancer cells w h i l e preserving enough o f the normal haematopoietic progenitor and stem cells to effect haematopoietic reconstitution. B o n e m a r r o w p u r g i n g u s i n g p h o t o d y n a m i c therapy ( P D T ) represents an ideal application o f this technology. T h e ex associated w i t h light delivery and  vivo nature o f p u r g i n g eliminates problems  in vivo p h a r m a c o k i n e t i c s . V a r i o u s photosensitisers have  been proposed f o r p u r g i n g i n c l u d i n g the b e n z o p o r p h y r i n derivatives, phthalocyanines a n d Merocyanine 540 ( M C 5 4 0 ) .  2 9 0  '  2 9 1  In this laboratory, the mono- a c i d r i n g A analogue o f  b e n z o p o r p h y r i n derivative ( B P D ) w a s s h o w n to be a p r o m i s i n g p u r g i n g agent because o f preferential accumulation b y leukaemic cells i n addition to their higher susceptibility to P D T mediated b y B P D . an  2 9 2  J a m i e s o n has also demonstrated the effectiveness o f B P D p u r g i n g i n  ex vivo m u r i n e p u r g i n g m o d e l .  2 9 4  In a d d i t i o n , B P D has an unique absorption peak at  63  690 n m w h i c h is outside the absorption range o f h a e m o g l o b i n ; therefore, photoactivation o f B P D is not affected by the presence o f erythrocytes, a c o m m o n occurrence in haematological s a m p l e s .  3 1 7  C a n c e r therapies w h i c h c o m b i n e different drugs have been i n use f o r m a n y years. C o m b i n a t i o n therapy is especially effective i n the management o f l e u k a e m i a and l y m p h o m a . P D T has been used i n combinations w i t h traditional chemotherapeutic agents or other photosensitisers to achieve i m p r o v e d k i l l i n g o f cancerous c e l l s .  3 0 5  -  3 1 8  Ideally,  c o m b i n a t i o n therapy aims to utilise drugs w i t h different mechanisms o f action such that the probability o f the cancer d e v e l o p i n g drug resistance to all the drugs i n the regimen is m i n i m i s e d . In addition, drugs used in c o m b i n a t i o n therapy s h o u l d have additive anticancer c y t o t o x i c i t y but not adverse effects against n o r m a l tissues. In this chapter, B P D - mediated P D T was c o m b i n e d w i t h D o x i n various concentration as w e l l as sequence c o m b i n a t i o n s . T h e relative sensitivities o f the m u r i n e leukaemic cell line L I 2 1 0 and its n o r m a l counterpart, haematopoietic progenitor cells f r o m D B A / 2 m i c e , were determined u s i n g standard agar based c o l o n y assays. T h i s chapter addresses the question as to whether a c o m b i n a t i o n approach u s i n g D o x and B P D can be applied to P D T p u r g i n g and whether the differential susceptibility o f normal and leukaemic cells can be altered w i t h such a c o m b i n a t i o n approach.  64  3.3 RESULTS  Single agent cytotoxicity In order to establish baseline susceptibility profiles to single- agent treatments. L 1 2 1 0 cells and haematopoietic progenitor cells derived f r o m D B A / 2 m i c e were treated s i n g l y w i t h either B P D - mediated P D T or D o x (with red light irradiation). In figure 3.1, normal and leukaemic cells were incubated w i t h various concentrations o f B P D f o l l o w e d b y red light irradiation at 15 J / c m . C l o n o g e n i c i t y o f the cells was then e x a m i n e d u s i n g 2  standard agar based c o l o n y assays, w h i c h measured c o l o n y f o r m i n g units- l e u k a e m i c c e l l ( C F U - L ) and c o l o n y f o r m i n g units- granulocyte/ macrophage ( C F U - G M ) o f L I 2 1 0 and the D B A / 2 haematopoietic progenitors, respectively. T h e number o f c o l o n i e s w h i c h consisted o f 5 0 or more cells were enumerated at day 6 (leukaemic cells) or day 7 (normal progenitor cells) w i t h an inverted m i c r o s c o p e . Results are expressed as s u r v i v a l fraction relative to control cells that were exposed to red light in the absence o f B P D or D o x . U n d e r identical conditions, both L I 2 1 0 cells and D B A / 2 haematopoietic cells responded to treatment in a dose- dependent manner. H o w e v e r , the L 1 2 1 0 cells were m u c h more susceptible to B P D - mediated P D T cytotoxicity than the D B A / 2 haematopoietic cells. B P D at a dose o f 10 ng/ml B P D and red light (15 J/cm ) resulted in 4- l o g reduction o f L 1 2 1 0 2  c l o n o g e n i c cells w h i l e the same dose o f B P D o n l y reduced D B A / 2 progenitors b y 0.5 l o g . S i m i l a r differential susceptibility between L 1 2 1 0 and D B A / 2 progenitor cytotoxicity was observed, albeit to a l o w e r degree, at 5 ng/ml o f B P D . Nevertheless, the data (figure 3.1) clearly showed that the l e u k a e m i c c e l l line L 1 2 1 0 was m u c h more susceptible to B P D mediated P D T than n o r m a l murine haematopoietic cells. B P D alone in the absence o f direct light exposure was not c y t o t o x i c as determined b y the M T T assay, w h i c h measures m i t o c h o n d r i a l dehydrogenase activity in viable cells (figure 3.2). D o x o r u b i c i n h y d r o c h l o r i d e ( D o x ) , u n l i k e B P D and light, s h o w e d essentially no selective k i l l i n g o f L 1 2 1 0 cells over n o r m a l progenitor cells (figure 3.3). In addition, D o x c y t o t o x i c i t y o n L 1 2 1 0 cells was unaltered w i t h or without direct light exposure (figure 3.4).  65  Figure 3.1  The effect of B P D - mediated P D T on the clonogenicity o f normal DBA/2 haematopoietic progenitors and the leukaemic cell line L1210 Differential susceptibility o f L I 2 1 0 leukaemic cells and D B A / 2 haematopoietic progenitors to P D T mediated b y B P D . C e l l s received different doses o f B P D and were exposed to 15 J / c m o f red light (600- 9 0 0 n m ) . C o l o n y f o r m i n g units o f n o r m a l and leukaemic cells w e r e determined w i t h standard assays and c o l o n i e s were scored on day 6 ( C F U - L ) and day 7 ( C F U - G M ) o f culture u s i n g an inverted m i c r o s c o p e . O n l y colonies w i t h 50 o r more cells were counted. Untreated L 1 2 1 0 cells a n d D B A / 2 progenitors y i e l d e d 137.85 ± 9.57 a n d 97.35 ± 7.78 c o l o n i e s , w h i c h translated to clonogenicity o f 41 % and 0.126 % respectively. S u r v i v a l fractions o f treated cells were calculated based o n the n u m b e r o f colonies generated d i v i d e d b y the n u m b e r o f colonies f r o m the untreated c o n t r o l . F u r t h e r m o r e , dilution o r correction factors were incorporated into the calculations f o r L I 2 1 0 cells i n order to cover the 4- l o g s u r v i v a l fraction range. Data obtained f r o m 12 ( L 1 2 1 0 ) and 11 ( D B A / 2 progenitors) independent experiments is presented. E r r o r bars are derived f r o m standard errors o f the m e a n . 2  66  survival fraction  DBA/2 B M  0.0H O  0.00  H  0.0001  j—i—i—i—  •  •  2.5  •  •  T 7.5  [ B P D ] ng/ml  67  i  10  L1210  100 •  75  A  %OD595 of respective no B P D controls  %OD595 of controlno light  50 4 •O25  %OD595 of control • 15J/cm2 red light  J  10  100  [BPD] ng/ml  Figure  3.2  Cytotoxicity of B P D in the absence or presence of 15 J / c m 2 red light irradiation on L1210 cells as determined by the M T T viability assay L 1 2 1 0 cells were incubated w i t h different doses o f B P D f o r 1 h r f o l l o w e d by r e d light exposure at 15 J / c m o r n o i l l u m i n a t i o n . V i a b i l i t y o f the cells was determined after overnight incubation at 37° C u s i n g the M T T viability assay. O D o f cells that received n o B P D w a s arbitrarily designated to be 100 % . B P D - mediated P D T is o n l y cytotoxic w h e n activated b y light and inhibited 5 0 % o f the L 1 2 1 0 cells ( I C 5 0 ) at 6 ng/ml. C e l l s w h i c h h a d received B P D but no light remained viable. D a t a f r o m a single representative experiment is presented. 2  5 9 5  L 1 2 1 0  68  Figure 3.3  The effect of Dox and 15 J/cm 2 red light on the clonogenicity of DBA/2 haematopoietic progenitor cells and L1210 leukaemic cells Differential susceptibility o f L I 2 1 0 leukaemic cells and D B A / 2 haematopoietic progenitors to D o x a n d light is m i n i m a l . C e l l s received different doses o f D o x and e x p o s e d to 15 J / c m o f red light (600- 9 0 0 n m ) . C o l o n y f o r m i n g units o f normal and leukaemic cells were determined w i t h standard assays and colonies were scored o n d a y 6 ( C F U - L ) and day 7 ( C F U - G M ) o f culture u s i n g an inverted m i c r o s c o p e . Untreated L I 2 1 0 cells and D B A / 2 progenitors yielded 119.33 ± 9.67 and 97.35 ± 7.78 c o l o n i e s , w h i c h translated to clonogenicity o f 3 6 % a n d 0.126 % respectively S u r v i v a l fractions o f treated cells were calculated based o n the number o f colonies generated d i v i d e d b y the n u m b e r o f colonies f r o m the untreated control. Data obtained f r o m 5 ( L 1 2 1 0 ) a n d 6 ( D B A / 2 progenitors) independent experiments is presented. E r r o r bars are derived f r o m standard errors o f the m e a n . 2  /  69  70  100 - i  75  %OD595 of controlno light  J  %OD595 of respective no Dox controls 50^  25  %OD595 of control15J/cm2 red light  H  0  J_L J-U.  0  10  100  [DOX]uM  Figure  3.4  Cytotoxicity of Dox in the absence or presence of 15 J/cm 2 red light irradiation on L1210 cells as determined by the M T T viability assay L 1 2 1 0 cells were incubated w i t h different doses o f D o x f o l l o w e d b y r e d light exposure at 15 J / c m o r , n o i l l u m i n a t i o n . V i a b i l i t y o f the cells w a s determined after overnight incubation at 3 7 ° C using the M T T viability assay. OD o f cells that received n d D o x w a s arbitrarily designated to be 100 % . L i g h t activation subsequent to D o x incubation d i d not alter the viability o f L 1 2 1 0 cells. T h e concentrations o f D o x w h i c h inhibited 5 0 % o f L 1 2 1 0 cells therefore were s i m i l a r at 2 0 u M , i n the presence o r absence o f 15 J / c m red light exposure. Data f r o m a single representative experiment is presented. 2  5 9 5  2  71  Combination experiments with B P D - mediated P D T and Dox: simultaneous and sequenced treatments of D B A / 2 haematopoietic progenitor cells T o examine the possible cytotoxic interactions between B P D - mediated P D T and D o x , D B A / 2 bone m a r r o w cells were treated w i t h various c o m b i n a t i o n s o f the above t w o agents, simultaneously and sequentially. C o i n c u b a t i o n o f D B A / 2 haematopoietic progenitor cells w i t h 5.0 ng/ml B P D and 2.5 p M D o x f o l l o w e d by 15 J / c m red light exposure ( D o x / 2  P D T ) resulted i n a modest increase i n cytotoxicity c o m p a r e d to P D T or D o x single agent treatments (Figure 3.5). C y t o t o x i c i t y d i d not change appreciably f r o m the simultaneous regimen w h e n the cells were treated first w i t h P D T f o l l o w e d b y one hour incubation with 2.5 p M D o x ( P D T - > D o x ) . W h e n D B A / 2 haematopoietic progenitors were incubated with D o x for one hour prior to P D T treatment (Dox-> P D T ) , a moderate reduction o f the s u r v i v a l fraction o f n o r m a l C F U - G M f r o m 0.47 ± 0.08 to 0.15 + 0.04 or a 3.2- f o l d increase i n k i l l i n g c o m p a r e d to reverse sequence was observed ( P D T - > D o x ) . Therefore, preincubation w i t h D o x appeared to presensitise cells to the subsequent P D T treatment. A s s h o w n i n figure 3.6, s i m i l a r f i n d i n g s were made w h e n 5.0 ng/ml B P D was c o m b i n e d w i t h 1.25 p M D o x i n the different c o m b i n a t i o n s . Figures 3.7 and 3.8 s h o w that the sequence o f D o x - > P D T u s i n g 2.5 ng/ml o f B P D i n c o m b i n a t i o n s w i t h 2.5 o r 1.25 p M D o x effected superior k i l l i n g c o m p a r e d to P D T - > D o x and the simultaneous use o f D o x / P D T . It should be noted that a less than one l o g reduction i n c e l l numbers was mediated b y the treatments s h o w n i n figures 3.6 and 3.7 since a l o w e r dose o f B P D w a s u s e d .  Combination experiments with B P D - mediated P D T and Dox: simultaneous and sequenced treatments of L 1 2 1 0 leukaemic cells T h e clonogenicity of the leukaemic c e l l line L 1 2 1 0 was s i m i l a r l y affected by the combination treatments as normal haematopoietic progenitors. A s demonstrated i n figure 3.5- 3.8, preincubation o f L 1 2 1 0 cells w i t h either 1.25 or 2.5 u M o f D o x f o r one h o u r  y  72  increased the efficacy o f subsequent P D T - mediated c e l l k i l l i n g . T h e resultant cytotoxicity was significantly greater than the reverse sequence P D T - > D o x and simultaneous treatment. In addition, L I 2 1 0 cells were more susceptible to the D o x - > P D T c o m b i n a t i o n regimen than D B A / 2 haematopoietic progenitors. A s s h o w n i n figure 3.5, the s u r v i v a l fraction o f L 1 2 1 0 cells was significantly reduced f r o m 0.10 + 0.07 to 0.0035 ± 0 . 0 0 5 , o r a 28.8- f o l d increase i n k i l l i n g , when the cells were e x p o s e d to 2.5 p M D o x prior to P D T mediated b y 5.0 ng/ml P D T (Dox-> P D T ) c o m p a r e d to the reverse sequence P D T - > D o x .  Dox->PDT PDT->Dox  I  Combination  £3 DBA/2 BMMNC  H  PDT/Dox  L1210  Dox only • BPD only 0.001  T  T  0.01  0.1  survival fraction  Figure 3.5  Survival fraction of DBA/2 haematopoietic progenitors and L1210 cells to combinations of 5 ng/ml B P D and 2.5 p M Dox in conjunction with 15 J/cm 2 of red light exposureSimultaneous D o x / P D T and P D T - > D o x treatments u s i n g 5 ng/ml o f B P D and 2.5 p M o f D o x yielded s i m i l a r reductions i n the c l o n o g e n i c i t y o f D B A / 2 C F U - G M and L 1 2 1 0 C F U - L . H o w e v e r , L I 2 1 0 cells were more susceptible to the D o x - > P D T treatment than its nonmalignant counterpart, resulting i n the enlargement o f the therapeutic w i n d o w . Data obtained f r o m 6 ( D o x / P D T o f L 1 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T and P D T - > D o x o f L I 2 1 0 a n d D B A / 2 cells) independent experiments is presented. E r r o r bars are derived f r o m standard errors o f the m e a n .  73  Dox->PDT PDT-->Dox  ED DBA/2 BMMNC  Combination  HI L12I0  PDT/Dox Dox only BPD only 0.001  T  0.01  0.1  Survival fraction  Figure 3.6  Survival fraction of DBA/2 haematopoietic progenitors and L1210 cells to combinations of 5 ng/ml B P D and 1.25 p M Dox in conjunction with 15 J/cm 2 of red light exposure Simultaneous D o x / P D T and P D T - > D o x treatments u s i n g 5 ng/ml o f B P D and 1.25 p M o f D o x y i e l d e d similar reductions' i n the clonogenicity o f D B A / 2 C F U - G M and L 1 2 1 0 . . C F U - L . H o w e v e r , : I J 2 1 0 cells were more susceptible to the D o x - > P D T treatment than its nonrhalignant counterpart, resulting i n the enlargement o f the therapeutic w i n d o w . Data obtained f r o m 6 ( D o x / P D T o f L l 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T and P D T - > D o x o f L 1 2 1 0 a n d D B A / 2 cells) independent experiments i s presented. E r r o r bars are d e r i v e d f r o m standard errors o f the mean.  74  Dox->PDT PDT->Dox Combination  PDT/Dox Dox only  •  DBA/2 B M M N C  H  1.1210  i—i  BPD only 0.1  Survival fraction  Figure  3.7  Survival fraction of D B A / 2 haematopoietic progenitors L1210 cells to combinations of 2.5 ng/ml B P D and 2.5 Dox in conjunction with 15 J/cm 2 of red light exposure  and pM  Preferential k i l l i n g . o f - L l 2 1 0 cells . o f the D o x - > P D T treatment p r o t o c o l disappeared w h e n a ' l o w e r dose o f B P D was u s e d . . C l o n o g e n i c i t y o f n o r m a l C F U - G M and l e u k a e m i c C F U - L were reduced b y the same amount w h e n 2.5 ng/ml o f B P D was used w i t h 2.5 u M o f D o x . Data obtained f r o m 6 ( D o x / P D T o f L 1 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T and P D T - > D o x o f L 1 2 1 0 and D B A / 2 cells) independent experiments i s presented. E r r o r bars are d e r i v e d f r o m standard errors o f the m e a n .  75  Dox->PDT PDT->Dox  [3 DBA/2 BMMNC  Combination  H L1210  PDT/Dox Dox only BPD only  •iim  0.1 Survival fraction  Figure 3.8  Survival fraction of DBA/2 haematopoietic progenitors L1210 cells to combinations of 2.5 ng/ml B P D and 1.25 Dox in conjunction with 15 J/cm 2 of red light exposure  and pM  Preferential k i l l i n g o f L 1 2 1 0 cells o f the D o x - > P D T treatment protocol disappeared w h e n a l o w e r dose o f B P D w a s u s e d . C l o n o g e n i c i t y Of normal C F U - G M and leukaemic C F U - L were reduced s i m i l a r l y w h e n 2.5 ng/ml o f B P D was u s e d w i t h 1.25 u M o f D o x . D a t a obtained f r o m 6 ( D o x / P D T o f L 1 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T a n d P D T - > D o x o f L 1 2 1 0 a n d D B A / 2 cells) independent experiments is presented. E r r o r bars are derived f r o m standard errors o f the mean.  76  Differential susceptibility of L1210  cells and DBA/2 haematopoietic  progenitors to Dox-> P D T sequenced combination treatment T o further investigate the effects that treatments w h i c h c o m b i n e D o x and B P D mediated P D T had o n the cytotoxicity therapeutic w i n d o w between D B A / 2 haematopoietic progenitors and L 1 2 1 0 c e l l s , data f r o m the above c o m b i n a t i o n s were represented as ratios o f L 1 2 1 0 C F U - L s u r v i v a l fractions over D B A / 2 C F U - G M s u r v i v a l fractions (Figure 3.9). A therapeutic ratio o f 1 i m p l i e s equivalent k i l l i n g o f n o r m a l and l e u k a e m i c cells. O n the other hand, a ratio higher than 1 denotes preferential k i l l i n g o f the l e u k a e m i c cells in lieu o f n o r m a l haematopoietic cells. S i n g l e agent P D T treatment u s i n g B P D at 5.0 ng/ml was moderately effective i n the selective eradication o f L I 2 1 0 c l o n o g e n i c cells w i t h a therapeutic ratio o f 3.30. D o x , h o w e v e r , was not selective i n its k i l l i n g o f l e u k a e m i c c e l l s w i t h therapeutic ratios o f 0.94 and 0.98 at 2.5 u M and 1.25 m M , respectively. T h e advantage o f treating cells w i t h D o x p r i o r to P D T (Dox-> P D T ) was clearly demonstrated i n c o m b i n a t i o n s w h i c h u t i l i s e d 5.0 ng/ml B P D w i t h 2.5 p M or 1.25 p M D o x ; h o w e v e r , preferential k i l l i n g o f L 1 2 1 0 cells was not realised w h e n the l o w e r dose o f B P D at 2.5 ng/ml was used. C o m b i n a t i o n treatments w h i c h i n v o l v e d the simultaneous administration o f P D T and D o x ( D o x / P D T ) as w e l l as P D T before D o x ( P D T - D o x ) y i e l d e d s i m i l a r therapeutic ratios. A g a i n , advantageous therapeutic ratios were achieved o n l y at the higher B P D dose o f 5.0 ng/ml but not at 2.5 ng/ml.  Statistical analysis of combination treatments T h e significance o f results f r o m the c y t o t o x i c i t y assays was determined using the two- w a y analysis o f variance ( A N O V A ) test w i t h term significant at alpha (p- value) = 0.05. D a t a f r o m c o m b i n a t i o n treatments o f D B A / 2 haematopoietic progenitor cells and L 1 2 1 0 leukaemic cells were independently analysed by arbitrarily setting the concentration of D o x (1.25 and 2.5 p M ) as the constant and testing the s i g n i f i c a n c e o f the resultant s u r v i v a l fractions o f the t w o different doses o f B P D (2.5 and 5.0 ng/ml) and the three  77  c o m b i n a t i o n treatment regimens were used ( D o x / P D T , P D T - > D o x , D o x - > P D T ) . A s s h o w n i n table 3.1a, there were significant differences between 2.5 and 5.0 ng/ml B P D as w e l l as amongst the three c o m b i n a t i o n treatment regimens w h e n 2.5 u M D o x was used in c y t o t o x i c i t y assays i n v o l v i n g D B A / 2 haematopoietic progenitor c e l l s ; however, at the l o w e r dose o f D o x (1.25 u M ) , o n l y the c o m b i n a t i o n regimens were significantly different f r o m each other. N e x t , intergroup differences were c o m p a r e d w i t h the B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test, a more p o w e r f u l analysis t o o l w h i c h p r o v i d e d a m o r e detailed picture o f the s i g n i f i c a n c e o f differences between the groups. A s s h o w n i n table 3.1b, c y t o t o x i c i t y results f r o m c o m b i n a t i o n treatments w h i c h incorporated the higher dose o f D o x o f 2.5 u M were significantly different f r o m each other i n terms o f the dose o f B P D used (2.5 and 5.0 ng/ml). M o r e importantly, B o n f e r r o n i analysis revealed that the r e g i m e n D o x > P D T was significantly different f r o m the other c o m b i n a t i o n regimens, P D T - > D o x and D o x / P D T . A t the l o w e r D o x concentration o f 1.25 u M , significant intergroup difference was o n l y demonstrated between D o x - > P D T and P D T - > D o x . T h e same analyses were p e r f o r m e d on c l o n o g e n i c i t y data o f L 1 2 1 0 cells treated w i t h the different combinations. In table 3.2a, two- w a y A N O V A demonstrated significance o f the results o f c o m b i n a t i o n experiments w h i c h utilised the higher D o x dose o f 2.5 u M . E x p e r i m e n t s w h i c h used the l o w e r D o x dose o f 1.25 u M were o n l y significant i n terms o f treatment sequence but not i n the dose o f B P D used. A g a i n , B o n f e r r o n i (allpairwise) m u l t i p l e c o m p a r i s o n test was used to p r o v i d e more statistical details. T a b l e 3.2b shows that the D o x - > P D T c o m b i n a t i o n was significantly different f r o m P D T - > D o x and D o x / P D T w h e n 2.5 u M o f D o x was used. H o w e v e r , the three c o m b i n a t i o n s were not s i g n i f i c a n t l y different w h e n a l o w e r dose o f D o x was used.  78  so 40 J  Therapeutic ratio  30-^  20 J  El  5.0ng/ml BPD/2.5uM Dox  •  5.0ng/mlBPD/1.25uMDox  •  2.5ng/ml BPD/2.5uM Dox  •  2.5ng/ml BPD/1 .25|JM Dox  10J  f—  Q  DM  A X  Treatment regimen  Figure  3.9  z O  D i f f e r e n t i a l k i l l i n g o f L1210 cells over the d i f f e r e n t P D T / D o x c o m b i n a t i o n s  DBA/2  progenitors  in  Preferential k i l l i n g o f L 1 2 1 0 cells i n the various P D T / D o x combinations are expressed as therapeutic ratios a n d are derived f r o m the ratios o f s u r v i v a l fractions o f D B A / 2 C F U - G M over the s u r v i v a l fractions o f L 1 2 1 0 C F U L. T h e r e f o r e , a therapeutic ratio o f 1 denotes equal k i l l i n g o f normal and leukaemic cells. Ratios over 1 suggest preferential k i l l i n g o f the L 1 2 1 0 cells over n o r m a l D B A / 2 bone m a r r o w cells. Preferential k i l l i n g o f L 1 2 1 0 cells in the D o x - > P D T regimen was dependent o n the B P D concentration since the l o w e r B P D dose o f 2.5 ng/ml effected no enhanced c y t o t o x i c i t y .  79  Table 3 . 1 a  Two- way analysis of variance ( A N O V A ) of cytotoxicity data from DBA/2 haematopoietic progenitor cells treated with the three different drug combinations involving Dox and B P D mediated P D T : significance of treatment sequence and B P D dose  B P D dose  Treatment sequence  (2.5 v s 5.0  (Dox/PDT, PDT->  ng/ml)  D o x , Dox-> P D T )  2.5 u M D o x  +  1.25 p M D o x  -  T w o - w a y analysis o f variance (ANOVA) w a s u s e d to determine the significance o f the different experimental groups b y arbitrarily setting the D o x concentrations (2.5 a n d 1.25 u M ) as the constants and e x a m i n i n g the significance o f B P D doses (2.5 vs 5.0 ng/ml) as w e l l as the three treatment regimens (Dox/ P D T , P D T - > D o x , D o x - > P D T ) . T h e plus sign (+) w i t h i n shaded cell indicates significant difference w i t h probability level less than a predetermined a value o f 0.05 (p< 0.05).  80  Table 3.1b  Statistical analyses of data from the different Dox/PDT combinations from DBA/2 haematopoietic cells: Bonferroni (allpairwise) multiplecomparison testings of BPD concentrations and treatment sequences  B P D dose (2.5 vs 5.0  Treatment sequence  ng/ml) 2.5 u M Dox  iiiiiiif  iiliiiiif  siHlljili  Dox/FDT  PDT- > Dox  Dox->PDT  Dox/FDT PDT-> Dox Dox->PDT  1.25 u M Dox  ++  Dox/FDT  -H-  PDT- > Dox  Dox->PDT  Dox/FDT PDT- > Dox Dox->PDT  ++  B o n f e r r o n i (all-pairwise) m u l t i p l e c o m p a r i s o n analysis was performed on cytotoxicity data f r o m D B A / 2 haematopoietic progenitor cells treated w i t h the different combinations o f B P D - mediated P D T and D o x . A g a i n , the concentration o f D o x w a s arbitrarily designated the constant i n the analysis. T h e sign (<|>) w i t h i n the shaded cell i n the middle c o l u m n indicates an significant intergroup difference between the t w o B P D concentrations o f 2.5 and 5.0 ng/ml w i t h i n a constant D o x concentration. T h e plus sign (++) w i t h i n the cells i n the rightmost c o l u m n indicate intergroup significant difference between the represented treatment regimen w i t h i n a constant D o x concentration o f either 1.25 o r 2.5 u M .  81  Table 3.2a  Two- way analysis of variance ( A N O V A ) of cytotoxicity data from L1210 leukaemic cells treated with the three different drug combinations involving Dox and B P D - mediated P D T : significance of treatment sequence and B P D dose  B P D dose  Treatment sequence  (2.5 vs 5.0  (Dox/PDT, PDT->  ng/ml)  D o x , Dox-> P D T )  2.5 u M D o x  +"  +  1.25 p M D o x  +  -  T w o - w a y analysis o f variance ( A N O V A ) was used to determine the significance o f the different experimental groups by arbitrarily setting the D o x concentrations (2.5 and 1.25 u M ) as the constants and e x a m i n i n g the significance o f B P D doses (2.5 v s 5.0 ng/ml) as w e l l as the three treatment regimens ( D o x / P D T , P D T - > D o x , D o x - > P D T ) . T h e plus sign (+) w i t h i n shaded cell indicates significant difference w i t h probability level less than a predetermined a value o f 0.05 (p< 0.05).  82  Table 3.2b  Statistical analyses of data from the different Dox/ P D T combinations from L1210 leukaemic cells: Bonferroni multiple- comparison testings of B P D concentrations and treatment sequences  B P D dose (2.5 v s 5.0  Treatment sequence  ng/ml) 2.5 p M Dox  Dox/FDT  PDT- > Dox  Dox->PDT  Dox/FDT PDT- > Dox Dox->PDT  1.25 p M Dox  ++  Dox/FDT  ++  PDT- > Dox  Dox->PDT  Dox/ FDT PDT- > Dox Dox-> PDT  B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n analysis was performed o n cytotoxicity data f r o m , L 1 2 1 0 leukaemic.xells treated w i t h the different combinations o f B P D - mediated P D T and D o x . A g a i n , the concentration of. D o x w a s arbitrarily-designated as the constant i n the analysis. T h e <> j sign (<j>) w i t h i n the shaded cells i n the m i d d l e C o l u m n indicate ah intergroup significant difference between the t w o B P D concentrations o f 2.5 and 5.0 ng/ml w i t h i n a constant D o x concentration. T h e plus s i g n (++) w i t h i n the cells i n the rightmost c o l u m n indicate intergroup significant difference between the represented treatment regimen w i t h i n a constant D o x concentration o f either 1.25 o r 2.5 u M .  83  DISCUSSION  T h e utility o f b e n z o p o r p h y r i n derivative- mediated p h o t o d y n a m i c therapy ( P D T ) i n autologous bone m a r r o w p u r g i n g was extensively e x p l o r e d b y J a m i e s o n and colleagues.  2 9 2 - 2 9 4  These investigators f o u n d that the m o n o a c i d ring- A analogue o f  b e n z o p o r p h y r i n derivative ( B P D ) was selectively taken up b y l e u k a e m i c cells o f human and mouse o r i g i n . O n average, a six- f o l d increase in photosensitiser uptake over n o r m a l haematopoietic cells was observed. Further studies e x a m i n i n g the c y t o t o x i c i t y o f B P D mediated P D T o n the clonogenicity o f n o r m a l haematopoietic cells and leukaemic cells also demonstrated the selectivity o f this m o d a l i t y o f treatment. In a d d i t i o n , u s i n g the long- term bone m a r r o w assay ( L T B M C ) , w h i c h measures the g r o w t h o f less d e v e l o p e d cells o f the haematopoietic system, these authors demonstrated that B P D - mediated P D T had m i n i m a l toxicity on h u m a n early haematopoietic progenitor cells. Photosensitisers other than B P D , such as m e r o c y a n i n e 5 4 0 ( M C 5 4 0 ) and the phthalocyanines are also b e i n g considered for P D T - mediated p u r g i n g o f contaminated bone m a r r o w  2 7 5  >  2 9 1  .  P D T has also been used i n conjunction w i t h different treatment modalities such as heat a n d i o n i s i n g radiation f o r  in vivo a p p l i c a t i o n s .  3 0 3  -  3 1 9  '  3 2 0  In a d d i t i o n , various groups  have c o m b i n e d P D T w i t h chemotherapeutic agents, cytokines, and even m u l t i p l e photosensitisers i n an effort to i m p r o v e the e f f i c a c y and selectivity o f t r e a t m e n t .  305  '  318  >  321-323 D o x o r u b i c i n or D o x is an anthracycline- based antibiotic d e r i v e d f r o m the Streptomyces species and has been used i n the management o f various f o r m s o f c a n c e r .  3 2 4  D a u n o r u b i c i n and idarubicin are also members o f the anthracycline f a m i l y w i t h extensive uses in ahtileukaemia therapy. D o x was chosen because its p h o t o p h y s i c a l , p r o f i l e and :  c y t o t o x i c m e c h a n i s m s were unique f r o m those o f B P D . T h e  ex vivo nature  of  p h a r m a c o l o g i c a l p u r g i n g bypasses many o f the constraints i m p o s e d b y systemic pharmacokinetics  in vivo and  therefore permits greater latitude i n dose and sequence  experimentation i n c o m b i n a t i o n therapy. T h e p r i n c i p a l goal o f the present project was to increase the efficacy o f B P D mediated photodynamic p u r g i n g . In this system, w h e n either n o r m a l D B A / 2 haematopoietic progenitors or L 1 2 1 0 l e u k a e m i c cells were treated simultaneously w i t h P D T and D o x ( D o x / P D T ) , the resultant c y t o t o x i c i t y was roughly additive c o m p a r e d to P D T or D o x single agent  84  treatment o f the same dose. T h e same observations were made w h e n cells were treated first w i t h P D T f o l l o w e d by one hour incubation w i t h D o x ( P D T - > D o x ) . Interestingly, preincubation o f the cells w i t h D o x prior to P D T (Dox-> P D T ) significantly increased the cytotoxicity o f the treatment i n both normal and l e u k a e m i c cells. M o r e significantly, the L 1 2 1 0 c e l l line was m u c h more susceptible to the D o x - > P D T regimen than n o r m a l D B A / 2 haematopoietic progenitor cells w i t h a resultant enlargement o f the therapeutic w i n d o w . A s demonstrated in figure 3.9, the enhancement o f differential k i l l i n g o f L 1 2 1 0 cells was o n l y evident w h e n the higher B P D dose o f 5.0 ng/ml was used and the cells were e x p o s e d to D o x before P D T treatment. T h e results f o r D B A / 2 haematopoietic progenitors and L 1 2 1 0 leukaemic cells treated w i t h different c o m b i n a t i o n regimens incorporating the higher D o x concentration o f 2.5 u M were significantly different f r o m each other as demonstrated b y two- w a y A N O V A w i t h p< 0.05. Importantly, there was significant intergroup difference between the D o x - > P D T sequence and the other t w o c o m b i n a t i o n regimens o f P D T - > D o x and D o x / P D T in both n o r m a l and l e u k a e m i c c e l l s exposed to 2.5 u M o f D o x . T h i s p h e n o m e n o n c o u l d be attributed to several events w h i c h w i l l be the subject o f chapter 4 o f this thesis. It is possible that pretreatment o f cells w i t h D o x c o u l d increase the subsequent uptake o f the photosensitiser or reduce the amounts o f c e l l u l a r antioxidants such as glutathione. D a u n o r u b i c i n , a congenor o f D o x , increases ceramide p r o d u c t i o n through its m o d u l a t i o n o f the e n z y m e ceramide s y n t h a s e .  325  C e r a m i d e is a s p h i n g o l i p i d w h i c h  mediates several signal transduction pathways such as those downstreams o f the t u m o u r necrosis factor type I receptor ( T N F r ) and F A S ; furthermore, addition o f exogenous short chain ceramide analogue induced apoptotic c e l l d e a t h .  3 2 6  Separovic and colleagues recently  demonstrated ceramide production in response to P D T mediated b y the phthalocyanine photosensitiser P c 4 .  2 6 8  Pretreatment o f cells w i t h D o x c o u l d therefore raise intracellular  ceramide to a critical level w i t h exacerbation o f subsequent P D T cytotoxicity. R e c e n t l y , several groups have i m p l i c a t e d the transcriptional factor N F - K(3 i n anthracycline- mediated c y t o t o x i c i t y . Inhibition o f N F - K p nuclear translocation enhanced the c y t o t o x i c i t y o f d a u n o r u b i c i n , T N F a , and y- radiation in H T 1 0 8 0 h u m a n f i b r o s a r c o m a cells w h i c h i m p l i c a t e d N F - K(3 activation as part o f the n o r m a l response to environmental stresses w h i c h threaten c e l l u l a r s u r v i v a l .  3 2 7  H o w e v e r , contradictory studies have s h o w n that N F -  K(3 activation precedes c e l l death therefore the exact role o f this ubiquitous transcriptional  85  factor in cellular demise c o u l d be quite i d i o s y n c r a t i c .  328  D a s and W h i t e demonstrated that  both d o x o r u b i c i n and d a u n o r u b i c i n effected N F - K(3 activation v i a protein kinase C .  3 2 9  S i n c e P D T has been s h o w n to activate N F - Kp\ pretreatment o f c e l l s w i t h D o x c o u l d c o n c e i v a b l y alter the activation threshold o f the transcriptional factor and therefore the resultant c y t o t o x i c i t y .  2 6 4  In summary, c o m b i n a t i o n treatment o f D o x and B P D - mediated P D T preferentially affected the leukaemic c e l l line L 1 2 1 0 over n o r m a l D B A / 2 haematopoietic cells. Selective k i l l i n g was o n l y realised w h e n D o x was used p r i o r to P D T (Dox-> P D T ) and at a dose o f 5.0 ng/ml B P D and 15 J / c m r e d light. 2  86  CHAPTER  4: C O M B I N E D T R E A T M E N T  HAEMATOPOIETIC  AND LEUKAEMIC  AND PDT:  4.1  OF MURINE  CELLS WITH  MECHANISTIC  NORMAL  DOXORUBICIN  STUDY  ABSTRACT B e n z o p o r p h y r i n derivative m o n o a c i d ring- A ( B P D ) is a porphyrin- based  photosensitiser w i t h potential i n the c l i n i c a l application o f p h o t o d y n a m i c therapy ( P D T ) . P r e v i o u s l y , w e have demonstrated the effectiveness o f B P D i n P D T p u r g i n g o f contaminated leukaemic cells f r o m autologous haematopoietic stem c e l l harvests. In order to i m p r o v e its efficacy, w e c o m b i n e d B P D - mediated P D T w i t h d o x o r u b i c i n ( D o x ) and f o u n d that the sequence o f D o x - > P D T was superior to simultaneous D o x / P D T and the reverse sequence of P D T - > D o x . In addition, the m u r i n e l e u k a e m i c c e l l line L 1 2 1 0 was m u c h more susceptible to the D o x - > P D T regimen than n o r m a l D B A / 2 haematopoietic progenitor cells. In this chapter, w e studied some o f the interactions between D o x and B P D and specifically the possible mechanisms responsible for the augmentation o f P D T cytotoxicity effected by D o x preincubation. T h e unique photophysical and b i o l o g i c a l properties o f B P D and D o x permit their cytotoxic cooperativity i n the simultaneous D o x / P D T regimen. Fluorescent m i c r o s c o p y s h o w e d that B P D was restricted to the c y t o p l a s m and the p l a s m a membrane whereas D o x l o c a l i s e d to both c y t o p l a s m i c and nuclear compartments. D o x - mediated i n h i b i t i o n o f B P D photobleaching was observed at the excitation wavelength o f 4 4 0 n n i but not at 6 3 0 . n m . S i n c e a red light source emittingbetween 600- 9 0 0 n m was used i n the c y t o t o x i c i t y assays described in chapter 3, the presence o f D o x should not affect B P D photoactivation. Spectrofluorimetric measurements o f L 1 2 1 0 cell lysates revealed that uptake o f B P D was reduced i n the presence o f D o x ; however, preincubation of L 1 2 1 0 w i t h D o x restored B P D accumulation to levels of control cells incubated w i t h o n l y B P D . N e x t , we proceeded to e x a m i n e the c e l l u l a r level o f glutathione ( G S H ) , an ubiquitous protective t h i o l . L 1 2 1 0 cells incubated w i t h 5 u M o f D o x f o r 1 h resulted in slight but reproducible reduction o f c e l l u l a r G S H . T h e above factors, i n  87  c o n j u n c t i o n w i t h other mechanisms, c o u l d be responsible f o r the presensitisation o f L 1 2 1 0 cells by D o x .  4.2  INTRODUCTION N u m e r o u s factors can affect the e f f i c i e n c y of p h o t o d y n a m i c therapy ( P D T ) .  C o n s i d e r a b l y more variables are i n v o l v e d w h e n P D T is used  in vivo;  f o r e x a m p l e , the  extent o f first pass hepatic m e t a b o l i s m and the degree o f b i n d i n g to various serum proteins affect the delivery o f the photosensitiser to the target site. Other factors o f particular importance i n o n c o l o g y include tissue oncotic pressure, regional b l o o d f l o w , and the h y p o x i c state o f the tumour, w h i c h are governed b y tumour size. T h e above concerns are not unique to P D T and are l i k e w i s e o f importance i n the delivery of other forms o f therapies such as chemotherapeutic agents and a n t i b o d i e s .  330  A t the c e l l u l a r l e v e l ,  photosensitiser uptake is also governed by s i m i l a r factors w h i c h affect the uptake o f other chemotherapeutic compounds. F o r e x a m p l e , m o l e c u l a r size and partition coefficient of the photosensitiser influence the cellular accumulation o f the c o m p o u n d . E n v i r o n m e n t a l factors such as the rate o f cellular proliferation, intracellular and extracellular p H , c e l l size, and s e n i m concentration all influence the uptake o f the p h o t o s e n s i t i s e r .  259  -  331  The  photosensitiser initially associates w i t h the c e l l membrane then gradually relocalises to the different intracellular compartments such as the m i t o c h o n d r i a and l y s o s o m e s .  3 3 2  In  addition, private attributes such as charge characteristics and.presence or absence o f sidechain moieties further contribute to the intracellular localisation o f the c o m p o u n d . 3  33  C a t i o n i c dyes such as merocyanine 5 4 0 ( M C 540) and r h o d a m i n e 123 ( R h 123) preferentially accumulate in the m i t o c h o n d r i a as a result o f the electrochemical gradient whereas the porphyrin- based photosensitiser b e n z o p o r p h y r i n derivative m o n o a c i d ring- A ( B P D ) , w h i c h is l i p o p h i l i c , initially localises to the p l a s m a membrane and then rapidly to various c e l l u l a r organelles w i t h longer incubation. In addition to photosensitiser a c c u m u l a t i o n , other factors also determine the effectiveness o f P D T c y t o t o x i c i t y . O f particular importance is the ratio o f cellular pro- and antioxidants since P D T c y t o t o x i c i t y is  88  p r i m a r i l y mediated by reactive o x y g e n species ( R O I s ) and radical f o r m a t i o n s .  255  Therefore, the amount o f cellular antioxidants such as glutathione ( G S H ) and a - tocopherol can influence the extent o f P D T damage and their modulations c a n alter P D T susceptibility.  261  -  334  >  3 3 5  P D T c o m b i n a t i o n therapy, through the incorporation o f P D T w i t h other treatment modalities or therapeutic agents, attempts to effect modulation o n the c e l l or the organism i n order to m a x i m i s e cytotoxic effects on the target c e l l population. M a n i p u l a t i o n s o f the t i m i n g , c h e m i c a l and p h y s i c a l characteristics o f the photosensitiser a n d drug dose, light fluence rate, as w e l l as the, activation wavelength can substantially affect the efficiency o f P D T - mediated c y t o t o x i c i t y . Therefore, introduction o f another f o r m o f treatment into a P D T regimen can significantly increase its c o m p l e x i t y and also the interpretation o f data. H y p e r t h e r m i a and i o n i s i n g radiation have s i m i l a r cytotoxic m e c h a n i s m s as P D T and were naturally used i n different variations o f c o m b i n a t i o n s w i t h P D T . S e v e r a l groups have demonstrated the beneficial effects o f P D T prior to hyperthermia treatment and sequence s p e c i f i c i t y o f this r e g i m e n was a result o f t u m o u r and vascular effects o f P D T .  3 0 3  '  3 0 9  -  3 2 0  Chemotherapeutic agents such as etoposide and d o x o r u b i c i n are also used i n conjunction with P D T .  3 0 6  '  3 0 8  M a et al. observed that presence o f m i t o m y c i n C ( M M C ) d u r i n g  Photofrin®- mediated P D T significantly increased its antitumour effects  vivo. 304'  318  in vitro and in  S o m e o f the most innovative regimens i n v o l v e d c o m b i n a t i o n s o f different  photosensitisers and activation wavelengths; f o r e x a m p l e , C i n c o t t a a n d colleagues reported synergistic antitumour effects o f P D T  in vivo mediated b y B P D and 5- ethylamino- 9-  diethylaminobenzo[a] p h e n o t h i a z i n i u m c h l o r i d e ( E t N B S ) .  3 0 5  Coadministration o f  cytokines such as G M - C S F and T N F a potentiated the antitumour e f f i c a c y o f P D T in  vivo. 322'  336  Myers-er al. observed that the addition o f t h e . i m m u n o s t i m u l a n t , C.'parvum,  significantly enhanced P D T efficacy i n an a n i m a l bladder t u m o u r m o d e l . experiments highlighted the participatory role o f the i m m u n e system i n  3 3 7  These  in vivo antitumour  action mediated by P D T . V a r i o u s mechanisms have been proposed f o r the different f o r m s o f P D T c o m b i n a t i o n therapy. In regimens i n v o l v i n g c o m b i n a t i o n s w i t h other photosensitisers, t w o groups have noted that synergistic k i l l i n g o f tumour cells w i t h differential localisations o f the a g e n t s .  3 0 5  -  3 3 8  in vitro and in vivo was achieved  C o m b i n i n g photosensitisers o r  chemotherapeutic agents w i t h unique photophysical and b i o c h e m i c a l characteristics can  89  invariably ensure an increase in cellular targets. M a et al. reported that c e l l c y c l e arrest f o l l o w e d b y S- phase a c c u m u l a t i o n was responsible f o r the increased uptake o f Photofrin® i n the presence M M C .  3 0 4  Therefore, one can understand w h y simultaneous i n c u b a t i o n o f  cells w i t h M M C and Photofrin® was superior to administration o f M M C post- P D T (P>light-> M M C ) . In this chapter, w e attempted to study the interactions between B P D and D o x and s p e c i f i c a l l y , to investigate the m e c h a n i s m s responsible for the enhanced k i l l i n g o f L 1 2 1 0 cells w h e n D o x was administered p r i o r to B P D - mediated P D T (Dox-> P D T ) .  4.3  RESULTS  P h o t o p h y s i c a l p r o p e r t i e s of B P D a n d  Dox  T h e m o l e c u l a r structure o f b e n z o p o r p h y r i n derivative m o n o a c i d r i n g A (one r e g i o i s o m e r , B P D ) is g i v e n in figure 4 . 1 . B P D , a m e m b e r o f the p o r p h y r i n f a m i l y , is a second generation photosensitiser w h i c h is m o r e potent and purer than its predecessor Photofrin®. T h e structure o f the anthracycline antibiotic d o x o r u b i c i n ( D o x , a d r i a m y c i n ) is s h o w n in f i g u r e 4.2. D o x is a 14- h y d r o x y analogue o f d a u n o r u b i c i n isolated f r o m  Streptomyces peucetius (var. caesius) and  is used extensively i n anticancer chemotherapy.  T h e absorption spectra o f B P D and D o x i n 10 % HI- F C S / P B S are g i v e n i n f i g u r e 4.3 and T p g / m l (1-.376 p M ) o f B P D and 10 p M o f D o x were u s e d ' d u r i n g the'analysis: T h e presence o f the unique B P D absorption peak at 6 9 0 n m , i n addition to several other peaks above 6 0 0 n m , permits B P D photoactivation at higher wavelength and is an unique feature o f this photosensitiser. T h e spike observed at 656 n m , w h i c h is present in both D o x and B P D spectrum, is an artefact o f the deuterium l a m p used for the spectrophotometric scan. D o x has a prominent b r o a d absorption peak at 5 0 0 n m and a smaller peak at 5 7 9 n m . Fluorescence e m i s s i o n spectra (A,ex = 4 4 0 n m ) o f 10 ng/ml (13.76 n M ) B P D and 5 p M D o x are presented i n figure 4.4a. N o t e that this is the same B P D : D o x m o l a r ratio that was used in the c y t o t o x i c i t y assays described i n chapter 3. A t this ratio, D o x fluorescence e m i s s i o n  90  virtually concealed almost all o f the B P D profile save the unique B P D e m i s s i o n peak at 693 n m , w h i c h remained detectable even in a solution c o n t a i n i n g 363.5 f o l d more o f D o x . T h i s is o f particular significance for the interpretation o f the photobleaching data i n a subsequent section as w e l l in the appreciation o f the general distinctiveness between B P D and D o x . F i g u r e 4.4b, w h i c h restricts the e m i s s i o n spectra to 650- 7 0 0 n m , h i g h l i g h t s this unique property o f B P D .  91  COOH  Figure 4.1  COOCH3  Chemical structure of the benzoporphyrin derivative ring- A ( B P D , Verteporfin)  monoacid  T h e molecular structure o f one regioisomer o f b e n z o p o r p h y r i n m o n o a c i d r i n g A ( B P D , V e r t e p o r f i n ) is s h o w n .  92  derivative  Figure 4.2  Chemical structure of Doxorubicin (Dox) T h e m o l e c u l a r structure o f D o x o r u b i c i n ( D o x ) is s h o w n .  93  0.1250 0.1000  0.07504  1 pg/ml B P D  Absorbance  10 u M Dox  0.0500-T 0.0250  0.0000 400  500  600  700  wavelength (nm)  Figure 4.3  Absorption spectra of 1 pg/ml (1.376 Dox i n 10 % H I - F C S / PBS  p M ) B P D and 10 p M  T h e absorption spectra o f 1 pg/ml (1.376 p M ) B P D and 10 p M D o x i n P B S c o n t a i n i n g 1 0 % HI- F C S are presented. T h e 6 5 6 n m spike observed i n both samples was an artefact o f the deuterium l a m p f r o m the. spectrophotometer.  94  5 uM Dox only Fluorescence intensity  5 uM Dox & 10 ng/ml BPD  550  600  650  700  wavelength (nm)  Figure 4.4a Fluorescence emission spectra of B P D in the presence of D o x (550 - 700 nm) S u p e r i m p o s i t i o n o f the fluorescence e m i s s i o n profile o f 5 u M D o x a n d a mixture c o n s i s t i n g o f 5 u M D o x w i t h 10 ng/ml B P D i n P B S containing 1 0 % HI- F C S . B P D has an e m i s s i o n peak at 6 9 3 n m w h i c h permits its activation even i n the presence o f D o x . N o t e that the molar ratio o f B P D to D o x ( 1 : 363.5) is the same that w a s used i n the c y t o t o x i c i t y assays. Settings f o r the spectrofluorimeter: continuous wavelength ( C W ) excitation at 4 4 0 n m w i t h bandpass o f 4 n m , e m i s s i o n at 7 0 0 n m w i t h bandpass o f 4 n m , 1 nm/s scan rate, p h o t o m u l t i p l i e r voltage ( P M T ) was set at 6 5 0 V .  95  1.5-,  5 uM Dox 5 uM Dox & 10 ng/ml B P D  Fluorescence intensity 0.5H  650  1  660  I  i  I  1  670  680  690  700  wavelength (nm)  Figure 4.4b Fluorescence emission profile of B P D in the presence of D o x (650 - 700 nm) Superposition o f the fluorescence e m i s s i o n profile o f 5 u M D o x and a mixture c o n s i s t i n g o f 5 u M D o x w i t h 10 ng/ml B P D i n P B S containing 1 0 % HI- F C S . B P D has a e m i s s i o n peak at 6 9 3 n m , accentuated in this e m i s s i o n scan between 650- 7 0 0 n m , w h i c h permits its activation even in the presence o f D o x . N o t e that the molar ratio o f B P D to D o x (1: 363.5) is the same that w a s used i n the cytotoxicity assays. Settings f o r the spectrofluorimeter: continuous wavelength ( C W ) excitation at 4 4 0 n m w i t h bandpass o f 4 n m , e m i s s i o n at 7 0 0 n m w i t h bandpass o f 4 n m , 1 nm/s scan rate, p h o t o m u l t i p l i e r voltage ( P M T ) was set at 6 5 0 V .  96  Intracellular localisations of B P D and Dox in the human leukaemic cell line K562 T o further delineate the differences between B P D and D o x , intracellular localisations o f the two c o m p o u n d s were established by fluorescent m i c r o s c o p y . T h e c e l l line K 5 6 2 , o r i g i n a l l y derived f r o m the pleural effusions o f a c h r o n i c m y e l o g e n o u s l e u k a e m i a ( C M L ) patient i n blast crisis, was used f o r this study. F l u o r e s c e n c e was visualised w i t h an O l y m p u s N e w V a n o x m i c r o s c o p e attached to the A H 2 - F L transmitted fluorescence unit, a blue range barrier filter was used f o r both B P D and D o x observations. Pictures were taken w i t h the attached c a m e r a u s i n g F u j i c h r o m e 4 0 0 I S O f i l m . A s presented in figure 4.5, K 5 6 2 cells that were incubated w i t h 10 pg/ml B P D for 6 0 m i n s h o w e d intense yet d i f f u s e d c y t o p l a s m i c fluorescence around but not w i t h i n the nuclear poles. In addition, discrete and punctate fluorescence patterns, suggesting B P D a c c u m u l a t i o n i n c y t o p l a s m i c organelles, were also observed. T h i s is consistent w i t h the f i n d i n g s o f C i n c o t t a  et al. u s i n g the m u r i n e m a m m a r y sarcoma c e l l line E M T - 6 .  3 0 5  D o x , at 10 u M ,  demonstrated both nuclear and c y t o p l a s m i c fluorescence in a d i f f u s e d pattern (figure 4.6). Interestingly, nuclear fluorescence o f D o x was m u c h stronger than c y t o p l a s m i c fluorescence w h i c h i m p l i e s significant nuclear accumulation o f D o x . T h e fluorescence pattern o f D o x is i n agreement w i t h k n o w n properties o f this c o m p o u n d w h i c h targets the l i p i d membrane bilayers, various c e l l u l a r e n z y m e s , and the genetic materials o f t h e c e l l .  3 2 4  In figure 4.7, K 5 6 2 cells were incubated simultaneously w i t h 10 pg/ml B P D and 10 p M D o x for 6 0 m i n and observed under the fluorescent m i c r o s c o p e at the same settings as before. A l l the cells demonstrated the red c y t o p l a s m i c fluorescence pattern o f B P D w h i l e D o x nuclear fluorescence was observed i n some o f the cells. D i f f e r e n t i a l localisations o f B P D and D o x to the different compartments o f the K 5 6 2 c e l l line, in addition to their unique but parallel c y t o t o x i c mechanisms o f action, support their use in c o m b i n a t i o n P D T therapy.  97  Figure  4.5  Fluorescence with B P D  micrograph  of  K562  leukaemic  cells  incubated  K 5 6 2 C e l l s were resuspended at a concentration o f 1 x 1 0 cells/ml in serum- free D M E M and incubated w i t h 10 pg/ml o f B P D f o r 6 0 m i n . C e l l s were then w a s h e d once and resuspended in 1 m l o f m e d i u m . A d r o p containing 5 0 p i or 5 x 1 0 o f cells was placed o n a glass slide and subsequently covered w i t h a cover slip. The cells were observed under an O l y m p u s N e w V a n o x microscope with the A H 2 - F L transmitted light fluorescence attachment o n " B l u e " setting (excitation @ 380- 4 9 0 n m , observation @ 515 n m +). 6  4  98  Figure  4.6  Fluorescence with Dox  micrograph  of  K562  leukaemic  cells  incubated  K 5 6 2 C e l l s were resuspended at a concentration o f 1 x 1 0 cells/ml in serum- free D M E M and incubated w i t h 10 u M o f D o x f o r 6 0 m i n . C e l l s were then w a s h e d once and resuspended in ! m l o f m e d i u m . A drop containing 5 0 pi or 5 x 1 0 o f cells w a s placed on a glass slide and then covered w i t h a c o v e r slip. T h e cells were observed under an O l y m p u s N e w V a n o x m i c r o s c o p e w i t h the A H 2 - F L transmitted light fluorescence attachment on " B l u e " setting (excitation @ 380- 4 9 0 n m , observation @ 515 n m +). 6  4  99  Figure  4.7  Fluorescence micrograph with B P D and Dox  of  K562  leukaemic  cells  incubated  K 5 6 2 C e l l s were resuspended at a concentration o f 1 x 1 0 cells/ml in serum-free D M E M and incubated w i t h 10 pg/ml (13.76 p M ) B P D and 10 u M D o x for 6 0 m i n . C e l l s were then w a s h e d once and resuspended in 1 m l of m e d i u m . A drop c o n t a i n i n g 5 0 pi o f cells was p l a c e d on a glass slide and thereafter covered w i t h a cover s l i p . The cells were observed under an O l y m p u s N e w V a n o x m i c r o s c o p e w i t h the A H 2 - F L transmitted light fluorescence attachment o n " B l u e " setting (excitation @ 380- 4 9 0 n m , observation @ 515 n m +). 6  100  The  role of excitation wavelength (k ) ex  has on the photobleaching of B P D in  the presence of Dox T o further investigate the interactions between B P D and D o x , the t w o c o m p o u n d s were m i x e d together i n a solvent consisting o f 10 % fresh h u m a n p l a s m a i n phosphate buffered saline (10 % H P / P B S ) and photobleaching o f B P D i n the presence o f different concentrations o f D o x was measured. P h o t o b l e a c h i n g is the process o f irreversible autodegradation o f the excited photosensitiser and originates f r o m the triplet excited state of the c o m p o u n d as a result o f intersystem c r o s s i n g f r o m the activated singlet state. H o w e v e r , it is inherently a c o m p l e x phenomenon and can also occur f r o m the singlet state. B P D does not photobleach readily i n organic solvents such as methanol or dichloromethane; h o w e v e r , photobleaching is significantly enhanced i n the presence o f h u m a n p l a s m a or foetal c a l f serum (data not shown). T h i s is consistent w i t h f i n d i n g s by A v e l i n e et al.  219  In the present  study, B P D p h o t o b l e a c h i n g was used as a surrogate marker o f photosensitiser activation. A s demonstrated i n figure 4.8, the mean fluorescence intensity o f 10 ng/ml o f B P D at 693 nm ( M F I  6 9 3 nm  ) decreased steadily w h e n the c o m p o u n d was subjected to continuous light  exposure at the excitation wavelength ( X ) o f 4 4 0 n m (0 p M D o x , bottommost curve). In ex  fact, the M F I  6 9 3 n m  a t the end o f a 15 m i n exposure p e r i o d was 4 3 . 2 % o f the starting l e v e l .  Interestingly, B P D photobleaching was ameliorated w h e n increasing concentrations o f D o x were introduced into the reaction cuvette. T o control for any contributions of D o x fluorescence to the observed reduction o f B P D photobleaching, different concentrations o f D o x were subjected to the same photoactivation protocol and D o x fluorescence emissions at 593 n m (fluorescence e m i s s i o n peak unique to D o x ) and 693 n m (fluorescence e m i s s i o n peak unique to B P D ) were m o n i t o r e d f o r 15 m i n . In both cases, M F I was not s i g n i f i c a n t l y altered at the end o f the observation p e r i o d suggesting D o x itself was not affected by the light exposure and the reduction o f B P D photobleaching in the presence o f D o x was caused by some f o r m o f m o l e c u l a r interaction between D o x and B P D . F i g u r e 4.9 summarises the results of three independent B P D photobleaching experiments in terms o f percent reductions of M F I MFI  6 9 3 nm  6 9 3 n m  a t the end o f the 15 m i n exposure p e r i o d c o m p a r e d to starting  . In the experiments described above, p h o t o b l e a c h i n g or photoactivation o f B P D  was f o u n d to be inversely correlated w i t h the concentration o f D o x present w h e n A, o f 4 4 0 ex  101  n m was used. S i n c e a red light source emitting between the range of 600- 9 0 0 n m was used f o r B P D photoactivation in the cytotoxicity assays described i n chapter 3, the p h o t o b l e a c h i n g experiments were repeated w i t h ?i set at 6 3 0 n m . F i g u r e 4.10 compares ex  the effects o f D o x had o n B P D p h o t o b l e a c h i n g between ^ o f 4 4 0 n m and 6 3 0 n m . T h e e x  data s h o w that at A, of 6 3 0 n m , reduction o f B P D p h o t o b l e a c h i n g by D o x was m u c h less ex  o b v i o u s than at Xex o f 4 4 0 n m . In fact, p h o t o b l e a c h i n g o f B P D was d r a m a t i c a l l y l o w e r at the f o r m e r wavelength. F i g u r e 4.11 illustrates that 1 m M of D o x in 10 % HI- F C S / P B S does not appreciably interfere w i t h light transmission above 6 0 0 n m . A t 6 3 0 n m , light transmittance was 89.39 % and reached a plateau at 7 2 0 n m (97.83 % ) . S i n c e a m u c h l o w e r concentration o f D o x (5- 10 p M ) was used i n the c y t o t o x i c i t y assays (chapter 3) and in the spectrofluorimetric analyses described i n this section, the presence o f D o x s h o u l d have minima] negative effect on light transmission to the B P D molecules in the solution w h e n A, o f above 6 0 0 n m was used. T h e results, i n s u m , suggest that the presence o f D o x ex  d i d not affect the efficiency o f red light activation o f B P D during the c y t o t o x i c i t y assay and the observed attenuation o f B P D activation by D o x at 4 4 0 n m was most probably secondary to shielding o f light b y the abundance o f D o x i n solution.  102  1.25-,  10 uM Dox  M e a n fluorescence intensity @ 693 n m  5.0 uM Dox  0.75-f* =«ta*t; (5=  2.5 uM Dox 0.5n.  1.25 uM Dox 0 uM Dox  0.25900 time (s)  Figure 4.8  Photobleaching of 10 ng/ml BPD in the presence of different concentrations of Dox (A,EX = 440 nm) B P D fluorescence intensity at 6 9 3 n m w a s m o n i t o r e d f o r 15 m i n w h i l e the sample was photoactivated at 4 4 0 n m i n a solvent c o n s i s t i n g o f 10 % h u m a n plasma/ P B S . T h e presence o f increasing concentrations o f D o x i n the reaction significantly reduced the degree o f B P D p h o t o b l e a c h i n g . Settings f o r the spectrofluorimeter: continuous wavelength ( C W ) excitation at 4 4 0 n m w i t h bandpass o f 16 n m , e m i s s i o n at 7 0 0 n m w i t h bandpass o f 4 n m , M F I at 6 9 3 n m c o n t i n u o u s l y monitored f o r 9 0 0 s, photomultiplier voltage ( P M T ) was set at 6 5 0 V  103  100  n  75 % BPD fluoresecence intensity of original  J  50  J  25  J  [ T  _  1  0  % BPDfluorescence@ 693 nm  1—•—•—|—•—•—p 1.25  2.5  5  —r10  [Dox] uM  Figure 4.9  Summary of B P D photobleaching in the presence concentrations of Dox (440 nm excitation)  of different  Photobleaching o f B P D as measured by its fluorescence intensity at 693 n m was proportionally decreased i n the presence o f increasing concentrations o f D o x . M i x t u r e s consisting o f 10 ng/ml o f B P D and various concentrations o f D o x were m i x e d i n 10 % h u m a n plasma/ P B S a n d were subjected to continuous wavelength ( C W ) activation and fluorescence e m i s s i o n at 693 n m was m o n i t o r e d f o r 9 0 0 s. Percent photobleaching o f B P D was obtained by d i v i d i n g B P D fluorescence at the end o f the 9 0 0 s exposure p e r i o d b y B P D fluorescence at the b e g i n n i n g o f the m o n i t o r i n g p e r i o d . Data f r o m 3 independent experiments is presented. E r r o r bars are derived f r o m standard errors o f the mean.  104  100-,  440  630  Excitation wavelength (nm)  Figure 4.10 The effect of A,ex on B P D photobleaching absence of 5 p M Dox  in the presence and  T h e inhibitory effect o f D o x had o n B P D photobleaching was observed at the lower excitation wavelength (kex)  o f 4 4 0 n m but was not evident at the  higher /V o f 6 3 0 n m . B P D photobleaching w a s assessed b y c o m p a r i n g MFI at the end o f 9 0 0 s o f exposure to that at the b e g i n n i n g o f the monitoring p e r i o d . Results f r o m a single representative experiment are shown. ex  6 9 3  n m  105  100  n  95 90 Percent light transmittance  85 80 75 70 I  600  1  1  1  1  i  1  650  1  1  1  i  1  1  700  1  1  i  750  wavelength (nm)  Figure 4.11 Light transmittance of 1 m M Dox in 10 % H I - F C S / PBS 1 m M o f D o x does not appear to significantly, interfere w i t h .light transmittance i n a solution o f 10 % HI- F C S / P B S . T h e r e f o r e , the presence o f 5- 10 u M o f D o x i n a solution containing B P D s h o u l d have n o adverse effects o n the photoactivation o f the photosensitiser w i t h A. above 6 0 0 n m . ex  106  Uptake of BPD by L1210 cells as affected by the presence of Dox in different combination regimens Spectrofluorimetric measurements o f lysates f r o m L I 2 1 0 cells that were incubated w i t h either B P D and D o x are presented i n figures 4.12a and 4.12b. Sensitive detection o f the two c o m p o u n d s was a c c o m p l i s h e d by measuring B P D and D o x mean fluorescence intensity ( M F I ) at their unique fluorescence e m i s s i o n peaks (Xem) at 593 n m ( D o x ) and 693 n m ( B P D ) . A l l experiments were p e r f o r m e d u s i n g 1 x 10 cells as described i n the 6  experimental procedure section. A  linear correlation was observed between M F I at  Xem  of  693 n m and the concentration o f B P D (figure 4.12a). Interestingly, fluorescence intensities o f L I 2 1 0 c e l l lysates were s i m i l a r between samples that were incubated w i t h the photosensitiser for 10 m i n and 60 m i n . F i g u r e 4.12b shows that a linear relationship also existed between M F I at ^  e m  o f 593 n m and the concentration o f D o x ; in addition, uptake o f  the drug was also quite rapid because o f the similarities between the M F I measurements f r o m the 10 m i n and 6 0 m i n c e l l lysate samples. F i g u r e 4.13 illustrates the effects that 10 u M D o x coincubation ( D o x / B P D ) o r 10 u M D o x preincubation (Dox-> B P D ) had on the uptake o f B P D . L 1 2 1 0 cells that were coincubated w i t h D o x ( D o x / B P D ) appeared to take up less B P D than control cells incubated w i t h o n l y B P D ; this p h e n o m e n o n became more prominent w i t h increasing concentrations o f the photosensitiser. In s u m m a r y , B P D uptake by L I 2 1 0 cells was not affected by prior i n c u b a t i o n w i t h D o x ; however, presence o f D o x (10 p M ) during B P D incubation appeared to reduce the uptake o f the photosensitiser.  107  lOrnin incb 6 0 m i n incb  [lBPD]ng/ml  Figure  4.12a  Uptake of B P D by L1210 cells as determined spectrofluorimetric measurements of cell lysates  by  MFI o f lysates f r o m 1 x 1 0 L 1 2 1 0 cells was directly correlated w i t h the concentration o f B P D . U p t a k e o f B P D was rapid since M F I of lysates f r o m cells incubated w i t h B P D f o r 10 m i n were s i m i l a r to those o f the 6 0 m i n group. 6 9 8  6  n m  6 9 8  108  n m  lOmin incb 60min incb  [DOX] u M  Figure 4.12b  Uptake of Dox by L1210 cells as determined spectrofluorimetric measurement of cell lysates  by  MFI o f lysates f r o m 1 x 1 0 L 1 2 1 0 cells was directly correlated w i t h the concentration o f D o x used d u r i n g the i n c u b a t i o n . U p t a k e o f D o x w a s rapid since M F I o f lysates f r o m cells incubated w i t h D o x f o r 10 m i n were s i m i l a r to those o f the 6 0 m i n test group. 5 9 3  6  n m  5 9 3  n m  109  OA-,  0.3-\  [BPD] ng/ml  Figure 4.13-  Uptake of BPD by L1210 cells when incubated simultaneously (BPD/ Dox) or after Dox (Dox-> BPD) L 1 2 1 0 cells appeared to take up less B P D i n the presence o f 10 u M o f D o x compared to the respective B P D o n l y c o n t r o l s : R e d u c t i o n o f B P D mean fluorescence intensity at 6 9 8 n m ( M F I ) w a s more prominent at higher B P D doses. P r e i n c u b a t i o n o f cells w i t h J O u M o f D o x appeared to have n o inhibitory effects o n the subsequent uptake o f the photosensitiser. Data obtained f r o m 3 independent experiments is presented. E r r o r bars are d e r i v e d f r o m standard errors o f the m e a n . 6 9 8 nm  110  Depletion of cellular glutathione (GSH)  in L1210  cells by preincubation  with Dox T o further investigate the effects o f D o x preincubation had on subsequent P D T cytotoxicity on L 1 2 1 0 cells, c e l l u l a r glutathione ( G S H ) levels were measured after 1 h incubation w i t h D o x . A g a i n , the same experimental conditions as the c y t o t o x i c assays described i n chapter 3 were used f o r this set o f experiments in order to ensure relevancy o f the data. Results are s u m m a r i s e d in figure 4.14. Preincubation o f L I 2 1 0 cells w i t h D o x resulted in a dose- related, albeit i n s i g n i f i c a n t (student's t- test, p >  0.05),  reduction o f  c e l l u l a r G S H . F o r e x a m p l e , 11.93 ± 0.76 p M G S H / 1 x 1 0 cells was obtained after 1 h 6  incubation w i t h 5 p M D o x c o m p a r e d to 13.54 + 1.51 n m o l e G S H / 1 x 1 0 cells f r o m the 6  untreated control group or 88.16 % of the untreated control. U s i n g the T i e t z e enzymatic method, that preincubation o f L I 2 1 0 cells w i t h 5 p M o f D o x d i d not s i g n i f i c a n t l y reduced the amount o f c e l l u l a r G S H . Therefore, this ruled out the role of G S H i n the enhancement o f P D T cytotoxicity v i a D o x preincubation.  Ill  20-,  [GSH] u M per 1 X 1 0 6 L 1 2 1 0 cells A  0  2.5  5  10  20  [Dox] u M  Figure  4.14 Cellular glutathione levels in L1210 cells after 1 h incubation with different doses of Dox Incubated w i t h D o x f o r 1 h resulted in a dose- related reduction o f cellular glutathione ( G S H ) i n L 1 2 1 0 c e l l s . G S H w a s determined u s i n g the Tietze enzymatic assay. Data f r o m a three independent experiments i s presented. E r r o r bars are derived f r o m standard errors o f the mean.  112  4.4  DISCUSSION T h e efficiency o f P D T is influenced by multiple intrinsic and extrinsic factors.  O b v i o u s l y , variables such as first- pass hepatic m e t a b o l i s m and relative tissue distribution are o f concern f o r for  in vitro P D T .  in vivo applications  of P D T ; however, the picture is no less c o m p l i c a t e d  B i o p h y s i c a l and b i o c h e m i c a l properties o f the photosensitiser such as  h y d r o p h o b i c i t y , extinction coefficient, singlet o x y g e n y i e l d , as w e l l as its absorption p r o f i l e are some o f the examples of the determinants o f P D T behaviour  vivo.  3,33  in vitro and in  Susceptibility to P D T c y t o t o x i c i t y is additionally established by cellular g r o w t h  rate, expression o f surface receptors such as the receptor f o r l o w density lipoprotein ( L D L r ) , and relative concentrations o f prooxidants and antioxidants i n the c e l l .  2 5 5  C o m b i n a t i o n therapy has been i n use for the management o f various forms o f m e d i c a l conditions; i n addition, it is indispensable i n the treatment m a n y o n c o l o g i c diseases. T h e p r i n c i p a l goals o f c o m b i n a t i o n therapy are to use agents w i t h different mechanisms of action so as to reduce the probability o f drug resistance development; furthermore, the c y t o t o x i c potentials o f the drugs should c o m p l e m e n t and not n u l l i f y each other. Ideally, the side effects o f the drugs should not be additive such that the patient but not the tumour remains tolerant to the drug c o m b i n a t i o n . A successful e x a m p l e o f such therapy is the use o f corticosteroid w i t h vincristine for the treatment of paediatric acute lymphoblastic leukaemia ( A L L ) .  1 1  S i n c e the advent o f the c l i n i c a l use o f P D T , numerous groups have  attempted to c o m b i n e P D T w i t h other treatment modalities such as hyperthermia, i o n i s i n g radiation, and surgery, w i t h different chemotherapeutic agents or w i t h different combinations of photosensitisers. H e n d e r s o n and colleagues i n i t i a l l y documented that the c o m b i n a t i o n of Photofrin®- mediated P D T and hyperthermia was synergistic in tumour k i l l i n g only when P D T was used before hyperthermic treatment ( P D T - > heat); the authors further demonstrated that pretreatment w i t h hyperthermia resulted in extensive haemorrhage o f the tumour site w h i c h interfered w i t h the subsequent delivery o f the photosensitiser as w e l l as reduced the amount o f light reaching the target s i t e .  3 0 3  A d d i t i o n a l studies supported  the early findings o f the influence that treatment sequence had on the efficacy o f P D T / hyperthermia c o m b i n a t i o n t h e r a p y .  309  Significantly, Chen  etal.  noticed that P D T resulted  in tumour acidity and h y p o x i a w h i c h sensitised the tumour to subsequent h y p e r t h e r m i a .  113  339  T h e experiments described above were p e r f o r m e d on i m p l a n t e d tumours i n m u r i n e hosts, w h i c h introduced  in vivo factors  affecting p h a r m a c o k i n e t i c s i n addition to unique  determinants such as tumour vasculature and o x y g e n a t i o n status. P a r a d o x i c a l l y , the reverse sequence o f heat-> P D T was f o u n d to be m o r e c y t o t o x i c than P D T - > heat o n L 1 2 1 0 cells in  vitro. 340 P D T was also used i n combinations w i t h chemotherapeutic agents i n c l u d i n g etoposide V P 16, t a x o l , v i n c r i s t i n e , and ET-18- O C H , an a l k y l - l y s o p h o s p h o l i p i d to effect 3  additive antitumour e f f e c t s .  3 0 6  '  3 4 1  -  3 4 2  T h e b i o r e d u c t i v e a l k y l a t i n g agent m i t o m y c i n C  ( M M C ) has also been extensively studied in P D T c o m b i n a t i o n therapy. M a et al. reported that simultaneous administration o f M M C and Photofrin® f o l l o w e d b y light exposure 16 h later ( M M C / P - > light) resulted in synergistic k i l l i n g o f the h u m a n c o l o n adenocarcinoma c e l l line W i D r  in vitro and in vivo i n  nude m i c e ; the authors also c o n c l u d e d that M M C  effected c e l l c y c l e i n h i b i t i o n w h i c h increased the t u m o u r S- phase fraction and consequently the uptake o f P h o t o f r i n ® .  304  '  318  T h i s is consistent w i t h earlier f i n d i n g s b y C h r i s t e n s e n et  al. that susceptibility to P D T i n several c e l l lines ( N H I K 3 0 2 5 , H e L a S , N H I K 1922, and 3  V 7 9 ) is correlated w i t h S- p h a s e .  3 4 3  B a a s et al. repeated the experiment o n the E 0 9 mouse  tumour m o d e l and c o n f i r m e d the sequence- specific synergy o f the M M C / P - > light regimen since M M C given after P D T d i d not enhance t u m o u r k i l l i n g .  3 4 4  T h e sequence c o m b i n a t i o n  o f M M C / P - > light was later successfully used i n a l i m i t e d c l i n i c a l trial on patients w i t h s k i n metastases o f p r i m a r y m a m m a r y c a r c i n o m a s .  323  C l e a r l y , numerous parameters can affect  the interpretation o f the different variations o f P D T c o m b i n a t i o n therapy, c h i e f a m o n g them include the photosensitiser and drug used, the sequence o f treatment, and the experimental system (tumour type,  in vitro  or  in vivo).  In this study, D o x was observed to effect'sequence- specific potentiated k i l l i n g o f the murine leukaemic c e l l line L 1 2 1 0 but not n o r m a l D B A / 2 haematopoietic progenitors w h e n used i n c o m b i n a t i o n w i t h B P D - mediated P D T . T h e sequence o f D o x - > P D T was f o u n d to be superior to simultaneous D o x / P D T , the reverse c o m b i n a t i o n sequence o f P D T > D o x , as w e l l as single agent P D T or D o x treatments. In a d d i t i o n , effectiveness o f the D o x - > P D T sequence was f o u n d to be B P D dose dependent since enhancement was not observed at 2.5 ng/ml but at 5.0 ng/ml B P D (chapter 3 o f this thesis). S e v e r a l other groups have also studied the use o f D o x i n P D T c o m b i n a t i o n therapy. N i a h a b e d i a n and colleagues successfully used a c o m b i n a t i o n o f haematoporphyrin derivative- mediated P D T w i t h D o x  114  to treat B A L B / c m i c e carrying the E M T - 6 t u m o u r .  3 0 8  T h e y attributed the potentiation o f  cytotoxicity to photochemical activation o f D o x by the red laser light (630 nm) used f o r P D T or alternatively to hyperthermic enhancement o f D o x as a result of laser irradiation. T h e first explanation was dubious because D o x w i t h an absorption peak o f 5 7 9 n m absorbs light m i n i m a l l y b e y o n d 6 0 0 n m .  3 4 5  Interestingly, L a n k s et al. was able to demonstrate  photodynamic enhancement o f D o x cytotoxicity in the L 9 2 9 c e l l line 514.1 n m and 488 n m . discrepancy between  3 4 6  in vitro at  the A  ex  of  O n e year earlier, C o w l e d et al. p u b l i s h e d a report c i t i n g the  in vitro and in vivo results  o f haematoporphyrin derivative ( H P D ) -  mediated P D T c o m b i n a t i o n therapy i n v o l v i n g D o x .  3 0 7  T h e authors f o u n d that the  simultaneous P D T / D o x regimen was superior to P D T - > D o x sequence treatment o n subcutaneously implanted L e w i s l u n g c a r c i n o m a  in vivo; h o w e v e r ,  they f o u n d that D o x  inhibited P D T cytotoxicity when the same regimen ( P D T / D o x ) was tested  in vitro  same c e l l line. In addition, they s h o w e d that D o x inhibited the uptake o f H P D  on the  in vitro.  In this study, fluorescence m i c r o s c o p y was used to establish the intracellular localisations o f B P D and D o x . U s i n g the h u m a n leukaemic c e l l line K 5 6 2 as a m o d e l , B P D was f o u n d to localise to the cytosol and the c e l l membrane whereas D o x was f o u n d in both c y t o p l a s m i c and nuclear compartments. W e believe that differential localisations o f the two c o m p o u n d s c o u p l e d w i t h their slightly different cytotoxic mechanisms contributed to the observed additivity in c e l l k i l l i n g o f the P D T / D o x regimen. W e also performed a series o f spectrofluorimetric experiments to further study the m o l e c u l a r interactions between these t w o c o m p o u n d s . B P D photobleaching, m o n i t o r e d as mean fluorescence intensity at 6 9 3 n m (MFI  6 9 3 n m  ) , was used as a surrogate m a r k e r o f the degree o f its photoactivation. U s i n g  p h y s i o l o g i c a l l y relevant concentrations o f B P D and D o x , w e determined that D o x inhibited B P D activation w h e n X  e x  o f 4 4 0 n m was u s e d (figure 4.9- 4.11). T h i s was p r o b a b l y  caused by interference o f B P D light absorption by D o x w h i c h was present at 363.5 f o l d m o l a r excess of B P D . W h e n an A . o f 6 3 0 n m was used, the presence o f D o x had a ex  negligible effect on B P D photobleaching. T h i s is expected because D o x absorbs w e a k l y b e y o n d 6 0 0 n m and B P D , even at a concentration 363.5 f o l d less than that of D o x , has three distinct absorption peaks above 6 0 0 n m . T h e f i n d i n g s f r o m the photobleaching experiments suggest that in P D T c o m b i n a t i o n therapy, the photophysical characteristics o f the c o m p o u n d and their possible interactions must be investigated thoroughly. S i n c e a red light source emitting between 600- 9 0 0 n m was used for the c y t o t o x i c i t y experiments i n  115  chapter 3, the presence o f D o x s h o u l d not affect the photoactivation o f B P D i n the D o x / P D T regimen. C o w l e d et al. f o u n d that D o x at 2 0 0 ug/ml (345 u M ) completely inhibited the uptake o f H P D i n the L e w i s l u n g c a r c i n o m a and R a j i c e l l lines  in vitro. 301  We  observed that 10 u M o f D o x decreased the accumulation o f B P D in the D o x / B P D r e g i m e n . H o w e v e r , L 1 2 1 0 cells that were preincubated w i t h 10 u M D o x f o l l o w e d b y 1 h incubation w i t h B P D (Dox-> B P D ) d i s p l a y e d normal levels o f the associated photosensitiser. M i l d i n h i b i t i o n o f B P D uptake b y the presence o f D o x c o u l d e x p l a i n the additive but not synergistic k i l l i n g o f L 1 2 1 0 cells. O n the other hand, " n o r m a l i s e d " B P D a c c u m u l a t i o n in D o x - > P D T probably accounted for the significant improvement i n c y t o t o x i c i t y o f this regimen. Nevertheless, one must note that the difference in B P D a c c u m u l a t i o n between the t w o regimens ( D o x / B P D vs D o x - > B P D ) was o n l y evident at the higher B P D doses w h i c h therefore questions the relevancy o f uptake m o d u l a t i o n i n the c o m b i n a t i o n experiments. A l s o , w e believe that the moderate effect on B P D uptake by D o x preincubation c o u l d not, by itself, account f o r the i m p r o v e m e n t in c e l l k i l l i n g seen at 5 ng/ml B P D i n the D o x - > P D T sequence. T o explain the superiority o f the D o x - > P D T sequence c o m p a r e d to D o x / P D T and P D T - > D o x regimen, w e postulated that pretreatment o f L 1 2 1 0 cells m i g h t result in the depletion o f cellular glutathione ( G S H ) w h i c h w o u l d then predispose the cells to subsequent oxidative damage mediated b y P D T . G S H , . a n abundant tripeptide s u l p h y d r y l , is i n v o l v e d i n a variety o f c e l l u l a r p r o c e s s e s .  347  A l o n e or i n c o n j u n c t i o n w i t h the e n z y m e s  glutathione S- transferase and selenium- glutathione peroxidase, G S H scavenges free radicals, reduces H 0 2  2  and organic peroxides. X e n o b i o t i c s and other chemotherapeutic  agents such as cisplatin can cause oxidative stress to tumour cells w h i c h is buffered b y cellular G S H and numerous studies have s h o w n an inverse correlation between the concentration o f G S H and susceptibility to chemotherapy. C a n c e r cells w h i c h exhibit the m u l t i d r u g resistance ( M D R ) phenotype often have higher amounts o f G S H or demonstrate heightened G S H m e t a b o l i s m .  3 4 8  C o n v e r s e l y , a r t i f i c i a l depletion o f c e l l u l a r G S H is  achievable v i a the use o f buthionine s u l p h o x i m i n e ( B S O ) w h i c h s p e c i f i c a l l y inhibits yglutamylcysteine synthase, a rate- l i m i t i n g e n z y m e i n the G S H synthesis cascade. B S O has been successfully used, in c o n j u n c t i o n w i t h v e r a p a m i l , to o v e r c o m e the M D R phenotype i n the M C F - 7 breast c a r c i n o m a c e l l l i n e .  2 4 5  c y t o t o x i c i t y are enhanced b y B S O .  3 5 0  3 4 9  '  Furthermore, d o x o r u b i c i n and cisplatin S i m i l a r l y , G S H w o u l d be expected to p l a y a  116  significant role in determining cellular susceptibility to P D T because o f the central role o f o x i d a t i v e stress i n P D T c y t o t o x i c i t y .  2 6 1  -  3 3 4  -  3 5 1  In this study, superior k i l l i n g o f L 1 2 1 0 b y  the D o x - > P D T sequence c o u l d not be related to m o d u l a t i o n o f cellular G S H b y D o x . U s i n g the T i e t z e enzymatic assay, pre- incubation o f L 1 2 1 0 cells w i t h 2.5 p M D o x d i d not effect a significant reduction o f c e l l u l a r G S H . Nevertheless, G S H reduction was observed i n a dose- dependent manner w i t h respect to the concentrations o f D o x used. In s u m m a r y , the current assay method (Tietze assay) d i d not s h o w a significant alteration o f cellular G S H w h i c h c o u l d potentially be secondary to the l o w resolution o f the assay itself. A m o r e sensitive m e t h o d o f measurement, such as one that is based on the fluorescence detection o f G S H - b o u n d m o n o c h l o r o b i m a n e or m o n o b r o m o b i m a n e , c o u l d p o s s i b l y reveal any alterations o f c e l l u l a r G S H as a result o f D o x p r e i n c u b a t i o n .  411  T h e unique photophysical characteristics o f B P D and D o x , i n addition to their distinct intracellular localisations, are responsible f o r the additivity in c y t o t o x i c i t y w h e n L 1 2 1 0 cells were treated simultaneously w i t h B P D - mediated P D T and D o x ( P D T / D o x ) . T h e apparent synergistic k i l l i n g o f L I 2 1 0 cells i n the D o x pretreatment regimen (Dox-> P D T ) d i d not effect apparent changes i n the uptake o f B P D nor reduction i n G S H ; therefore, other factors such as the generation o f ceramide and alteration o f activation threshold o f the nuclear transcription factor N F - K(3 c o u l d e x p l a i n the b i o l o g i c a l f i n d i n g s . A l t e r n a t i v e l y , s m a l l changes i n the above measured parameters, i n s u m , c o u l d potentially affect P D T c y t o t o x i c i t y in the D o x - > P D T regimen. Nevertheless, more sensitive detection assays are needed in order to ascertain the role o f G S H i n D o x pretreatment and other possible m e c h a n i s m s as list above must be c o n s i d e r e d .  117  C H A P T E R 5: S E L E C T I V E P R E P R O T E C T I O N O F N O R M A L HAEMATOPOIETIC C O M M I T T E D PROGENITIOR C E L L S F R O M PDT WITH N-ACSDKP: CYTOTOXICITY  5.7  STUDY  ABSTRACT  Several studies have demonstrated that the tetrapeptide N - A c S D K P (Seraspenide) selectively inhibits c e l l cycle progression o f n o r m a l haematopoietic cells thereby protecting them f r o m the cycle- specific c y t o t o x i c i t y o f chemoradiation. It was f o u n d that NA c S D K P , but not the control peptides N - A c S D K E and S D K P , protected D B A / 2 bone m a r r o w cells f r o m B P D - mediated P D T cytotoxicity. W h e n D B A / 2 bone m a r r o w cells were incubated w i t h 100 n M o f N - A c S D K P f o r 1.5 hours p r i o r to P D T , subsequent C F U - G M survivals were i m p r o v e d b y 1 . 5 - 2 folds over cells that were preincubated w i t h the control peptides or w i t h m e d i u m . T h e l e u k a e m i c c e l l line L 1 2 1 0 was not protected f r o m P D T w i t h N - A c S D K P preincubation. S i m i l a r results were observed in n o r m a l and l e u k a e m i c h u m a n haematopoietic cells and i n the l e u k a e m i c c e l l line K 5 6 2 . That is, N - A c S D K P effected selective photoprotection o f n o r m a l h u m a n C F U - G M f o r m a t i o n but not l e u k a e m i c C F U - L formation f r o m patient isolates or f r o m the K 5 6 2 c e l l line. Therefore, results f r o m the in  vitro  short- term c o l o n y assays suggest that N - A c S D K P c o u l d be used f o r the selective  protection o f the n o r m a l c o m m i t t e d progenitor cells during P D T p u r g i n g . W e then used the one step long- term bone m a r r o w culture ( L T B M C ) assay to assess whether the photoprotective effect o f N - A c S D K P also extends to earlier haematopoietic progenitor and stem cells. D i r e c t quantification o f c e l l numbers and C F U - G M p r o d u c t i o n over a fiveweek p e r i o d s h o w e d no difference between D B A / 2 bone m a r r o w cells treated w i t h P D T after preincubation w i t h 100 n M o f N - A c S D K P or w i t h m e d i u m . W e therefore c o n c l u d e d that the photoprotective effect o f N - A c S D K P as used here applied o n l y to c o m m i t t e d progenitor cells w h i c h g i v e rise to C F U - G M c o l o n i e s . H o w e v e r , this l e v e l o f protection, albeit l i m i t e d , c o u l d still be o f c l i n i c a l advantage i n the improvement o f the early phase o f engraftment mediated by the c o m m i t t e d progenitor populations.  118  5.2  INTRODUCTION  Haematopoiesis is a h i g h l y d y n a m i c yet coordinated process c o n t r o l l e d b y a plethora o f positive and negative regulators; the bone m a r r o w , site o f p r i n c i p a l haematopoiesis i n humans, must be able to function i n a steady state e q u i l i b r i u m i n order to replace mature b l o o d cells that are lost to turnovers f o r the lifespan o f the o r g a n i s m . In addition, the bone m a r r o w must be able to support and maintain heightened haematopoiesis in the course o f increased demands such as d u r i n g infections or b l o o d l o s s .  2 0 0  -  3 5 2  The  soluble factors w h i c h p o s i t i v e l y regulate haematopoiesis are also termed g r o w t h factors, c y t o k i n e s , or c o l o n y s t i m u l a t i n g factors ( C S F s ) . T h e first C S F i d e n t i f i e d w a s erythropoietin ( E P O ) b y R e i s s m a n n and colleagues; h o w e v e r , m u r i n e granulocytemacrophage c o l o n y stimulating factor ( G M - C S F ) was the first haematopoietic c y t o k i n e to be c l o n e d .  3 5 3  '  3 5 4  Identification o f C S F s w a s also aided b y the maturation o f the  hierarchical p a r a d i g m o f haematopoiesis and technical developments i n the isolation o f stem c e l l s as w e l l as their in vitro p r o p a g a t i o n .  352  '  3 5 5  >  3 5 6  Industrial- scale p r o d u c t i o n s o f  various recombinant h u m a n C S F s have revolutionised c l i n i c a l m e d i c i n e , especially i n the management o f cancer p a t i e n t s .  357  E P O , G - C S F and G M - C S F are such e x a m p l e s and are  indicated f o r the amelioration o f anaemia and l e u k o p a e n i a , r e s p e c t i v e l y .  358  In addition,  thrombopoietin ( T P O ) , a recently isolated c y t o k i n e , shows great p r o m i s e i n m a n a g i n g the thrombocytopaenic c o n d i t i o n i n cancer patients u n d e r g o i n g m y e l o a b l a t i v e t h e r a p y .  359  R a t i o n a l use o f C S F s i n the c l i n i c is still far f r o m realisation, however. R e c e n t l y , several multicentre trials have demonstrated the abilities o f G- and G M - C S F s to i m p r o v e objective criteria without significant improvements i n the c l i n i c a l endpoints o f the p a t i e n t s . 3 6 0  1 1 3  '  1 1 4  '  H o e l z e r suggested that C S F s c a n play important and relevant roles i n the c l i n i c , g i v e n  the appropriate i n d i c a t i o n s .  361  Maintenance o f a d y n a m i c e q u i l i b r i u m i n haematopoiesis also requires the activities of m u l t i p l e feedback mechanisms w h i c h negatively modulate the system, some o f w h i c h include soluble regulatory or i n h i b i t o r y factors, adhesion m o l e c u l e s , and intrinsic p r o g r a m m i n g w i t h i n the c e l l s .  2 0 0  Inhibitory factors such as t u m o u r necrosis factor- a  ( T N F - a ) , t r a n s f o r m i n g g r o w t h factor- (3 ( T G F - (3), and interleukin- 1 (IL- 1) have been well characterised.  362  M a c r o p h a g e i n h i b i t o r y factor- l a ( M I P - l a ) is a m e m b e r o f the C-  C c h e m o k i n e f a m i l y and was o r i g i n a l l y identified as a stem c e l l inhibitor b y G r a h a m and  119  colleagues.  201  M I P - l a also mediates the i n h i b i t o r y effects o f interferon- a ( I F N - a ) on  C M L progenitors, through the restoration o f [31 integrin mediated adhesion o f the l e u k a e m i c progenitor cells to the bone m a r r o w stroma, hence effecting restitution o f the g r o w t h i n h i b i t o r y signals f r o m the s t r o m a .  2 1 5  A new class o f peptide- based haematopoietic inhibitors w h i c h i n c l u d e N - A c S D K P , p E E D C K , and S K & F 108636 were i d e n t i f i e d w i t h various m e t h o d s .  3 6 3  -  3 6 4  The  b i o c h e m i c a l as w e l l as the b i o l o g i c a l properties o f N - A c S D K P has since then been characterised i n d e t a i l .  2 0 5  N u m e r o u s studies have s h o w n that the peptide as w e l l as  cytokine inhibitors selectively affect o n l y n o r m a l haematopoietic cells, a fact o f c l i n i c a l significance.  204  '  3 6 5  -  3 6 6  H a e m a t o p o i e t i c inhibitors act p r i n c i p a l l y through the prevention o f  stem c e l l c y c l e entry f r o m Gj/G,  to S phase; therefore, these factors can protect  haematopoietic cells f r o m cycle- s p e c i f i c c h e m o t h e r a p y .  2 1 0  -  3 6 7  -  3 6 8  F u r t h e r m o r e , the  inhibitors also effect other cellular changes that result in the protection o f n o r m a l haematopoietic cells f r o m y- radiation, and hyperthermia, i n addition to chemotherapy. F o r e x a m p l e , IL- 1 and T N F - a i n d u c e the e x p r e s s i o n o f protective e n z y m e s .  369  -  3 7 0  N-  A c S D K P has been demonstrated to selectively protect normal human haematopoietic progenitors f r o m P D T - mediated phototoxicity b y the first generation photosensitiser Photofrin®.  209  In this chapter, the photoprotective effects o f N - A c S D K P or Seraspenide was further investigated on P D T mediated b y the second generation photosensitiser B P D . Relative susceptibility o f D B A / 2 haematopoietic progenitors and L 1 2 1 0 l e u k a e m i c cells to P D T was studied after preincubation w i t h N - A c S D K P , or the control peptides S D K P and N - A c S D K E . In addition, the long- term bone m a r r o w culture ( L T B M C ) assay was used to study whether the photoprotective effect also extended to earlier n o r m a l haematopoietic progenitors. T o c o m p l e m e n t the m u r i n e studies, N - A c S D K P was used i n P D T experiments i n v o l v i n g normal and leukaemic human haematopoietic cells.  120  5.3  RESULTS  Photoprotective effect of N-AcSDKP on DBA/2 haematopoietic progenitors F i g u r e 5.1 demonstrates that preincubation of D B A / 2 bone m a r r o w cells w i t h 100 n M o f N - A c S D K P f o r 1.5 hr protected the haematopoietic progenitors f r o m subsequent B P D - mediated P D T , as measured by the short- term c o l o n y assay. In addition, 100 n M o f the control peptides N - A c S D K E and S D K P , as w e l l as tissue culture m e d i u m , d i d not offer protection. T h e photoprotective effect mediated by 100 n M N - A c S D K P was especially prominent at B P D concentrations o f 2.5, 5, and 10 ng/ml w i t h a p p r o x i m a t e l y 1 . 5 - 2 f o l d higher survival o f D B A / 2 C F U - G M c o m p a r e d to cells preincubated w i t h 100 n M o f control peptides or m e d i u m . C o n t r o l peptides were not tested at the highest B P D concentrations o f 15 and 2 0 ng/ml; h o w e v e r , N - A c S D K P was still able to offer approximately 2- f o l d protection of the progenitors in c o m p a r i s o n to m e d i u m preincubation. U n l i k e its effects o n h u m a n haematopoietic c e l l s , N - A c S D K P d i d not suppress m u r i n e C F U - G M f o r m a t i o n s .  2 0 4  W e s h o w e d that p r e i n c u b a t i o n f o r 1.5 hr w i t h  100 n M o f N - A c S D K P , S D K P , and N - A c S D K E d i d not decrease the numbers o f day 7 c o l o n i e s in standard agar based c o l o n y assays (figure 5.2).  Absence of N-AcSDKP mediated photoprotection of L1210 cells T h e murine leukaemic c e l l line L 1 2 1 0 was not protected f r o m subsequent P D T by 1.5 hr o f preincubation w i t h 100 n M N - A c S D K P . A s s h o w n i n figure 5.3, cells that were preincubated w i t h N - A c S D K P d i d not s h o w any s u r v i v a l advantage post- P D T in c o m p a r i s o n w i t h cells that were preincubated w i t h the control peptides N - A c S D K E and S D K P or in the absence o f any peptides. F i g u r e 5.4 shows that neither the haematopoietic inhibitory peptide N - A c S D K P nor the control peptides had any inhibitory effects on the number o f day 6 L 1 2 1 0 c o l o n i e s . T h e above results suggest that N - A c S D K P has no photoprotective effects on L 1 2 1 0 c e l l line.  121  Figure  5.1  Selective cytoprotective effects of N - A c S D K P on DBA/2 haematopoietic p r o g e n i t o r cells subjected to B P D - m e d i a t e d PDT D B A / 2 haematopoietic progenitors were preincubated w i t h 100 n M o f N A c S D K P (Seraspenide), 100 n M o f the control peptides S D K P o r N A c S D K E , or m e d i u m f o r 1.5 h r f o l l o w e d b y P D T treatment. C l o n o g e n i c i t y was determined w i t h the standard agar- based c o l o n y assay and colonies ( C F U - G M ) were scored o n day 7 o f culture u s i n g an inverted m i c r o s c o p e . O n l y colonies w i t h 4 0 o r more cells were c o u n t e d . Percent s u r v i v a l o f treated cells w a s calculated based o n the number o f colonies generated d i v i d e d b y the number o f c o l o n i e s f r o m the n o B P D control w i t h i n the same peptide g r o u p Average number f r o m the 0 ng/ml B P D control samples o f the f o u r pretreatment groups is 100.815 day 7 c o l o n i e s f r o m 7 0 0 0 0 cells plated. Data obtained f r o m f i v e independent experiments (n= 5) is presented. E r r o r bars are d e r i v e d f r o m standard errors o f experimental means. Results are significant w i t h p< 0.05 as determined b y t w o wayanalysis o f variance test ( A N O V A ) .  122  i50-ri  [BPD] ng/ml  123  03  lOOnM N - AcSDKP  •  lOOnM SDKP  •  lOOnM N - A c S D K E  Q  no peptide  150  Yl  #Day 7 CFU- G M per 7 0 0 0 0 cells  CU Q  Q  <  Q  on  <  1  1  2;  Peptide treatment  Figure 5.2  Effects of N - A c S D K P , control peptides N - A c S D K E or S D K P , and medium on the clonogenicity of DBA/2 haematopoietic progenitor cells D B A / 2 haematopoietic progenitor cells were incubated w i t h 100 n M o f N A c S D K P , 100 n M o f the control peptides N - A c S D K E and S D K P , o r m e d i u m prior to m o c k P D T treatment, i.e. exposure to 15J/cm red light i n the absence o f the photosensitiser B P D . D a y 7 C F U - G M formations i n each group were assessed u s i n g the standard agar- based c o l o n y assay. A n inverted microscope was used to enumerate colonies c o n s i s t i n g o f 5 0 or more cells. T h e n u m b e r o f day 7 C F U - G M w a s not significantly altered i n each o f the peptide- treated g r o u p . Data f r o m five independent experiments is presented (n= 5). S o u r c e o f error is d e r i v e d f r o m the standard errors o f experimental means. 2  124  F i g u r e 5.3  Absence of c y t o p r o t e c t i v e effects of N - A c S D K P on l e u k a e m i c cell line subjected to B P D - m e d i a t e d P D T  L1210  L 1 2 1 0 leukaemic cells were preincubated w i t h 100 n M o f N - A c S D K P (Seraspenide), 100 n M o f the control peptides S D K P or N - A c S D K E , or m e d i u m f o r 1.5 hr f o l l o w e d b y P D T treatment. C l o n o g e n i c i t y was determined w i t h the standard agar- based c o l o n y assay and colonies ( C F U L ) were scored o n day 6 o f culture u s i n g an inverted m i c r o s c o p e . O n l y colonies w i t h 50 or m o r e c e l l s were counted. L o g reductions o f treated cells were calculated w i t h respect to the no B P D control w i t h i n the same peptide g r o u p . Data obtained f r o m three independent experiments (n= 3) is presented. E r r o r bars are d e r i v e d f r o m standard errors o f experimental means. Results are significant w i t h p< 0.05 as determined b y t w o wayanalysis o f variance test ( A N O V A ) .  125  126  # of Day 6 C F U - L per 335 cells  Peptide treatment  Figure 5.4  Effects of N-AcSDKP, control peptides N-AcSDKE or S D K P , and medium on the clonogenicity of L1210 leukaemic cells L I 2 1 0 l e u k a e m i c cells were incubated w i t h 100 n M o f N - A c S D K P , 100 n M o f the control peptides N - A c S D K E and S D K P , or m e d i u m o n l y p r i o r to m o c k P D T treatment, i.e. exposure to 15J/cm red light i n the absence o f B P D . D a y 6 C F U - L f o r m a t i o n i n each g r o u p were assessed u s i n g the standard agar- based c o l o n y assay. A n inverted m i c r o s c o p e w a s used to enumerate colonies c o n s i s t i n g o f 5 0 or more c e l l s . T h e n u m b e r o f day 6 C F U - L w a s not significantly altered i n each o f the peptide- treated g r o u p . Data f r o m three independent experiments is presented (n= 3). S o u r c e o f error is derived f r o m the standard errors o f experimental means. 2  127  S t a t i s t i c a l analyses of p r o g e n i t o r assay d a t a T w o - w a y analysis o f variance ( A N O V A ) and B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n tests were e m p l o y e d to test f o r the significance o f data f r o m the progenitor assays. T w o - w a y A N O V A demonstrated significant differences i n c o l o n y survivals (p <0.05) between the three B P D doses o f 2.5, 5, a n d 10 ng/ml i n both D B A / 2 and L 1 2 1 0 cells (data not shown). M o r e importantly, the A N O V A test demonstrated that the P D T survival data o f D B A / 2 cells preincubated w i t h the different peptides were significantly different f r o m each other yet there was no significant difference amongst P D T - treated L 1 2 1 0 cells preincubated w i t h the different peptides. T h i s suggested that selective photoprotective action was b e i n g exerted o n the normal D B A / 2 haematopoietic cells, but not o n the leukaemic cell line L 1 2 1 0 . T o further delineate the difference between the various groups, a more p o w e r f u l statistical analytical tool was used. T h e B o n f e r r o n i (allpairwise) multiple c o m p a r i s o n test was used to reveal whether intergroup differences exist between the different peptide ( N - A c S D K P , S D K P , N - A c S D K E , a n d m e d i u m ) and B P D (2.5, 5, 10 ng/ml) groups i n both D B A / 2 haematopoietic cells and L 1 2 1 0 l e u k a e m i c cells. Results f r o m n o r m a l c e l l s are presented i n tables 5.1a and 5.1b. T a b l e 5.1a shows that a significant intergroup difference exists between 10 ng/ml B P D and the t w o l o w e r doses o f 5 and 2.5 ng/ml. There w a s n o significant difference between 5 a n d 2.5 n g / m l . T a b l e 5.1b shows that the haematopoietic inhibitory peptide N - A c S D K P acted selectively o n the n o r m a l haematopoietic cells since progenitor assay data o f N - A c S D K P preincubated cells treated w i t h P D T was significantly different f r o m P D T - treated cells that were preincubated w i t h the control peptides N - A c S D K E , S D K P , .as w e l l as w i t h m e d i u m c o n t r o l . D a t a f r o m the L 1 2 1 0 experiments are presented i n tables 5.2a and 5.2b. A l l three B P D doses are significantly different f r o m each other (table 5.2a). M o r e importantly, there w a s n o significant intergroup difference in P D T survival data amongst L 1 2 1 0 cells preincubated w i t h the different peptides (table 5.2b). Therefore, N - A c S D K P selectively photoprotected normal D B A / 2 haematopoietic progenitor cells but not L l 2 1 0 l e u k a e m i c cells f r o m B P D mediated P D T .  128  Table 5.1a- Bonferroni (all- pairwise) multiple comparison test of clonogenicity data of DBA/2 haematopoietic progenitors: significance of B P D doses  [ B P D ] ng/ml  10  5  2.5  10  —  ++  ++  5  ++  —  —  2.5  ++  —  —  T a b l e 5.1a B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test o f absolute numbers o f day 7 C F U - G M o f D B A / 2 haematopoietic progenitor cells treated w i t h 10, 5, and 2.5 ng/ml o f B P D and 15J/cm o f red- light irradiation. T h e plus signs (++) w i t h i n the shaded cells indicate intergroup significant differences between the represented B P D concentration regardless o f the type o f peptide used d u r i n g the preincubation. 2  129  Table 5.1b- Bonferroni (all- pairwise) multiple comparison test of clonogenicity data of D B A / 2 haematopoietic progenitors: significance of peptides  Peptide  N-AcSDKP  SDKP  N-AcSDKE  no peptide  N-AcSDKP  —  ++  ++  ++  SDKP  ++  —  —  —  N-AcSDKE  ++  —  —  —  no peptide  ++  —  —  —  F i g u r e 5.1b B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test o f absolute numbers o f day 7 C F U - G M o f D B A / 2 haematopoietic progenitor cells preincubated w i t h 100 n M o f N - A c S D K P , 100 n M o f control peptides S D K P or N - A c S D K E , or m e d i u m o n l y f o r 1.5 hr f o l l o w e d b y B P D - mediated P D T . T h e plus sign (++) w i t h i n the shaded cells indicate intergroup significant differences in the survival o f D B A / 2 haematopoietic progenitor cells between the represented peptide used regardless o f the concentration o f B P D used.  130  Table 5.2a- Bonferroni (all- pairwise) multiple comparison test of clonogenicity data of L1210 leukaemic cells: significance of B P D doses  [ B P D ] ng/ml  10  5  10  ++  ++ ': ++  5  ++  ++  ++  2.5  ++  ++  '++.-  2.5  T a b l e 5.2a B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test o f absolute numbers o f day 6 C F U - L o f L 1 2 1 0 l e u k a e m i c cells treated w i t h 10, 5, and 2.5 ng/ml o f B P D and 15J/cm o f red- light irradiation. T h e plus sign (++) w i t h i n the shaded cells indicate intergroup significant differences between the represented B P D concentration regardless o f the type o f peptide used d u r i n g the preincubation. 2  131  Table 5.2b- Bonferroni (all- pairwise) multiple comparison test of clonogenicity data of L1210 leukaemic cells: significance of peptides  Peptide  N-AcSDKP  SDKP  N-AcSDKE  no peptide  N-AcSDKP  —  —  —  —  SDKP  —  —  —  —  N-AcSDKE  —  —  —  —  no peptide  —  —  —  —  T a b l e 5.2b B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test of absolute numbers of day 6 C F U - L o f L 1 2 1 0 l e u k a e m i c cells preincubated w i t h 100 n M o f N - A c S D K P , 100 n M o f control peptides S D K P or N - A c S D K E , or m e d i u m o n l y f o r 1.5 hr f o l l o w e d b y B P D mediated P D T . There was no significant difference in c y t o t o x i c response o f L 1 2 1 0 cells preincubated w i t h N - A c S D K P , the c o n t r o l peptides, or m e d i u m .  132  N - A c S D K P protection of D B A / 2 haematopoietic cells does not extend to earlier progenitors and stem cells A one step long- term bone m a r r o w culture ( L T B M C ) assay was used to assess whether N - A c S D K P - mediated photoprotection also extends to earlier D B A / 2 haematopoietic progenitors or even stem cells. In this L T B M C assay, freshly harvested bone m a r r o w cells were again preincubated w i t h 100 n M N - A c S D K P or tissue culture m e d i u m for 1.5 hr prior to B P D - mediated P D T . T h e cells were then m i x e d w i t h L T B M C culture m e d i u m and plated into duplicate w e l l s of 24 w e l l tissue culture plates and assessed as described i n the  experimental procedures section. D u r i n g the course o f in vitro culture,  stromal precursors adhered to the bottom surface o f the wells and established a stromal microenvironment s i m i l a r to that f o u n d in the bone m a r r o w ; haematopoietic progenitor cells and stem cells undergo maturation i n close contact w i t h the stromal layer (figure 5.5). T h e one- step L T B M C assay uses cells f r o m a single harvest for both the establishment o f the stroma as w e l l as f o r subsequent haematopoiesis. C o l o n y formations f r o m long- term progenitors are apparent at w e e k four and f i v e o f the assay whereas colonies f r o m w e e k one to three harvests are derived f r o m the m o r e mature progenitor c e l l p o p u l a t i o n s .  371  In table 5.3, results in the f o r m o f c e l l concentrations f r o m w e e k l y harvestings o f a f i v e w e e k L T B M C assay are presented. C e l l numbers of L T B M C suspension harvests f r o m both peptide- treated and control groups inversely correlated w i t h B P D concentrations and was most o b v i o u s i n cultures f r o m w e e k one to three (figure 5.6). L a t e r time points (week 4 and 5, data.not s h o w n ) . s h o w e d less definitive difference; the.lack o f correlation between harvested c e l l number and B P D dose later i n culture was caused by dehiscence o f cells f r o m the stromal layer. In a d d i t i o n , contributions o f cells f r o m the dehisced stromal layer greatly inflated the c e l l counts in the control as w e l l as the peptide- treated group in w e e k four and five. N- A c S D K P does not significantly affect suspension c e l l numbers in the P D T - treated cells, especially in samples harvested at w e e k one to three, suggesting that 100 n M N - A c S D K P d i d not appear to offer photoprotection to the population(s) o f cells measured by the L T B M C  assay.  133  S u s p e n s i o n cells f r o m the w e e k l y harvests were subsequently put into short- term c o l o n y assay w h i c h measures the late haematopoietic progenitors such as those that f o r m C F U - G M s . A s s h o w n i n table 5.4, there was again an inverse correlation between the numbers o f c o l o n i e s f r o m the harvested cells and the dose o f B P D . H o w e v e r , the numbers were not significantly different between the peptide- treated and the control group; therefore, results f r o m the L T B M C assay suggest that N - A c S D K P - mediated photoprotection seen in late haematopoietic progenitors does not extend to earlier progenitor cells.  134  Table 5.3- Suspension cell numbers of weekly harvests of long- term bone marrow culture of DBA/2 haematopoietic cells preincubated i n the presence or absence of 100 n M N - A c S D K P followed by B P D - mediated P D T No [BPD]  20  Denticle control  100 nM N-AcSDKP  10  5  0  15000 ±  17300±  23000 ±  27400 ±  5600±  15600±  21600 ±  28300 ±  300  6300  800  3400  500  2700  2500  6900  3900 ±  5400 ±  8000 ±  9800 ±  1600 ±  7500 ±  6600 ±  9500 ±  300  800  1100  300  200  2000  400  1200  5600 ±  6700 ±  12000 ±  14000 ±  3900 ±  2700 ±  6100 ±  23600 ±  1600  3000  400  5900  800  1400  600  900  20  10  5  0  ng/ml week 1  week 2  week 3  T a b l e 5.3 Freshly isolated bone m a r r o w cells f r o m D B A / 2 m i c e were preincubated w i t h 100 n M N - A c S D K P or m e d i u m f o r 1.5 hr p r i o r to B P D - mediated P D T . Treated cells were resuspended in L T B M C m e d i u m and plated into 24 w e l l tissue culture plates i n duplicates. A t w e e k l y intervals, nonadherent cells f r o m each w e l l were harvested by gentle agitation and aspiration u s i n g an E p p e n d o r f pipetter. T h e number o f cells f r o m each sample was counted under a haemocytometer by E o s i n Y staining o f an aliquot o f cells f r o m each w e l l . T h e concentrations o f cells f r o m the samples were then calculated. E a c h number, rounded o f f to two significant figures, represent an average o f t w o independent samples (n= 2) and source o f error is standard error o f the m e a n .  135  Figure  5.5  O n e step long- t e r m bone m a r r o w culture ( L T B M C ) bone m a r r o w cells  of  DBA/2  F r e s h l y harvested bone m a r r o w cells f r o m D B A / 2 mice were resuspended in long- term bone m a r r o w culture ( L T B M C ) m e d i u m ( M y e l o C u l t 5300) containing 10" M hydrocortisone and dispensed into 24 w e l l tissue culture plates in 1.0 m l v o l u m e s containing 1 x 1 0 cells. Establishment o f the stromal layer b y stromal precursor cells was f o l l o w e d b y initiation and maintenance o f haematopoiesis. T h e nonadherent cells w h i c h contain haematopoietic cells at various phases o f maturation were harvested w e e k l y v i a gentle agitation and aspiration o f the m e d i a , leaving the stroma behind and intact. T h i s picture was taken at week 2 o f a five week L T B M C culture u s i n g a N i k o n F 3 camera connected to the Zeiss Axiovert 35 inverted m i c r o s c o p e . The l O x objective w i t h phase contrasting setting 1 ( P I ) w a s selected. N o t e the confluent stromal layer as w e l l as the clusters o f cells that appeared to rest on top o f the it; active haematopoiesis occurs at these clusters. 6  6  136  Figure 5.6  Comparison of week 1 long- term bone marrow culture (LTBMC) harvests of nonadherent cells from samples preincubated with 100 n M N - A c S D K P or medium followed by PDT G r a p h i c representation o f the first r o w o f table 5.3' compares the cell numbers o f the harvested nonadherent populations o f w e e k 1 L T B M C . C e l l numbers were similar between the peptide- treated and control groups that were exposed to 0, 5, 10 ng/ml o f B P D and 15 J / c m red light. T h e difference observed at 2 0 ng/ml d i d not extend to later harvests (week 2- 5). 2  137  Table 5.4- Numbers of C F U - G M progenitor colonies generated from nonadherent cells harvested weekly from long- term bone marrow culture of DBA/2 haematopoietic cells preincubated i n the presence or absence of 100 n M N - A c S D K P followed by B P D - mediated P D T No [BPD]  20  peptide control  100 n M N-AcSDKP  10  5  0  6.80 ±  12.30 ±  16.00 ±  19.80 ±  2.20 +  11.20 ±  14.50 ±  13.00 ±  1.20  2.30  1.70  2.20  0.50  0.20  0.50  1.00  1.80 ±  2.80 ±  5.70 ±  7.20 ±  0.70 ±  5.80 ±  7.50 +  11.50 ±  0.80  0.20  3.00  3.50  0.70  3.80  3.80  0.20  7.70 ± 0  7.30 ±  23.50 ±  15.70 +  2.30 ±  19.70 ±  32.50 ±  1.00  15.20  3.00  0.30  13.70  2.80  20  10  0  5  ng/ml week 1  week 2  week 3  • '5.80 ± 4.20  T a b l e 5.4 C e l l s f r o m the w e e k l y harvestings o f nonadherent populations o f L T B M C were resuspended at a standard v o l u m e and g r o w n i n standard short- term c o l o n y assays. T h e numbers o f day 7 C F U - G M c o n s i s t i n g o f 4 0 or more c e l l s were enumerated u s i n g an inverted m i c r o s c o p e . E a c h number, r o u n d e d o f f to one significant f i g u r e , represent an average o f two independent samples (n= 2) and source o f error is standard error o f the mean.  Selective photoprotection of normal human bone marrow cells but not leukaemic cells from P D T by N - A c S D K P H u m a n bone m a r r o w cells or primary l e u k a e m i c cells f r o m a n e w l y diagnosed C M L patient were preincubated w i t h 100 n M N - A c S D K P or tissue culture m e d i u m f o r 1.5  138  hr prior to P D T mediated by 10 ng/ml B P D and 15 J / c m red light. T h e treated cells were 2  then put into short- term c o l o n y assays w h i c h measured late h u m a n haematopoietic progenitors w h i c h give rise to C F U - G M or C M L l e u k a e m i c precursor cells w h i c h give rise to C F U - L. In a d d i t i o n , the h u m a n C M L c e l l l i n e K 5 6 2 was also e x a m i n e d . N u m b e r s o f colonies f r o m the peptide group were d i v i d e d b y the n u m b e r o f colonies in the tissue culture m e d i u m group to obtain a photoprotection ratio. A ratio o f more than 1 signifies photoprotection and the results is s h o w n i n f i g u r e 5.7. C e l l s f r o m t w o independent h u m a n bone m a r r o w samples benefited f r o m preincubation w i t h 100 n M N - A c S D K P w i t h photoprotection ratios o f 4.5 and 8. H o w e v e r , the l e u k a e m i c c e l l line K 5 6 2 and c e l l s f r o m the p r i m a r y l e u k a e m i c c e l l sample d i d not benefit f r o m N - A c S D K P because both samples demonstrated photoprotection ratios o f 1. Results f r o m the h u m a n experiments m i r r o r e d those o f the earlier m u r i n e assays; that is, N - A c S D K P selectively protected late haematopoietic progenitors but not leukaemic cells f r o m B P D - mediated P D T .  139  10-,  BM1 Ratio of photo- protection conferred by lOOnM  6H  •  K562-1  N-AcSDKP *  2H  1 HuBMMNC  Figure 5.7-  BM2  K562  A  K562-2  4  CML  CML  Cytoprotection conferred by 100 n M of N - A c S D K P against subsequent B P D - mediated P D T was specific for normal human bone marrow cells Preincubation o f normal h u m a n bone m a r r o w cells w i t h l O O . n M o f N A c S D K P f o r 1.5 hr selectively protected them f r o m subsequent P D T treatment mediated by 10 ng/ml o f B P D and 15 J / c m o f red light. T h e human myelogenous l e u k a e m i c c e l l line K 5 6 2 and p r i m a r y untreated h u m a n C M L cells d i d not r e s p o n d to the photoprotective effects o f N - A c S D K P . The ratios o f photoprotection o f the respective cell type were derived f r o m the ratio o f the percentage o f colonies f r o m P D T - treated cells that were preincubated w i t h 100 n M N - A c S D K P over the percentage o f colonies o f P D T - treated cells that were preincubated w i t h m e d i u m . 2  140  DISCUSSION  T h e process o f haematopoiesis is a h i g h l y regulated process i n v o l v i n g both positive and negative s i g n a l s .  2 0 0  V a r i o u s c o l o n y stimulating factors or cytokines have been  identified and c l o n e d and some o f these factors s h o w promise in the c l i n i c a l management o f the cancer p a t i e n t .  357  F u n c t i o n a l haematopoiesis also depends on its efficient control v i a  negative regulators such as IL- 1, T G F - p\ and c h e m o k i n e s such as M I P - l a .  3 6 2  A new  class o f oligopeptide haematopoietic inhibitors w h i c h i n c l u d e the tetrapeptide N-AcetylSer- A s p - L y s - P r o ( A c S D K P ) and p y r o G l u - G l u - A s p - C y s - L y s ( p E E D C K ) was recently characterised.  205  N - A c S D K P is d e r i v e d f r o m proteolytic p r o c e s s i n g o f the N- terminus o f  t h y m o s i n (34, a protein w h i c h b i n d s F- a c t i n .  3 7 2  >  3 7 3  S e r u m l e v e l o f N - A c S D K P changes  during chemotherapy in leukaemic patients and i n m i c e after treatment w i t h cytosine arabinoside (ara- C ) .  3 7 4  -  3 7 5  N u m e r o u s studies have demonstrated that N - A c S D K P  selectively inhibits cell c y c l e progression o f n o r m a l haematopoietic cells, preventing the entry f r o m G Q / G , to S- p h a s e .  2 0 4  -  3 6 5  S e l e c t i v e c y c l e i n h i b i t i o n o f n o r m a l c e l l s by N-  A c S D K P protects them f r o m chemotherapy, hyperthermia, radiation, as w e l l as P D T treatments.  2 0 6 - 2 0 9  H o w e v e r , other m e c h a n i s m s mediated b y N - A c S D K P c o u l d be i n v o l v e d  as w e l l . L a r g e scale production o f the peptide f o r p r o p o s e d trials for h u m a n applications p r o m p t e d subsequent h u m a n p h a r m a c o k i n e t i c s t u d i e s .  376  N - A c S D K P therefore j o i n s the  other haematopoietic inhibitory cytokines and peptides and becomes part o f the armamentorium in the new f i e l d o f stem cell protection, i n w h i c h b i o l o g i c a l factors are used to effect selective protection o f normal haematopoietic cells f r o m chemotherapy and radiation t h e r a p y .  210  -  3 6 7  <  3 6 8  -  3 7 7  Ih a d d i t i o n , other candidates such as the thiol- based  c o m p o u n d amifostine s h o w e d p r o m i s e in ameliorating the h a r m f u l side effects o f chemotherapy on normal haematopoietic c e l l s . in the  ex vivo p u r g i n g o f  3 7 8  F o r e x a m p l e , incorporation o f amifostine  autologous bone m a r r o w f r o m breast cancer patients w i t h 4-  h y d r o p e r o x y c y c l o p h o s p h a m i d e significantly i m p r o v e d subsequent haematological recovery.  3 7 9  In this study, N - A c S D K P was used to effect selective photoprotection on n o r m a l haematopoietic cells. A previous study has s h o w n that N - A c S D K P c o u l d be used to protect normal h u m a n bone m a r r o w cells f r o m P D T mediated b y the first generation  141  photosensitiser Photofrin®.  209  S i n c e B P D is a m u c h m o r e potent and pure preparation than  Photofrin®, this study aimed to investigate whether N - A c S D K P also extended its protective potential to B P D . F r e s h l y isolated bone m a r r o w cells f r o m D B A / 2 m i c e or L 1 2 1 0 l e u k a e m i c cells were incubated w i t h 100 n M o f N - A c S D K P f o r 1.5 h p r i o r to exposure to P D T mediated by B P D . C o l o n y f o r m i n g units o f granulocyte/ macrophage ( C F U - G M ) and c o l o n y f o r m i n g units o f leukaemic cells ( C F U - L ) were determined u s i n g standard c o l o n y assays. N e i t h e r the tetrapeptide N - A c S D K P nor the c o n t r o l peptides S D K P and NA c S D K E i n h i b i t e d the growth o f C F U - G M and C F U - L. T h e absence o f i n h i b i t o r y effect on late m u r i n e progenitors supported earlier experimental f i n d i n g s u s i n g C B A / H derived haematopoietic c e l l s .  3 8 0  N - A c S D K P protected D B A / 2 late haematopoietic progenitors f r o m  P D T , especially at the l o w e r doses o f B P D o f 2.5 and 5.0 ng/ml. T h e c o n t r o l peptides NA c S D K E and S D K P as w e l l as the m e d i u m control had no protective effects on the cells. T h e same protective effect was not observed in L 1 2 1 0 cells preincubated w i t h N - A c S D K P or the c o n t r o l peptides. In addition, B o n f e r r o n i (all- pairwise) m u l t i p l e c o m p a r i s o n test o f the data f r o m the progenitor assays demonstrated significant intergroup differences between P D T - treated D B A / 2 haematopoietic cells that were preincubated w i t h N - A c S D K P and the P D T - treated cells that were preincubated w i t h the control peptides or m e d i u m . N o significant intergroup difference was observed i n P D T - treated L 1 2 1 0 cells preincubated w i t h the different peptides and one therefore can conclude that N - A c S D K P selectively protected D B A / 2 haematopoietic progenitor cells but not L 1 2 1 0 leukaemic cells f r o m B P D mediated P D T . Furthermore, one step long- term bone m a r r o w culture ( L T B M C ) assay revealed that the photoprotective effect on the late progenitors measured b y the c o l o n y assays d i d not extend to earlier progenitors. C e l l numbers o f w e e k l y harvests as w e l l as the ^numbers o f C F U - G M produced f r o m the harvests were not s i g n i f i c a n t l y different between ;  the peptide and control group treated w i t h P D T at different doses. B o t h harvested c e l l number and c o l o n y number correlated inversely w i t h the dose o f B P D and this correlation was more o b v i o u s in the earlier harvests o f w e e k 1 to 3. T h e loss o f adherence o f the stromal layer d u r i n g harvesting o f the suspension cells contributed to the inflated c e l l numbers and therefore c o l o n y numbers i n the harvests o f w e e k s 4 and 5 (data not s h o w n ) , w h i c h also contributed to the loss o f correlation between the B P D doses and the results o f the L T B M C assay. A n apparent contradiction exists between the results i n F i g u r e 5.1 and table 5.4. S p e c i f i c a l l y , short- term agar c o l o n y assay o f D B A / 2 bone m a r r o w c e l l s  142  demonstrated the photoprotective effect o f 100 n M N- A c S D K P (figure 5.1); however, the numbers o f C F U - G M d e r i v e d f r o m w e e k 1 L T B M C harvests o f c o n t r o l and peptide treated cells s h o w e d n o sigificant difference. In fact, the same trend was noted in the direct c e l l counts o f the w e e k 1 harvests (table 5.3). N - A c S D K P at 100 n M was able to selectively protect n o r m a l h u m a n bone m a r r o w cells f r o m B P D - mediated P D T . T h i s c o n f i r m s earlier results by C o u t t o n and colleagues u s i n g Photofrin®- mediated P D T .  2 0 9  T h e procedures described here d i f f e r e d somewhat  f r o m the ones used i n the above mentioned paper. S p e c i f i c a l l y , w e were able to achieve photoprotection o f the n o r m a l human haematopoietic progenitors b y 1.5 h incubation w i t h 100 n M o f N - A c S D K P whereas a 2 0 h incubation p e r i o d was used in C o u t t o n ' s paper. W e also observed i n h i b i t o r y activity against h u m a n in vitro C F U - G M in w h i c h human bone m a r r o w cells incubated w i t h 100 n M N - A c S D K P f o r 1.5 h resulted i n the p r o d u c t i o n o f 54- 67 % o f day 14 c o l o n i e s c o m p a r e d to the no peptide controls. In a d d i t i o n , the inhibitory effects o f N - A c S D K P were not u n i f o r m i n the different h u m a n bone m a r r o w samples w h i c h suggested idiosyncratic susceptibility to the peptide. Photoprotection against B P D was not observed w h e n N - A c S D K P was used d u r i n g p r e i n c u b a t i o n o f K 5 6 2 cells or p r i m a r y C M L cells. T h e utility o f selective photoprotection mediated b y N - A c S D K P is still o b v i o u s even though it does not extend to earlier haematopoietic progenitor and stem cells. N o r m a l haematopoietic cells, especially the earlier progenitor and stem cells, already exhibit a great resistance to B P D - mediated P D T as demonstrated in the L T B M C assay performed here and in earlier results o f the h u m a n L T B M C a s s a y s .  292  Pretreatment o f m u r i n e bone  m a r r o w cells effected significant protection o f the C F U - G M p o p u l a t i o n f r o m P D T . T h i s permits B P D dose escalation in purging without significant damage to the haematopoietic c e l l compartment responsible f o r long- term reconstitution (early progenitors and stem cells) and cells that are responsible for short- term and rapid engraftment ( C F U - G M s ) .  143  CHAPTER 6: PREPROTECTION OF NORMAL HAEMATOPOIETIC CELLS WITH N-ACSDKP: MECHANISTIC STUDY  6.1 ABSTRACT Preincubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M o f N - A c S D K P selectively protected o f the c o m m i t t e d granulocyte/macrophage progenitors ( C F U - G M ) but not cells o f earlier lineage f r o m P D T . Peptide pretreatment, however, d i d not protect the m u r i n e l e u k a e m i c c e l l line L 1 2 1 0 . F A C S analysis w a s used to determine B P D uptake i n the cells responsible for short- term haematopoietic reconstitution. D o u b l e l a b e l l i n g o f D B A / 2 bone m a r r o w cells preincubated w i t h control m e d i u m or 100 n M o f N - A c S D K P w i t h anti- C D 3 4 antibody and 10 ng/ml B P D demonstrated n o significant difference i n mean fluorescent intensity ( M F I ) o f B P D i n the C D 3 4 p o p u l a t i o n . In addition, peptide incubation d i d not +  alter the proportion o f C D 3 4 c e l l s . T h e b i o a c t i v i t y o f N - A c S D K P w a s v e r i f i e d b y standard +  ara- C suicide assay. Incubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M N - A c S D K P f o r 1.5 h o r 5.0 h resulted i n substantial protection o f C F U - G M f o r m a t i o n , v i a prevention o f S- phase entry, f r o m subsequent exposure to 5 0 o r 100 p M o f ara- C . S i n c e 1.5 h incubation w i t h N - A c S D K P w a s able to effect c y c l e i n h i b i t i o n i n C F U - G M progenitors, the observed photoprotective effect c o u l d therefore be the result o f N - A c S D K P - mediated c y c l e i n h i b i t i o n . T h i s p o s s i b i l i t y was p r o v e n b y pretreating the bone m a r r o w cells w i t h 5 0 u M A r a - C w h i c h resulted i n the destruction o f S- phase cells. T h e s u r v i v i n g cells were more tolerant to subsequent PDT'Cytotoxicity, suggesting that quiescent C F U - G M progenitors are more resistant to P D T than their active counterpart. E x a m i n a t i o n o f cellular glutathione content revealed no significant difference between the peptide- treated cells and the control cells. Therefore, the specific m e c h a n i s m s responsible for N - A c S D K P - mediated photoprotection are still not clear. H o w e v e r , it is possible that N - A c S D K P - mediated c e l l c y c l e i n h i b i t i o n c o u l d affect m u l t i p l e pathways (i.e. drug uptake, levels o f protective thiols, subcellular localisation o f target proteins, etc) that together result ultimately i n protection from P D T .  144  6.2  INTRODUCTION  Selective protection o f n o r m a l haematopoietic stem cells and progenitor cells d u r i n g in vivo and ex vivo antineoplastic radiochemotherapy is a therapeutic goal l o n g sought after b y c l i n i c i a n s (and patients alike). Other n o r m a l tissues, such as the regenerating epithelium o f the gastrointestinal tract and hair f o l l i c u l a r cells, are also susceptible to the side effects o f therapy; i n addition, organ- specific toxicities f r o m potent yet indispensable drugs such as d o x o r u b i c i n and vincristine l i m i t the utility o f these agents. Nevertheless, bone m a r r o w toxicity encourages the development o f life- threatening episodes o f thrombocytopaenia, anaemia, as w e l l as i m m u n o s u p p r e s s i o n and therefore remains one o f the major l i m i t i n g factors i n the use o f therapeutically efficient doses o f radiochemotherapy. Therefore, one ideally w o u l d l i k e to selectively protect n o r m a l haematopoietic cells during therapy so as to enlarge the therapeutic w i n d o w . In vivo bone m a r r o w protection can be effected through the use o f positive haematopoietic regulators, such as granulocyte and granulocyte- macrophage c o l o n y stimulating factor (G- C S F , G M C S F ) and erythropoietin ( E P O ) , w h i c h stimulate the proliferation plus differentiation o f n o r m a l haematopoietic cells to counteract therapy- i n d u c e d m y e l o s u p p r e s s i o n .  200  The  usefulness o f these factors in the c l i n i c a l setting, especially i n effecting myeloprotection f r o m dose and schedule intensification o f r a d i o c h e m o t h e r a p y , is c o n t r o v e r s i a l .  1 1 4  -  1 1 5  >  3 6 0  Nevertheless, j u d i c i o u s and rational use c o u p l e d w i t h i m p r o v i n g c l i n i c a l and scientific k n o w l e d g e w i l l increase the a p p l i c a b i l i t y o f these factors i n cancer t h e r a p y .  361  Conversely,  cytokines such as tumour necrosis factor- a ( T N F a ) and interleukin- 1 (IL- 1), the c h e m o k i n e macrophage i n f l a m m a t o r y factor- l a ( M I P - l a ) , and s m a l l peptides such as acetyl- N- Ser- A s p - L y s - P r o ( N - A c S D K P ) and p G l u - G l u - A s p - C y s - L y s ( p E E D C K ) can also effect stem c e l l protection through the i n h i b i t i o n o f c e l l c y c l i n g and possibly other means.  2 1 0  -  3 6 8  T h e p h o s p h o r y l a t e d s u l p h y d r y l c o m p o u n d a m i f o s t i n e ( W R - 2721) has been  demonstrated to afford haematopoietic protection to murine hosts exposed to y- radiation or c y t o t o x i c agents, probably v i a the m o d u l a t i o n o f cellular thiol c o n c e n t r a t i o n s .  381  A m i f o s t i n e , i n combination w i t h G- C S F , can significantly accelerate m y e l o p o i e t i c recovery i n lethally irradiated m i c e .  3 8 2  -  3 8 3  145  H a e m a t o p o i e t i c stem c e l l transplantation is essentially a f o r m o f  ex vivo bone  m a r r o w protection and the transplantation o f bone marrow- derived stem cells constituted one o f the earliest application o f such t h e r a p y . 3 7  3 8 4  A u t o l o g o u s haematopoietic stem c e l l  transplantations (bone m a r r o w or peripheral b l o o d stem cells) eliminate the need for H L A c o m p a t i b l e allogeneic donors and hence reduce the r i s k o f graft- versus- host d i s e a s e .  152  Nevertheless, contamination o f autologous stem c e l l harvests b y neoplastic cells leads to an increase i n the incidence o f disease relapse p o s t t r a n s p l a n t a t i o n .  154  Ex vivo m a n i p u l a t i o n  or  p u r g i n g o f the harvests w i t h p h a r m a c o l o g i c a l agents attempts to eradicate a significant number o f the contaminating cancer cells w h i l e preserving enough o f the normal haematopoietic cells for reconstitution. T h e same p r i n c i p l e that is applicable for stem c e l l protection is also suitable for  ex vivo p u r g i n g in  in vivo  that the procedure attempts to  enlarge the therapeutic w i n d o w v i a the selective protection o f n o r m a l haematopoietic cells, w h i c h permits dose escalation o f the p u r g i n g r e g i m e n .  2 0 5  -  2 1 0  M e c h a n i s t i c a l l y , the current p a r a d i g m o f stem c e l l protection mediated by inhibitory molecules revolves around the c e l l c y c l e . M o s e r and P a u k o v i t s , in their r e v i e w article, conceded that cytostatic drug- induced haematopoietic damage is effected on the proliferating compartments o f the bone m a r r o w w h i c h contain the mitotically active haematopoietic progenitor and precursor cells, whose depletions result in the recruitment o f n o r m a l l y quiescent stem cells into c y c l e .  3 6 7  U n f o r t u n a t e l y , the c y c l i n g stem cells, i n an  effort to replenish the progenitors, in turn b e c o m e vulnerable to damage and repeated rounds o f chemotherapy can lead to the irreversible impairment or .quantitative impairment o f the stem c e l l p o o l .  3 8 5  Gardner et al. recently demonstrated that chemotherapy causes the  exhaustion o f the pluripotent p r i m i t i v e haematopoietic stem c e l l ( P H S C ) p o o l , resulting in prolonged;marro.W'reconstitutive d e f e c t s .  1386  S t e m c e l l inhibitprs.such.as M I P - l a , N-  A c S D K P and p E E D C K selectively prevent cell c y c l e entry o f normal haematopoietic stem cells and precursor cells. N - A c S D K P was o r i g i n a l l y characterised to prevent the recruitment o f m u r i n e P H S C s into the D N A synthetic or S- phase after ara- C chemotherapy as determined b y spleen c o l o n y f o r m i n g units ( C F U - S) and administration o f the peptide protected m i c e f r o m lethal doses o f ara- C .  3 6 3  -  3 8 7  Furthermore, N - A c S D K P  was s h o w n to inhibit the S- phase entry o f h u m a n haematopoietic progenitor and precursor cells  in vitro  in short- term progenitor assays and long- term bone m a r r o w c u l t u r e .  3 8 8  L e u k a e m i c cells do not appear to respond to the regulatory effects o f N - A c S D K P and  146  -  3 8 9  therefore are not afforded protection b y the p e p t i d e .  2 0 4  -  3 6 5  M I P - l a was f o u n d to be  another mediator o f stem cell protection w i t h s i m i l a r target populations and mechanisms as N-AcSDKP.  2 0 1  -  2 0 2  >  3 6 6  Interestingly, C a s h m a n et al. presented e v i d e n c e that the N -  A c S D K P - mediated inhibitory effect c o u l d be abrogated by the simultaneous addition o f M I P - 1/3, an antagonist o f M I P - l a therefore suggesting that M I P - l a is a.downstream mediator o f N - A c S D K P b i o a c t i v i t y .  2 0 4  A different f o r m o f stem c e l l protection described b y Z u c a l i and colleagues concerns the cytoprotective effects o f T N F a and f L - 1 on h u m a n and murine haematopoietic cells f r o m i o n i s i n g radiation and the p u r g i n g agent 4- h y d r o p e r o x y c y c l o p h o s p h a m i d e (4HC).  3 9 0  '  3 9 1  F u r t h e r m o r e , they presented evidence that c y t o k i n e i n d u c t i o n o f the e n z y m e s  manganese- superoxide dismutase ( M n S O D ) and aldehyde dehydrogenase ( A L D H ) was responsible f o r their cytoprotective e f f e c t s .  369  -  3 7 0  >  3 7 7  M n S O D detoxifies the h i g h l y  reactive superoxide anion, generated d u r i n g cellular exposure to i o n i s i n g radiation or the d r u g d o x o r u b i c i n , v i a its transmutation into h y d r o g e n p e r o x i d e .  3 9 2  A L D H , whose  transcription i n h u m a n m a r r o w cells is increased three- f o l d w i t h IL- 1 or T N F a , detoxifies 4- H C .  3 6 9  S t e m c e l l protection can be mediated by c e l l c y c l e i n h i b i t i o n , induction o f enzymes, and augmentation o f the cellular thiol p o o l or, conversely through the use o f positive regulators w h i c h n u m e r i c a l l y expand b l o o d cells to counteract the deleterious effects o f therapy. In this chapter, w e e x a m i n e d some o f the m e c h a n i s m s responsible f o r the selective photoprotection o f normal haematopoietic cells by N - A c S D K P ; s p e c i f i c a l l y , the effects o f N - A c S D K P on c e l l c y c l i n g , photosensitiser uptake, and cellular glutathione were investigated.  147  6.3  RESULTS  B P D uptake by D B A / 2 bone marrow cells preincubated with 100 n M N AcSDKP Freshly isolated femoral bone m a r r o w cells f r o m D B A / 2 m i c e were incubated i n the presence or absence o f 100 n M N - A c S D K P i n serum- free I M D M f o r 1.5 h f o l l o w e d b y incubation w i t h 10 ng/ml B P D f o r 0.5 h. F A C S analysis was then used to assess f o r the percentage o f C D 3 4 - positive cells. C D 3 4 is a surface s i a l o m u c i n expressed b y populations o f haematopoietic progenitor and stem c e l l s .  3 9 3  N e x t , the l e v e l o f B P D uptake o r  association i n the peptide- treated population was determined b y analysing B P D mean fluorescence intensity ( M F I ) i n C D 3 4 cells (described i n the Experimental procedure +  chapter). A s s h o w n i n table 6.1, preincubation w i t h N - A c S D K P d i d not alter the proportion o f C D 3 4 - expressing cells i n the peptide- treated group (16.13 + 0 . 9 4 % ) compared to the culture m e d i u m - treated control group (16.83 + 0.43 % ) . F u r t h e r m o r e , there was n o significant difference i n B P D M F I between the peptide- treated group (4.67 + 0.25) and the c o n t r o l group (4.84 + 0.18). Therefore, in vitro preincubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M N - A c S D K P f o r 1.5 h d i d not appreciably alter the proportion o f C D 3 4 c e l l s nor d i d it change the degree o f B P D association w i t h i n the +  C D 3 4 p o p u l a t i o n . Nevertheless, as demonstrated i n chapter 5, the same peptide +  preincubation protocol was able to effect cytoprotection o f D B A / 2 C F U - G M progenitors from P D T .  148  Figure 6.1a-c Representative F A C S histograms of B P D uptake i n control and N - A c S D K P - treated C D 3 4 - expressing murine bone marrow mononuclear cells ( B M M N C ) Preincubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M N - A c S D K P f o r 1.5 h d i d not significantly alter the relative proportion o f C D 3 4 cells n o r B P D uptake i n these cells. T w o c o l o u r F A C S analysis o f cells labelled w i t h biotin- C D 3 4 antibody (clone R A M 3 4 ) / F I T C - strepavidin and 10 ng/ml B P D . R A M 3 4 , a m o n o c l o n a l antibody o f rat o r i g i n , is specific f o r the mouse haematopoietic stem cell antigen C D 3 4 . F A C S analysis was based o n 100000 events per s a m p l e . N - A c S D K P does not significantly alter the percentage o f C D 3 4 c e l l s (Figure 6.1a) and the peptide does not affect B P D association and uptake i n B M M N C s (Figure 6.1b). In a d d i t i o n , N - A c S D K P does not alter B P D association a n d uptake i n C D 3 4 c e l l s (Figure 6.1c). +  +  +  149  No peptide control  100 nM N- AcSDKP  Table 6.1 B P D uptake in the CD34 expressing subpopulation of D B A / 2 bone marrow cells  % CD34 bone inhibitor  marrow  no peptide  treatment  expressing  BPD  MFI in CD34 cells +  cells  100 n M N -  no peptide  AcSDKP  100 n M N AcSDKP  expt. 1  15.30  17.10  4.33  4.48  expt. 2  18.00  17.40  5.15  4.98  expt. 3  15.10  16.00  4.52  5.05  Mean  16.13  16.83  4.67  4.84  S.E.  0.94  0.43  0.25  0.18  T a b l e 6.1 Preincubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M N - A c S D K P f o r 1.5 h d i d not significantly alter the relative proportion o f C D 3 4 cells o r B P D uptake i n these cells. T w o c o l o u r F A C S analysis o f cells labelled w i t h biotin- C D 3 4 antibody (clone R A M 3 4 ) / F I T C - strepavidin and 10 ng/ml B P D . R A M 3 4 , a m o n o c l o n a l antibody o f rat o r i g i n , is specific f o r the m o u s e haematopoietic stem cell antigen C D 3 4 . F A C S analysis was based o n 100000 events per sample. Data f r o m three independent experiments are presented and source o f error is d e r i v e d f r o m the standard error o f the mean. +  151  Inhibition of the proliferative activity of murine CFU- GM progenitors by 100 nM N-AcSDKP F i g u r e 6.2 shows that incubation o f freshly harvested D B A / 2 bone m a r r o w cells w i t h 100 n M N - A c S D K P f o r 1.5 h resulted i n the i n h i b i t i o n o f S- phase entry o f the C F U G M progenitors. C e l l s that were exposed to N - A c S D K P became resistant to the c y t o t o x i c activity o f ara- C , an S- phase specific t o x i n . I n h i b i t i o n o f S- phase entry w a s also demonstrated i n c e l l samples that were incubated w i t h N - A c S D K P f o r 5.0 h (figure 6.3). Interestingly, the absolute numbers o f day 7 c o l o n i e s were not appreciably reduced b y N A c S D K P preincubation. W e therefore demonstrated that the fraction o f S- phase D B A / 2 C F U - G M progenitors c o u l d be reduced w i t h in vitro exposure to 100 n M N - A c S D K P f o r 1.5 h. In addition, our data suggests that the observed photoprotective effect mediated b y N - A c S D K P c o u l d be due to its ability to inhibit c e l l c y c l e progression into the D N A synthetic or S- phase. W e proceeded to examine the relationship between c e l l c y c l i n g a n d P D T susceptibility i n D B A / 2 C F U - G M progenitors.  Cell cycle inhibition of DBA/2 haematopoietic progenitor cells mediated by 100 nM N-AcSDKP E x p o s u r e o f freshly harvested D B A / 2 bone m a r r o w cells to 100 n M N - A c S D K P f o r 1.5 h effected cell c y c l e i n h i b i t i o n o f the C F U - G M fraction, rendering the cells resistant to the S- phase specific c y t o t o x i c i t y o f ara- C . T h e observation also suggest that the photoprotective effect reported i n chapter 5 w a s mediated v i a the inhibition o f c y c l e entry. F i g u r e 6.4 further illustrates the relationship between cell c y c l i n g and susceptibility to B P D - mediated P D T i n the C F U - G M progenitor p o p u l a t i o n . Harvested m a r r o w cells were incubated w i t h 5 0 p M ara- C f o r 1.0 h w h i c h , as s h o w n i n figure 6.2, resulted i n the destruction o f approximately 5 0 % o f the C F U - G M progenitor p o p u l a t i o n . A r a - C effected  152  selective cytotoxicity to cells in S- phase w h i c h resulted i n the enrichment o f quiescent cells ( G / G , ) . Subsequent exposure to B P D - mediated P D T revealed differential susceptibility 0  to P D T c y t o t o x i c i t y . E v e n though 5 0 u M ara- C caused a reduction o f the absolute number o f day 7 C F U - G M , the s u r v i v i n g fraction was s i g n i f i c a n t l y more resistant to P D T than the control cells not pretreated w i t h ara- C (figure 6.4).  153  125-,  HXH 75Day 7 CFU- G M  50-  25  A  0 I 100 [Ara-C] u M  Figure 6.2  Effect of 1.5 h preincubation with 100 n M N - A c S D K P the cycling status of C F U - G M from D B A / 2 mice  had on  C F U - G M formation is protected f r o m ara- C cytotoxicity by N - A c S D K P ; h o w e v e r , exposure to the peptide alone had n o effects o n the number o f C F U - G M . D B A / 2 bone m a r r o w cells were incubated w i t h 100 n M N A c S D K P for 1.5 h in serum free I M D M at 3 7 ° C . T h e cells were washed and incubated w i t h 0, 5 0 , or 100 u M o f ara- C f o r 1 h and put into standard agar- based c o l o n y assay in triplicates. C o l o n i e s , w h i c h consisted o f 50 or more c e l l s , were counted on day 7 o f the experiment w i t h an inverted m i c r o s c o p e . A p p r o x i m a t e l y 7 0 0 0 0 cells were seeded per plate w h i c h translated to a plating efficiency o f 0 . 1 2 9 % . Data obtained f r o m 3 independent experiments is presented. E r r o r bars are derived f r o m standard error o f the mean ( S E M ) .  154  100-,  75 J  100 n M  50 4 D7 CFU- G M  N - A c S D K P - 5hr no peptide control- 5hr  25  0-  mm. 100 [Ara-C] u M  Figure 6.3  Effect of 5.0 h preincubation with 100 n M N - A c S D K P had on the cycling status of C F U - G M from D B A / 2 mice C F U - G M formation is protected f r o m ara- C cytotoxicity b y ' N - A c S D K P ; h o w e v e r , exposure to the peptide alone had n o effects o n the number o f C F U - G M . D B A / 2 bone m a r r o w cells were incubated w i t h 100 n M N A c S D K P for 5.0 h i n serum free I M D M at 3 7 ° C . T h e cells were washed and incubated w i t h 0, 5 0 , or 100 p M o f ara- C f o r 1 h and put into standard agar- based c o l o n y assay i n triplicates. C o l o n i e s , w h i c h consisted o f 5 0 or more c e l l s , were counted o n day 7 o f the experiment w i t h an inverted m i c r o s c o p e . A p p r o x i m a t e l y 7 0 0 0 0 cells were seeded per plate w h i c h translated to a plating efficiency o f 0.129 % . Data obtained f r o m 3 independent experiments is presented. E r r o r bars are derived f r o m standard error o f the mean ( S E M ) .  155  150 - I  Figure 6.4  Correlation between cell cycling and susceptibility mediated P D T in D B A / 2 C F U - G M progenitors  to B P D -  C F U - G M progenitors preincubated w i t h 50 u M were less susceptible to  ;  BPDr mediated PDT. E v e n though ara- C resulted i n a. reduction o f the  absolute number o f day 7 C F U - G M , cells that were preincubated w i t h 50 p M ara- C s h o w e d higher relative s u r v i v a l f r o m subsequent PDT treatment than control cells preincubated w i t h m e d i u m . C o n t r o l DBA/2 progenitors y i e l d e d 185.67 ± 14.57 c o l o n i e s f r o m 70000 cells plated (0.264 % c l o n i n g efficiency) whereas progenitors cells treated w i t h 50 u M ara- C yielded 169.33 + 9.07 colonies f r o m 140000 cells plated (0.121 % c l o n i n g e f f i c i e n c y ) . Results f r o m 3 independent experiments are presented. E r r o r bars are derived f r o m standard errors o f the mean ( S E M ) .  156  Glutathione level in D B A / 2 bone marrow cells treated with 100 n M N AcSDKP T h e amount o f intracellular glutathione ( G S H ) was established u s i n g the T i e t z e m e t h o d as described in the Experimental procedure chapter. T h e G S H content f r o m c e l l extracts d e r i v e d f r o m 2 x 1 0 D B A / 2 bone m a r r o w cells incubated w i t h 0 ( 5 1 . 6 7 + 11.46 6  p M G S H ) , 10 (47.01 ± 12.59 p M G S H ) , o r 100 n M (51.77 ± 15.54 p M G S H ) N A c S D K P for 1.5 h were not s i g n i f i c a n t l y different f r o m one another (figure 6.5). W e therefore c o u l d not attribute the photoprotective effect o f N - A c S D K P to an increase o f the intracellular antioxidant G S H . H o w e v e r , a m o r e sensitive m e t h o d o f measurement, such as one that is based on the fluorescence detection o f G S H - b o u n d m o n o c h l o r o b i m a n e o r m o n o b r o m o b i m a n e , c o u l d p o s s i b l y reveal any alterations o f c e l l u l a r G S H as a result o f N A c S D K P incubation. In addition, w e d i d not consider other c e l l u l a r antioxidants w h i c h c o u l d mediate photoprotection i n this experiment.  157  [N- A c S D K P ] n M  Figure  6.5  Cellular glutathione content in DBA/2 bone incubated with 0, 10, or 100 n M N - A c S D K P  marrow  cells  Incubation o f D B A / 2 bone m a r r o w cells w i t h N - A c S D K P f o r 1.5 h had n o effect, o n cellular glutathione ( G S H ) content as determined b y the Tietze m e t h o d . C e l l extracts f r o m 2 x 1 0 cells incubated w i t h .0, 10, 1 0 0 ' n M N A c S D K P were processed as described i n the Experimental Procedure chapter and G S H content i n u M w a s measured. R e s u l t s f r o m t w o independent experiments are s h o w n . Standard errors o f the means ( S E M ) are used as source o f error. 6  158  6.4  DISCUSSION  B o n e m a r r o w toxicity is one o f the major l i m i t i n g factors i n the effective use o f radiochemotherapy i n cancer control, and dose escalation i n therapy is i n v a r i a b l y a c c o m p a n i e d b y life- threatening episodes o f m a r r o w aplasia and end- organ toxicities. Interruptions o f treatment regimens are frequent occurrences i n the c l i n i c and ultimately contribute to inefficient control o f cancer. B o n e m a r r o w t o x i c i t y is especially problematic i n allogeneic and autologous haematopoietic stem c e l l transplantations but it is, h o w e v e r perverse, a necessary part o f the therapy. H i g h dose radiochemotherapy is required d u r i n g the c o n d i t i o n i n g regimen to prepare the host f o r the acceptance o f donor (allogeneic)  in vivo.  haematopoietic cells and the destruction o f a significant n u m b e r o f neoplastic cells  L o n g - term defects i n m a r r o w reconstitutive capability as a result o f the intensive therapy are evident i n h u m a n patients and c o u l d be due to irreversible damage to the m a r r o w m i c r o e n v i r o n m e n t w h i c h remain o f host o r i g i n .  3 8 5  In fact, N o v i t z k y and M o h a m m e d  documented functional aberrations i n the haematopoietic progenitor populations o f patients 8 years after their t r a n s p l a n t a t i o n s .  394  In addition, the p r o b l e m o f iatrogenic damage to  n o r m a l haematopoietic cells is also encountered  ex vivo i n the p u r g i n g o f autologous  stem  c e l l harvests f r o m cancer patients. Overzealous p u r g i n g w i l l result i n effective destruction o f the contaminating neoplastic cells, at the expense o f n o r m a l haematopoietic cells i n the harvest. T h i s can result i n faulty engraftment manifested either b y a temporal delay i n achieving haematopoietic normalisation or an absence o f permanent e n g r a f t m e n t .  220  Haematopoietic stem c e l l protection attempts to selectively preserve normal haematopoietic cells f r o m c y t o t o x i c damage, w h i c h theoretically permits dose escalation o f therapy-to effect efficient destruction o f the neoplastic cells  in vivo or ex vivo. 210  A t the present m o m e n t ,  toxicities to other n o r m a l tissues such as the l i n i n g o f the gastrointestinal tract and hair f o l l i c u l a r cells are not obviated b y stem c e l l protection. H o w e v e r , improvements i n chemotherapeutic agents and treatment protocols i n addition to developments o f n o v e l protective c o m p o u n d s w i l l help to ameliorate the iatrogenic damage to n o r m a l tissues and alleviate organ- specific t o x i c i t y . F o r example, s o d i u m 2- mercaptoethanesulfonate ( M E S N A ) , i n c o n j u n c t i o n w i t h prophylactic hydration, is used to reduce the i n c i d e n c e o f acute haemorrhagic cystitis i n patients receiving c y c l o p h o s p h a m i d e t r e a t m e n t .  159  395  S t e m c e l l protection effected b y negative regulators o f haematopoiesis is a p r o m i s i n g area o f research. O f special significance are t w o l o w molecular- weight oligopeptide inhibitors, the pentapeptide p G l u - G l u - A s p - C y s - L y s ( p E E D C K ) and the tetrapeptide acetyl- N- Ser- A s p - L y s - P r o ( N - A c S D K P ) .  2 0 5  The acknowledged principal  m e c h a n i s m o f protection is the i n h i b i t i o n o f c e l l c y c l e entry into S- phase, where the c e l l becomes vulnerable to a plethora o f c y t o t o x i c a g e n t s .  367  A n o t h e r apparently unique feature  o f these inhibitors is in the ability to preferentially affect normal haematopoietic cells hence s p e c i f i c i t y in their protective a c t i o n .  2 0 4  -  3 6 5  A l r e a d y , several groups have demonstrated that  N - A c S D K P mediated stem c e l l protection in vivo i n m i c e as w e l l as m o n k e y s and d o g s .  205  -  387, 396  In this study, we s h o w e d that exposure o f freshly harvested D B A / 2 bone m a r r o w cells to 100 n M ( 1 0 " M ) N - A c S D K P f o r 1.5 h in serum free I M D M resulted i n the 7  i n h i b i t i o n o f S- phase entry o f C F U - G M progenitors, manifested b y resistance to cyclespecific toxicity o f ara- C . N - A c S D K P , h o w e v e r , d i d not alter the total n u m b e r o f C F U G M . Together, this demonstrated that i n h i b i t i o n o f S- phase entry was achieved without toxicity to the C F U - G M progenitors. S i g n i f i c a n t l y , the incubation p r o t o c o l was identical to the one used in the cytotoxicity experiments described i n chapter 5, suggesting a possible explanation for the observed photoprotection o f D B A / 2 C F U - G M . O u r results are in agreement w i t h those p u b l i s h e d b y W i e r e n g a and K o n i n g s .  3 9 7  U s i n g a slightly different  p r o t o c o l , they s h o w e d that i n c u b a t i o n w i t h 10~ to 10~ M o f N - A c S D K P f o r 8 h in the 7  12  presence o f 30 % foetal c a l f serum and 1 u M captopril resulted in protection o f C F U - G M f r o m the S- phase- specific t o x i n h y d r o x y u r e a . T h e authors rationalised, that due to r a n d o m distribution o f cells over the c e l l c y c l e , an incubation p e r i o d o f 8 h was necessary to realise optima] inhibitory effect on the C F U - G M progenitors; this in turn necessitated i n c l u s i o n o f serum i n the m e d i u m and the addition o f 1 u M captopril. G r i l l o n et al. s h o w e d that the bioactivity o f N - A c S D K P is s i g n i f i c a n t l y reduced by serum proteases, w h o s e proteolytic activities c o u l d be abrogated b y the addition o f captopril, a metalloproteinase i n h i b i t o r .  3 9 8  S i n c e we were able to achieve c y c l e i n h i b i t i o n w i t h an incubation p e r i o d o f 1.5 h, w e d i d not i n c l u d e serum or captopril in the tissue culture m e d i u m . I n a d d i t i o n , w e observed no adverse effects on C F U - G M numbers resulting f r o m our incubatory c o n d i t i o n up to 5 h. Other groups c o u l d not demonstrate N - A c S D K P - mediated i n h i b i t i o n o f the proliferative activity o f m u r i n e C F U - G M in vitro. > 3S0  399  W e , h o w e v e r , b e l i e v e that the discrepancies  160  c o u l d be due to the different experimental conditions as w e l l as the source o f bone m a r r o w cells. T h e observed photoprotection o f D B A / 2 C F U - G M progenitors c o u l d therefore be a result o f N - A c S D K P - mediated c e l l c y c l e i n h i b i t i o n . W e s h o w e d data supporting this hypothesis b y depleting the S- phase fraction f r o m harvested bone m a r r o w cells w i t h 5 0 u M o f ara- C f o l l o w e d by P D T . O v e r a l l , the absolute numbers o f day 7 C F U - G M i n the group that was pretreated w i t h ara- C were approximately 5 0 % o f the control g r o u p ; nevertheless, the relative percent s u r v i v a l in response to different doses o f B P D were significantly higher in the experimental group pretreated w i t h ara- C . C e l l s i n S- phase were susceptible to ara- C c y t o t o x i c i t y w h i c h resulted in the enrichment o f non S- phase cells in the survival fraction and the C F U - G M progenitors i n ara- C- enriched fraction were f o u n d to have higher tolerance to B P D - mediated P D T c y t o t o x i c i t y (figure 6.4). Studies have s h o w n that cells i n S and G / M phases are more susceptible to P D T than cells 2  that are i n the quiescent G</ G , p h a s e .  3 4 3  - °4 0  4 0 1  F u r t h e r m o r e , G a n t c h e v and colleagues  have documented that P D T c y t o t o x i c i t y f r o m l o w dose Photofrin® and light exposure resulted in preferential damage to cells in S- phase; whereas h i g h photosensitiser load resulted i n general cytotoxicity without cell c y c l e s e l e c t i v i t y .  402  O b o c h i et al. recently  demonstrated that activated murine splenic l y m p h o c y t e s take up more B P D than resting l y m p h o c y t e s , w h i c h subsequently translated to increased susceptibility to P D T .  2 8 5  In addition, previous studies i n this laboratory have s h o w n that p r i m a r y l e u k a e m i c cells and leukaemic c e l l lines take up more B P D than n o r m a l haematopoietic c e l l s .  2 9 3  Increased uptake o f photosensitiser b y proliferating cells c o u l d be related to c e l l v o l u m e and the surface expression o f l o w density l i p o p r o t e i n r e c e p t o r s .  2 5 7  -  2 5 9  -  4 0 3  W e therefore  proceeded to evaluate the uptake o f B P D b y . D B A / 2 bone marrow-.cells preincubatediwith 100 n M N - A c S D K P u s i n g t w o c o l o u r fluorescent activated c e l l sorting ( F A C S ) analysis. A rat m o n o c l o n a l antibody, R A M 3 4 , was used to label cells w h i c h express the m u r i n e equivalence o f the h u m a n haematopoietic stem and progenitor c e l l antigen C D 3 4 .  4 0 4  Morel  et al. isolated C D 3 4 - expressing murine haematopoietic cells f r o m f e m o r a l bone m a r r o w labelled w i t h R A M 3 4 w h i c h resulted in significant enrichments o f various types o f p r i m i t i v e haematopoietic c e l l s .  3 9 3  In vitro  incubation o f D B A / 2 bone m a r r o w cells w i t h 100  n M N - A c S D K P d i d not alter the proportion o f C D 3 4 c e l l s (16.83 + 0.43 % o f the peptide+  treated group vs 16.13 + 0.94 % o f m e d i u m control). T h i s was expected since the k n o w n  161  characteristics o f N - A c S D K P do not include the alterations o f the percentage o f C D 3 4 cells +  nor the surface expression of the C D 3 4 g l y c o p r o t e i n . In addition, it is d o u b t f u l whether the short incubatory p e r i o d o f 1.5 h has any effects on these parameters. U n f o r t u n a t e l y , w e also s h o w e d no significant difference in the level o f B P D mean fluorescence intensity ( M F I ) i n C D 3 4 c e l l s between the N - A c S D K P - treated cells (4.67 + 0.25) and the c o n t r o l +  cells (4.84 + 0.18). T h e absence o f demonstrable difference i n B P D uptake between the t w o groups, h o w e v e r , does not d i m i n i s h the s i g n i f i c a n c e o f our earlier f i n d i n g s . W e have s h o w n i n chapter 4 that uptake o f B P D is rapid and reaches an e q u i l i b r i u m q u i c k l y ; therefore, it is not surprising that B P D uptake or association was s i m i l a r between the t w o groups. In the experiments described by O b o c h i and colleagues, m u r i n e splenocytes were incubated w i t h 2.5 pg/ml o f c o n c a n a v a l i n A ( C o n A ) for 72 h w h i c h resulted i n blast c e l l f o r m a t i o n and increased expressions o f the activation markers IL- 2 R (5- f o l d x control) and C D 7 1 (2.67- f o l d x c o n t r o l ) .  285  B o h m e r and colleagues demonstrated a direct  correlation between c e l l size and photosensitiser u p t a k e .  2 5 9  C o n A was used to effect  m a x i m a l stimulation and to highlight the difference between resting and activated l y m p h o c y t e s in terms o f B P D uptake and susceptibility to B P D - mediated P D T . In our system, B P D uptake or association was evaluated between t w o populations o f C F U - G M progenitors w h i c h were m u c h closer i n their b i o p h y s i c a l and b i o c h e m i c a l characteristics. Furthermore, H u n t et al. have s h o w n discordance between B P D uptake and P D T susceptibility i n p u r i f i e d D B A / 2 peritoneal macrophages subjected to different forms o f c e l l activation.  405  S p e c i f i c a l l y , they reported the highest B P D uptake i n cells stimulated w i t h 10  ug/ml l i p o p o l y s a c c h a r i d e ( L P S ) ; interestingly, cells activated w i t h interferon- y (IFN- y, . 100 U/ml f o r 7 2 h) or m e d i u m control took u p equivalent levels o f the photosensitiser. 'Susceptibility to P D T , however, was highest i n the I F N - y stimulated cells f o l l o w e d by resting cells ( m e d i u m control) and lastly, L P S - stimulated cells. T h e authors postulated that the d i c h o t o m y between uptake and cytotoxicity c o u l d be due to a multitude o f factors i n c l u d i n g quenching of reactive o x y g e n species, enhancement o f P D T c y t o t o x i c i t y f r o m nitric o x i d e generated v i a I F N - y stimulation, and distinct cellular targets in the differentially stimulated cells. O f the last point, F A C S analysis does not distinguish between cells w i t h different intracellular localisations o f photosensitiser and therefore cells w i t h s i m i l a r B P D M F I s c o u l d have different intracellular distributions o f the photosensitiser. T h e group o f W i l s o n et al. at the Ontario C a n c e r Institute noticed s i m i l a r total cellular uptake of  162  Photofrin  by the P D T - resistant c e l l line RTF- 8 A and the parental l i n e REF- 1; they,  however, f o u n d significant reduction in the m i t o c h o n d r i a l a c c u m u l a t i o n o f the photosensitiser in the P D T resistant c e l l l i n e .  4 0 6  '  4 0 7  W e believe that the unique intracellular  m i l i e u d u r i n g the S- phase o f the c e l l c y c l e presents n o v e l targets f o r P D T . T h e expressions o f S- phase specific protein products such as c y c l i n D and the S- phase specific assembly o f the D N A replication c o m p l e x e s are such e x a m p l e s .  4 0 8  '  4 0 9  B e r g and M o a n , i n their  recent review article, postulated that microtubules are cycle- specific targets of P D T cytotoxicity  4 1 0  A n o t h e r possible m e c h a n i s m responsible for N - A c S D K P - mediated photoprotection is through the m o d u l a t i o n o f the cellular thiol p o o l , w h i c h c o u l d effect defence against oxidative damage. W e c o u l d not detect any difference in the intracellular concentrations o f glutathione ( G S H ) between N - A c S D K P - treated cells and control cells u s i n g the T i e t z e method. A n o t h e r assay technique u s i n g either the G S H - specific fluorescent probes m o n o c h l o r o b i m a n e or m o n o b r o m o b i m a n e in conjunction w i t h F A C S analysis c o u l d p r o v i d e a more sensitive m e t h o d o f single c e l l measurement o f G S H .  4 1 1  N - A c S D K P - mediated protection o f D B A / 2 bone m a r r o w progenitor cells f r o m P D T cytotoxicity is correlated w i t h the ability o f the peptide to prevent c e l l cycle progression. A r t i f i c i a l depletion o f S- phase cells w i t h ara- C resulted i n an enrichment o f cells w h i c h are significantly more resistant to P D T than their proliferating counterpart. Attempts to elucidate the mechanisms responsible for N - A c S D K P - mediated photoprotection i n c l u d e d the examination B P D uptake and G S H content i n cells after peptide incubation. H o w e v e r , neither o f the t w o parameters s h o w e d significant difference between the N - A c S D K P and the control group. W e believe that other c e l l cycle- specific factors such as the expression o f n o v e l proteins and assembly o f phase- specific protein complexes and therefore n o v e l targets m a y contribute to the observed heightened P D T cytotoxicity i n proliferating cells.  163  CHAPTER  7:  DISCUSSION  P u r g i n g o f autologous haematopoietic stem c e l l harvests w i t h light- activated drugs is g a i n i n g increasing exposure i n the c l i n i c a l setting as a result o f the e x p l o s i v e growth o f the f i e l d o f photodynamic therapy ( P D T ) and the need f o r new and effective p u r g i n g modalities. P u r g i n g is sometimes necessary i n autologous haematopoietic stem c e l l transplantations because o f the probable presence o f contaminating neoplastic cells i n the harvest, w h i c h can contribute to posttransplantation r e l a p s e s .  154  D e v e l o p m e n t o f the  second generation photosensitiser b e n z o p o r p h y r i n derivative m o n o a c i d ring- A ( B P D ) , w h i c h has many b i o p h y s i c a l and b i o c h e m i c a l characteristics c o n d u c i v e to effective P D T , has significantly i m p r o v e d the efficacy as w e l l as broadened the indications o f this treatment m o d a l i t y .  2 6 9  '  2 8 4  In a d d i t i o n , B P D - mediated P D T has been s h o w n to effect  significant yet selective destructions o f murine and h u m a n leukaemic cells  ex vivo  m u r i n e bone m a r r o w p u r g i n g m o d e l .  2 9 1  '  292  >  in vitro and i n an  2 9 4  In this thesis, augmentation o f B P D - mediated P D T c y t o t o x i c i t y was described using t w o independent approaches: c o m b i n a t i o n o f P D T w i t h d o x o r u b i c i n ( D o x ) and selective preprotection o f normal haematopoietic cells w i t h the stem c e l l inhibitory peptide N - A c S D K P . T h e findings f r o m the above experiments w i l l help to advance o u r understanding o f the apparent selective c y t o t o x i c i t y o f B P D has on l e u k a e m i c cells. In addition, factors w h i c h c o u l d influence  PDT COMBINATION  in vitro P D T  c y t o t o x i c i t y w i l l be discussed.  THERAPY  P D T combination therapy attempts to optimise photocytotoxicity through the interaction o f the light- activated photosensitiser w i t h another drug or treatment modality such as hyperthermia or i o n i s i n g radiation. H o w e v e r , one cannot discount the p o s s i b i l i t y that P D T also can endow the other partner o f the regimen w i t h enhanced cytotoxic activity. C l e a r l y , c o m b i n a t i o n therapy requires mutual cooperativity between the different participants w i t h i n the regimen and the effectiveness o f a particular regimen is predicated o n multiple factors. C h i e f a m o n g them are the type o f photosensitiser or drug and the f o r m o f  164  treatment m o d a l i t y selected, the temporal order o f drug administration i.e. simultaneous or sequenced, a n d the type o f m o d e l system and t u m o u r  (in vitro  vs  in vivo, m u r i n e  vs  human) used i n the experiment. Adequate delivery o f the chemotherapeutic agent or photosensitiser to the desired target organ remains one o f the c h i e f obstacle i n  in vivo drug  therapy. Parenteral and nonparenteral administration present t w o different routes o f drug d e l i v e r y ; i n addition, b i n d i n g to various serum protein components and hepatic m e t a b o l i s m further c o m p l i c a t e the delivery o f drug i n an o r g a n i s m . A s mentioned i n a previous chapter, bone m a r r o w p u r g i n g , b e i n g an constraints encountered  in vivo.  ex vivo (or in vitro) In a d d i t i o n , ex  procedure, bypasses m a n y o f the  vivo P D T  eliminates the p r o b l e m s o f  ensuring adequate light delivery to the appropriate organ. In studies performed f o r this thesis, the c o m b i n a t i o n i n v o l v i n g D o x treatment prior to B P D - mediated P D T (Dox-> P D T ) effected significant cytotoxicity on L 1 2 1 0 cells whereas the reverse sequence P D T - > D o x and simultaneous D o x / P D T treatment were not as efficacious as the first regimen (chapter 3). T h e enhancement i n c y t o t o x i c i t y was dependent o n the concentration o f B P D ; s p e c i f i c a l l y , enhanced k i l l i n g o f L 1 2 1 0 cells was achieved w i t h 5.0 but not 2.5 ng/ml B P D . A d d i t i o n a l l y , even though D o x - > P D T also effected heightened cytotoxicity against D B A / 2 haematopoietic progenitors, L l 2 1 0 cells were f o u n d to be m u c h more susceptible to the D o x - > P D T sequence than n o r m a l haematopoietic cells. Subsequent investigations revealed that the photophysical properties o f D o x and B P D are unique such that D o x d i d not interfere w i t h the photoactivation o f B P D at X  e x  above 6 0 0 n m (chapter 4). T h e presence o f D o x appeared to reduce the uptake o f  B P D whereas preincubation o f L 1 2 1 0 cells w i t h D o x d i d not affect the subsequent uptake o f B P D ( D o x / P D T ) . In addition, preincubation w i t h 2.5 u M or 5 u M D o x d i d not significantly reduCe cellular glutathione ( G S H ) . T h e factors behind the superiority o f the D o x - > P D T regimen are still not clear and m u l t i p l e mechanisms are responsible.  N-ACSDKP-  MEDIATED  PHOTOPROTECTION  FROM  BPD  Haematopoietic stem cell protection attempts to selectively protect normal b l o o d cell progenitors and precursors f r o m treatment- related c y t o t o x i c i t y . M a n y o f mediators o f  165  protection are the negative regulators o f haematopoiesis w h i c h effect negative i n h i b i t i o n on the g r o w t h o f haematopoietic c e l l s .  2 1 0  -  3 6 8  F o r some o f the negative mediators, s p e c i f i c a l l y  the peptides N - A c S D K P and p E E D C K , the protective m e c h a n i s m appeared to rely on the selective i n h i b i t i o n o f cell c y c l i n g in normal haematopoietic cells w h i c h therefore results in the sparing o f above population f r o m cycle- specific t o x i c i t y o f t r e a t m e n t .  367  C o u t t o n and  colleagues later demonstrated that N - A c S D K P also effected selective protection o f n o r m a l h u m a n C F U - G M progenitors f r o m Photofrin® - mediated P D T .  2 0 9  In chapter 4, preincubation o f D B A / 2 bone m a r r o w cells w i t h 100 n M o f the tetrapeptide N - A c S D K P resulted in statistically significant photoprotection f r o m B P D mediated P D T . T h e control peptides N - A c S D K E and S D K P , as w e l l as tissue culture m e d i u m c o n t r o l , d i d not alter sensitivity to P D T . T h e protective effect, however, was restricted to the late c o m m i t t e d progenitor population w h i c h was measured b y the C F U G M assay. C e l l s o f earlier lineages, as measured b y the L T B M C assay, were not protected b y N - A c S D K P . T h e L I 2 1 0 murine l e u k a e m i c c e l l line was not responsive to N - A c S D K P as w e l l as the t w o control peptides and therefore was not protected f r o m P D T . F i n d i n g s f r o m the murine experiments were extended into human cells. S p e c i f i c a l l y , n o r m a l h u m a n late haematopoietic progenitors were photoprotected by preincubation w i t h N - A c S D K P whereas the l e u k a e m i c cell line K 5 6 2 and primary l e u k a e m i c cells f r o m C M L patients were not protected by N - A c S D K P preincubation. T o further e x a m i n e the mechanisms responsible, I measured B P D uptake (or association) i n a subpopulation o f D B A / 2 bone m a r r o w cells responsible f o r short- term as w e l l as long- term haematopoiesis. Interestingly, B P D uptake was not statistically different between C D 3 4 - expressing cells f r o m the N - A c S D K P preincubated and the c o n t r o l sample. In addition, no significant difference in c e l l u l a r G S H between peptide- treated and c o n t r o l c e l l s was noted. U s i n g the traditional ara- C suicide assay, I s h o w e d that preincubation o f D B A / 2 cells w i t h 100 n M N - A c S D K P f o r 1.5 h d i d prevent the progression o f c e l l c y c l e into S- phase. Therefore, the observed photoprotective effect can be attributed to c e l l c y c l e i n h i b i t i o n . I was able to prove this b y depleting the harvested D B A / 2 bone m a r r o w sample o f S- phase cells w i t h 5 0 p M ara- C f o l l o w e d b y P D T . S- phase depletion resulted in cells w i t h higher survival to P D T w h i c h s h o w e d that susceptibility to P D T was related to c e l l c y c l e and N - A c S D K P effected its photoprotective effect v i a the i n h i b i t i o n o f c e l l c y c l i n g . H o w e v e r , the exact downstream  166  mechanisms are still not clear still w e were not able to show significant difference i n the uptake o f B P D and i n the amount o f the c e l l u l a r antioxidant G S H .  FACTORS AFFECTING  PDT  CYTOTOXICITY  Thiols, a - tocopherol, and haeme oxygenase C e l l u l a r G S H is correlated w i t h susceptibility to a variety o f chemotherapeutic agents, p o s s i b l y v i a detoxification o f reactive o x y g e n species ( R O I s ) and l i p i d peroxides.  2 4 5  -  4 1 2  -  4 1 3  In vitro  and  in vivo P D T  c y t o t o x i c i t y also appeared to be m o d u l a t e d  by the amount o f G S H and the degree o f its m e t a b o l i s m .  3 3 4  '  3 5 1  A n o t h e r important c e l l u l a r  antioxidant is a- tocopherol, a f o r m o f v i t a m i n E. W e l l s and colleagues have demonstrated a correlation between a- tocopherol l e v e l and resistance to D o x  4 1 4  G o m e r and colleagues  initially described the i n d u c t i o n o f haeme oxygenase 1 ( H O I ) transcription after Photofrin® - mediated P D T  4 1 5  Lin  etal  recently described the induction o f H O I expression and P D T  resistance ( M C 5 4 0 ) after prolonged incubation o f L 1 2 1 0 cells w i t h h a e m i n (ferriprotoporphyrin I X ) ; the role o f H O I i n the i n d u c t i o n o f P D T resistance is not clear, however.  4 1 6  N F - K B and its role in P D T cytotoxicity A variety o f s t i m u l i , i n c l u d i n g c y c l o h e x i m i d e , double- stranded R N A , c a l c i u m ionophore, T N F - a , active p h o r b o l ester, interleukin- 1, l i p o p o l y s a c c h a r i d e and l e c t i n , can mediate the i n d u c t i o n o f the transcriptional factor N F - k a p p a B (NF- K p )  4 1 7  In a d d i t i o n ,  B o l a n d et al. s h o w e d the anthracycline d a u n o r u b i c i n also activates N F - K p i n the H L - 6 0  167  p r o m y e l o c y t i c and Jurkat T l y m p h o m a c e l l l i n e s .  4 1 8  Activation of N F - K p involves  separation f r o m its c y t o p l a s m i c b i n d i n g partner, I K P , f o l l o w e d b y nuclear translocation the i n d u c t i o n o f KP- dependent gene e x p r e s s i o n  4 1 9  R y t e r and G o m e r i n i t i a l l y reported that  Photofrin- mediated P D T causes N F - K P activation i n the L l 2 1 0 m u r i n e l e u k a e m i c c e l l line.  2 6 4  . T h e i r observation was not s u r p r i s i n g i n light o f f i n d i n g s that reactive o x y g e n  species ( R O I s ) are essential as s i g n a l l i n g intermediates in the activation o f N F - K p b y a variety o f s t i m u l i .  4 2 0  "  4 2 2  S c h r e c k and colleagues also demonstrated that the radical  scavengers N - A c e t y l - cysteine ( N A C ) and the p y r r o l i d o n e derivative o f dithiocarbamate ( P D T C ) effectively i n h i b i t e d N F - K p activation upon cellular s t i m u l a t i o n . R O I s are also important intermediates m e d i a t i n g the activation o f N F - K P in response to stimulation o f the T c e l l surface receptor C D 2 8  4 2 3  T h r o u g h their studies on h u m a n B c e l l l i n e s , S c h i e v e n et  al. have i m p l i c a t e d tyrosine kinase in the R O I - mediated activation o f N F - K P  4 2 4  R e c e n t l y , several reports have demonstrated that N F - K p plays an important role i n determining cellular response to a variety o f c y t o t o x i c agents. S i g n i f i c a n t l y , W a n g and colleagues f o u n d that activation o f the transcription factor was paramount in the cytoprotection o f the h u m a n s a r c o m a c e l l line H T 1 0 8 0 f r o m T N F - a , i o n i s i n g radiation, and the d a u n o r u b i c i n . NF- K P in  3 2 7  P a r a d o x i c a l l y , several papers have reported c o n f l i c t i n g roles o f  in vitro e x c i t o t o x i c n e u r o d e s t r u c t i o n .  328  W i t h respect to the role o f N F - K p i n  determining cellular response to P D T , A n d e r s o n et al. i n i t i a l l y reported that the presence o f 0 . 1 - 1 0 m M s a l i c y c l i c a c i d ( S A ) d u r i n g M C 5 4 0 - mediated P D T s i g n i f i c a n t l y enhanced c y t o t o x i c i t y to L 1 2 1 0 and K 5 6 2 c e l l lines  4 2 5  In a d d i t i o n , S A and s o d i u m salicyclate ( A S A  or aspirin) are k n o w n i n h i b i t o r s o f N F - K P a c t i v a t i o n .  4 2 6  -  4 2 7  T h e r e f o r e , one can assume  that the m o d u l a t i o n o f N F - K P activation can also alter c e l l u l a r response to P D T . Unfortunately, TrauJ et al. later s h o w e d that S A i n d u c e d nonselective enhancement o f P D T k i l l i n g o f L 1 2 1 0 cells and n o r m a l haematopoietic stem cells but m o r e importantly, they f o u n d that S A displaces M C 5 4 0 f r o m serum a l b u m i n w h i c h resulted in the increase o f free photosensitiser available f o r b i n d i n g .  4 2 8  H o w e v e r , the question o f the role that N F - K P  plays in P D T remain opened. W e used the proteasome i n h i b i t o r M G 1 3 2 ( c a r b o b e n z o x y l l e u c i n y l - l e u c i n y l - leucinal), w h i c h inhibits the proteasome degradation o f I K P and hence nuclear translocation o f N F - K P .  3 2 7  P r e l i m i n a r y results s h o w e d that 1 h p r e i n c u b a t i o n o f  Jurkat cells w i t h 4 0 u M M G 1 3 2 resulted i n moderate yet consistent enhancement o f B P D mediated P D T c y t o t o x i c i t y (data not shown). I f o u n d that the same experimental p r o t o c o l  168  also potentiated d o x o r u b i c i n k i l l i n g in Jurkat cells. Interestingly, M G 1 3 2 preincubation protected the human C M L c e l l line K 5 6 2 f r o m B P D - mediated P D T . I a m in the process o f e x a m i n i n g the amount o f IK(3 in c e l l lysates f r o m the above two c e l l lines and w i l l continue to investigate the role o f N F - K(3 in P D T - mediated c e l l k i l l i n g . I also tried to introduce M G 1 3 2 into the P D T c o m b i n a t i o n experiments described i n chapter 3. S i n c e W a n g et al. reported that M G 1 3 2 protected the c e l l line H T 1 0 8 0 f r o m daunorubicin c y t o t o x i c i t y , I was interested in whether the peptide aldehyde c o u l d affect the k i l l i n g o f L 1 2 1 0 cells i n the D o x - > P D T c o m b i n a t i o n .  3 2 7  O b l i q u e l y , I was curious o f the  role o f N F - K(3 activation during the D o x pretreatment phase o f D o x - > P D T , w h i c h resulted in significant enhancement o f cytotoxicity o f the regimen. Unfortunately, the L I 2 1 0 c e l l line was f o u n d to be extremely sensitive to M G 1 3 2 and prevented the incorporation o f the inhibitor into the e xi s ti n g P D T experiments i n L I 2 1 0 .  Summary A s l o n g as there is a need for the therapeutic transfusions o f autologous haematopoietic stem cells in the o n c o l o g i c setting, there is a need f o r p u r g i n g . T h e overall e f f i c i e n c y o f the different established p u r g i n g modalities is s i m i l a r to each other. Therefore, enhancements to a proven m o d a l i t y , such as B P D - mediated P D T p u r g i n g , s h o u l d increase its attractiveness to the c l i n i c i a n . O n the other h a n d , new and e x c i t i n g technologies are e m e r g i n g and m a y offer a quantum leap i n p u r g i n g p e r f o r m a n c e .  2 1 3  '  4 2 9  T h e u n d e r l y i n g theme o f this project is the i m p r o v e m e n t o f P D T cytotoxicity v i a t w o independent approaches. D o x treatment p r i o r to P D T (Dox-> P D T ) resulted in a significant improvement in the k i l l i n g o f the l e u k a e m i c c e l l line L I 2 1 0 but not o f D B A / 2 haematopoietic progenitor cells. T h e reverse sequence ( P D T - > D o x ) and simultaneous treatment ( D o x / P D T ) only effected moderate i m p r o v e m e n t in k i l l i n g . Therefore, one must be cautious in c o m b i n i n g P D T w i t h other drugs or treatment modalities i n the c l i n i c . S p e c i f i c a l l y , the sequence o f the c o m b i n a t i o n c o u l d significantly affect the outcome o f the treatment. T h e second approach i n v o l v e d the selective protection o f n o r m a l haematopoietic cells w i t h N - A c S D K P prior to P D T . M u r i n e and h u m a n l e u k a e m i c cells d i d not respond to  169  the tetrapeptide and therefore were not afforded subsequent photoprotection. T h e above findings s h o u l d also aid i n the c o n t i n u i n g understanding o f the m e c h a n i s m s o f P D T cytotoxicity.  170  REFERENCES 1. F r e i r e i c h E J , L e m a k N A . M i l e s t o n e s in l e u k e m i a research and therapy. B a l t i m o r e : T h e J o h n H o p k i n s U n i v e r s i t y Press, 1991. 2. O s i e r W . T h e P r i n c i p l e s and Practice o f M e d i c i n e . N e w Y o r k : D . A p p l e t o n & C o m p a n y , 1909. 3. N o w e l l P C , H u n g e r f o r d D A . A minute c h r o m o s o m e i n h u m a n c h r o n i c granulocytic l e u k e m i a (abstract). S c i e n c e 1960; 132:1497-1501. 4. 149.  Rabbitts T H . C h r o m o s o m a l translocations in h u m a n cancer. Nature 1994; 372:143-  5. W o g l o m W H . G e n e r a l r e v i e w o f cancer therapy. In: M o u l t o n F R , ed. A p p r o a c h e s to tumor chemotherapy. W a s h i n g t o n : A m e r i c a n A s s o c i a t i o n for the A d v a n c e m e n t o f S c i e n c e , 1947:1-10. 6. H a d d o w A , T i m m i s G M . M y l e r a n i n chronic m y e l o i d l e u k e m i a . L a n c e t 1953; 1:208-213. 7. Farber S. S o m e observations on the effect o f f o l i c a c i d antagonists o n acute l e u k e m i a and other f o r m s o f incurable cancer. B l o o d 1949; 4:160-167. 8. Z u b r o d C G . H i s t o r i c milestones i n curative chemotherapy. S e m i n O n c o l 1979; 6:490-505. 9. H i t c h i n g s G H , E l i o n G B . T h e chemistry and b i o c h e m i s t r y o f purine analogs. A n n N Y A c a d S c i 1954; 60:195-199. 10. F r e i r e i c h E J , B o d e y G P , H a r r i s J E , et a l . T h e r a p y f o r acute g r a n u l o c y t i c l e u k e m i a . C a n c e r R e s 1967; 27:2573-2577. 11. C a r b o n e P P , B o n o V , F r e i E, III, et al. C l i n i c a l studies w i t h v i n c r i s t i n e . B l o o d 1963;21:640-647. 12. S e l a w r y O S , F r e i E, III. P r o l o n g a t i o n o f r e m i s s i o n i n acute l y m p h o c y t i c l e u k e m i a by alteration i n dose schedule and route o f administration o f methotrexate (abstract). C l i n Res 1964; 12:231. ; 13. E l i o n G B , S i n g e r S, H i t c h i n g s G H . A n t a g o n i s t s o f n u c l e i c a c i d derivatives: synergism in c o m b i n a t i o n s o f b i o c h e m i c a l l y related antimetabolites. J*. B i o l . C h e m 1954; 208:477-488. 14. Farber S, T o c h R, Sears E M , et a l . A d v a n c e s i n chemotherapy o f cancer i n m a n . A d v . Cancer Res 1956;4:1-71. 15. Redpath J L . M e c h a n i s m s in c o m b i n a t i o n therapy: i s o b o l o g r a m analysis and sequencing. Int. J . R a d i a t . B i o l 1980; 38:355-356. 16. C h a n g T T , G u l a t i S, C h o u T C , et a l . C o m p a r a t i v e c y t o t o x i c i t y o f various drug combinations for h u m a n l e u k e m i c cells and n o r m a l hematopoietic precursors. C a n c e r Res 1987;47:119-122.  171  17. Z u b r o d C G . O r i g i n s and development o f chemotherapy research at the N a t i o n a l C a n c e r Institute. C a n c e r Treat. R e p 1984; 68:9-19. 18. M e n i t o v e JE. Platelet transfusions for a l l o i m m u n i z e d patients. C l i n c O n c o l 1983; 2:587-609. 19. S t r u m i a M M . E f f e c t of leukocyte cream injections i n the treatment o f neutropenias. A m . J . M e d . S c i 1934; 187:527-544. 20. M c C r e d i e K B , F r e i r e i c h E J . B l o o d c o m p o n e n t therapy. In: H o l l a n d JF, F r e i E, eds. C a n c e r M e d i c i n e . P h i l a d e l p h i a : L e a & F e b i g e r , 1973:1115-1129.  III,  21. V a l l e j o s C , M c C r e d i e K B , B o d e y G P , et a l . W h i t e b l o o d c e l l transfusions f o r control o f infections in neutropenic patients. T r a n s f u s i o n 1975; 15:28-33. 22. W i n s t o n D J , H o W G , H o w e l l C L , et a l . C y t o m e g a l o v i r u s infections associated w i t h l e u k o c y t e transfusions. A n n . Intern. M e d 1 9 8 0 ; 93:671-675. 23. B o d e y G P . Infections i n patients w i t h cancer. In: H o l l a n d JF, F r e i E, III, eds. C a n c e r M e d i c i n e . P h i l a d e l p h i a : L e a & F e b i g e r , 1982:1339-1372. 24. B a k e r R D . L e u k o p e n i a and therapy in l e u k e m i a as factors predisposing to fatal m y c o s e s : m u c o r m y c o s i s , a s p e r g i l l o s i s , and c r y p t o c o c c o s i s . A m . J . C l i n . P a t h o l 1 9 6 2 ; 37:358-363. 25. B o d e y G P . F u n g a l infection and fever o f u n k n o w n o r i g i n i n neutropenic patients. A m . J . M e d 1986; 8 0 ( S u p p l 5 C ) : 112-119. 26. B o r t i n M M . A c o m p e n d i u m o f reported h u m a n bone m a r r o w transplants. Transplantation 1 9 7 0 ; 9 : 5 7 1 - 5 8 7 . 27. Fabricious- M o e l l e r J . E x p e r i m e n t a l studies o f hemorrhagic diathesis f r o m X - ray sickness. C o p e n h a g e n : L e v i n & M u n k s g a a r d , 1922. 28. Jacobson L O , S i m m o n s E L , M a r k s E K , et a l . R e c o v e r y f r o m radiation injury. S c i e n c e 1 9 5 1 ; 113:510-511. 29. M o r r i s o n M , S a m w i c k A A . Intramedullary (sternal) transfusion o f h u m a n bone m a r r o w . J A M A 1940; 115:1708-1711. 30. F o r d C E , H a m e r t o n J L , B a r n e s D W H , et a l . C y t o l o g i c a l identification o f radiation c h i m e r a s . Nature 1956; 177;239-247. 31. L o r e n z E, U p h o f f D , R e i d T R , et a l . M o d i f i c a t i o n o f irradiation injury i n m i c e and g u i n e a pigs b y bone m a r r o w injections. J . N a t l . C a n c e r Inst 1 9 5 1 ; 12:197-201. 32. Santos G W , C o l e L J . E f f e c t o f d o n o r and host l y m p h o i d and m y e l o i d tissue injections in lethally X - irradiated m i c e treated w i t h rat bone m a r r o w . J . N a t l . C a n c e r Inst 1958; 21:279-293. 33. B i l l i n g h a m R E , B r e n t L. A s i m p l e m e t h o d f o r i n d u c i n g tolerance o f s k i n grafts in m i c e . Transplantation B u l l e t i n 1957; 4:67-71. 34. D u p o n t B. H L A factors and bone m a r r o w grafting. In: B u r c h e n a l J H , Oettgen H F , eds. C a n c e r : achievements, challenges and prospects f o r the 1980s. N e w Y o r k : G r u n e and Stratton, 1980:683-693.  172  35. A m o s B, W a r d F E , Z m i j e w s k i C M , et a l . Graft donor selection based u p o n single locus (haplotype) analysis w i t h i n f a m i l i e s . Transplantation 1968; 6:524-534. 36. van R o o d JJ, E i j s v o o g e l V P . H L - A identical phenotypes and genotypes i n unrelated i n d i v i d u a l s . L a n c e t 1970; 1:698-700. 37. T h o m a s E D , L o c h t e H L , Jr , L u W C , et a l . Intravenous i n f u s i o n o f bone m a r r o w i n patients r e c e i v i n g radiation and chemotherapy. N e w E n g l . J . M e d 1957; 257:491-496. 38. Gatti R A , M e u w i s s e n H J , A l l e n H D , et a l . I m m u n o l o g i c a l reconstitution o f sexl i n k e d l y m p h o p e n i c i m m u n o l o g i c a l deficiency. Lancet 1968; i i : 1366-1369. 39. H o w a r d M R . Selecting donrs f o r bone m a r r o w transplantation. I n : W h i t t a k e r J A , ed. L e u k a e m i a . O x f o r d : B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , 1992:568-593. 40. P o w l e s R L , C l i n k H M , Spence D . C y c l o s p o r i n A to prevent graft- versus- host disease. L a n c e t 1980; i:327-329. 41. Storb R, D e e g H J , W h i t e h e a d J , et a l . Methotrexate and c y c l o s p o r i n c o m p a r e d w i t h c y c l o s p o r i n alone for p r o p h y l a x i s o f acute graft versus host disease after m a r r o w transplantation f o r l e u k e m i a . N e w E n g l . J . M e d 1986; 314:728-735. 42. T u t s c h k a PJ. M a r r o w ablation: the need for space, i m m u n e suppression and malignant c e l l eradication. In: A t k i n s o n K , ed. C l i n i c a l bone m a r r o w transplantation: a reference textbook. C a m b r i d g e : C a m b r i d g e U n i v e r s i t y Press, 1994:13-18. 43. G l u c k m a n E, B r o x m e y e r H E , A u e r b a c h A D , et al. H e m a t o p i e t i c reconstitution i n a patient w i t h Fanconi's a n e m i a b y means o f u m b i l i c a l - c o r d b l o o d f r o m an H L A - i d e n t i c a l s i b l i n g . N e w E n g l . J . M e d 1989; 321:1174-1178. 44. T h o m a s E D , B u c k n e r C D , B a n a j i M , et a l . T o t a l b o d y irradiation i n preparation for m a r r o w engraftment. Transplantation Proceedings 1976; 8:591-593. 45. Santos G W , Haghshenass M . C l o n i n g o f syngeneic hematopoietic cells i n the spleens o f m i c e and rats pre- treated w i t h c y t o t o x i c drugs. B l o o d 1968; 32:629-637. 46. H o b b s J R . T h e Westminster bone m a r r o w transplantation team: bone m a r r o w transplantation f o r inborn errors. L a n c e t 1981; ii:735-739. 47. B a c h H H . Bone- m a r r o w transplantation i n a patient w i t h the W i s k o t t - A l d r i c h syndrome. L a n c e t 1968; ii:1364r!366. 48. T h o m a s E D , B u c k n e r C D , R u d o l p h R H , et a l . A l l o g e n e i c m a r r o w grafting f o r hematologic m a l i g n a n c y u s i n g H L - A matched donor- recipient s i b l i n g pairs. B l o o d 1971. Petridou E, T r i c h o p o u l o s D , D e s s y p r i s N , et al. Infant l e u k a e m i a after exposure to radiation f r o m C h e r n o b y l . Nature 1996; 382:352-353.  49.  50.  in utero  D o l l R, E v a n s H J , D a r b y S C . Paternal exposure not to b l a m e . Nature 1994;  367:678-80.  51. F o r d A M , R i d g e S A , C a b r e r a M E , et a l . In utero rearrangements i n the trithoraxrelated oncogene i n infant leukaemias. Nature 1993; 363:358-60. 52.  1051.  Greaves M . A natural history for pediatric acute l e u k e m i a . B l o o d 1993; 82:1043-  173  53. C a m p a n a D , P u i C H . Detection o f m i n i m a l residual disease in acute l e u k e m i a : m e t h o d o l o g i c advances and c l i n i c a l significance. B l o o d 1995; 85:1416-1434. 54. H a r r i s C C , H o l l s t e i n M . C l i n i c a l i m p l i c a t i o n o f the p53 tumor-suppressor gene. N e w E n g l . J . M e d 1993; 329:1318-1327. 55.  C l i n e M J . T h e m o l e c u l a r basis o f l e u k e m i a . N e w E n g l . J . M e d 1994; 330:328-336.  56.  B o r m a n S. C o m b i n a t o r i a l chemistry. C & E N 1996:28-54.  57. S e r v i c e R F . C o m b i n a t o r i a l chemistry hits the drug market. S c i e n c e 1996; 272:1266-1268. 58. M e y d a n N , Grunberger T , D a d i H , et a l . I n h i b i t i o n o f acute l y m p h o b l a s t i c l e u k a e m i a b y a Jak-2 inhibitor. Nature 1996; 379:645-8. 59. D r u k e r B J , T a m u r a S, B u c h d u n g e r E, et a l . E f f e c t s o f a selective i n h i b i t o r o f the A b l tyrosine kinase on the growth o f B c r - A b l p o s i t i v e cells. Nature M e d i c i n e 1996; 2:561566. 60. C h e s o n B D . T h e purine analogs- A therapeutic beauty contest. J C l i n O n c o l 1992; 10:868-871. 61. W a r r e l l R P , Jr., de T h e H , W Z Y , et a l . M e d i c a l Progress: A c u t e P r o m y e l o c y t i c L e u k e m i a . N e w E n g l J M e d 1993; 329:177-189. 62. Sachs L. R e g u l a t i o n o f membrane changes, differentiation, and m a l i g n a n c y i n carcinogenesis. H a r v e y Lectures 1974; 68:1-35. 63. Sachs L. T h e control o f hematopoiesis and l e u k e m i a : F r o m basic b i o l o g y to the c l i n i c . P r o c . N a t l . A c a d . S c i U S A 1996; 9 3 : 4 7 4 2 - 4 7 4 9 . 64. B r e i t m a n T R , S e l o n i c k S E , C o l l i n s SJ. Induction o f differentiation o f the h u m a n p r o m y e l o c y t i c l e u k e m i a c e l l line (HL- 60) b y retinoic a c i d . P r o c . N a t l . A c a d . S c i . U S A 1980; 7 7 : 2 9 3 6 - 2 9 4 0 . 65. C h e n G Q , J Z h u J , S h i X G , et a l . In v i t r o studies on c e l l u l a r and m o l e c u l a r mechanisms o f arsenic trioxide ( A s 2 0 3 ) in the treatment o f acute p r o m y e l o c y t i c l e u k e m i a : A s 2 0 3 induces N B 4 c e l l apoptosis w i t h d o w n r e g u l a t i o n o f B c l - 2 expression and m o d u l a t i o n o f P M L - R A R a l p h a / P M L proteins. B l o o d 1996; 88:1052-1061. 66. "Hanahan D , F o l k m a n J . Patterns and e m e r g i n g m e c h a n i s m s o f the angiogenic s w i t c h d u r i n g tumorigenesis. C e l l 1996; 86:353-364. 67. B i k f a l v i A , H a n Z C . A n g i o g e n i c factors are hematopoietic growth factors and v i c e versa. L e u k e m i a 1994; 8:523-529. 68. Perezatayde A R , S a l l a n S E , T e d r o w U , et al. S p e c t r u m o f t u m o r angiogenesis i n the bone m a r r o w o f c h i l d r e n w i t h acute l y m p h o b l a s t i c l e u k e m i a . A m . J P a t h o l o g y 1997; 150:815-821. 69. V a l l e r a D A . I m m u n o t o x i n s for ex v i v o bone m a r r o w p u r g i n g in h u m a n bone m a r r o w transplantation. In: F r a n k e l A E , ed. I m m u n o t o x i n s : K l u w e r A c a d e m i c P u b l i s h e r s , 1988:515-535.  174  70.  V a l l e r a D A . I m m u n o t o x i n s : w i l l their c l i n i c a l p r o m i s e be f u l f i l l e d ? B l o o d 1994;  83:309-317.  71. K a m i n s k i M S , Z a s a d n y K R , F r a n c i s IR, et a l . Iodine-131 - anti- B l radioimmunotherapy for B- c e l l l y m p h o m a . J C l i n O n c o l 1996; 14:1974-1981. 72. U c k u n F M , E v a n s W E , F o r s y t h C J , et a l . B i o t h e r a p y o f B- c e l l precursor l e u k e m i a by targeting genistein to C D 1 9 - associated tyrosine kinases. S c i e n c e 1995; 267:886-891. 73. M a t t h e w s D C , B a d g e r C C , F i s h e r D R , et al. Selective radiation o f h e m a t o l y m p h o i d tissue delivered by a n t i - C D 4 5 antibody. C a n c e r Research 1992; 52:1228-34. 74. M a t t h e w s D C , A p p e l b a u m F R , E a r y JF, et al. D e v e l o p m e n t o f a m a r r o w transplant regimen for acute l e u k e m i a using targeted hematopoietic irradiation delivered by 1311labeled anti-CD45 antibody, c o m b i n e d w i t h c y c l o p h o s p h a m i d e and total b o d y irradiation. B l o o d 1995; 85:1122-31. 75. Pietersz G A , W e n j u n L, Sutton V R , et a l . In vitro and i n v i v o antitumor activity o f a c h i m e r i c anti- C D 1 9 antibody. C a n c e r I m m u n o l o g y , Immunotherapy 1995; 41:53-60. 76. F i s c h e r A . T h e use o f m o n o c l o n a l antibodies i n allogeneic bone m a r r o w transplantation. B r . J H a e m a t o l 1 9 9 3 ; 83:531-534. 77. P u i C H , B e h m F G , C r i s t W M . C l i n i c a l and b i o l o g i c relevance o f i m m u n o l o g i c marker studies i n c h i l d h o o d acute l y m p h o b l a s t i c l e u k e m i a . B l o o d 1 9 9 3 ; 82:343-362. 78. R i b e i r o R C , O l i v e i r a M S P , F a i r c l o u g h D , et al. A c u t e megakaryoblastic l e u k e m i a in children and adolescents: a retrospective analysis o f 2 4 cases. L e u k . L y m p h o m a 1993; 10:299-306. 79. W i e r s m a S R , Ortega J , S o b e l E, et al. C l i n i c a l importance o f m y e l o i d - antigen expression i n acute l y m p h o b l a s t i c l e u k e m i a o f c h i l d h o o d . N e w E n g l . J M e d 1 9 9 1 ; 324:800-808. 80. C a m p a n a D. A p p l i c a t i o n sof cytometry to study acute l e u k e m i a : In vitro determination o f drug sensitivity and detection o f m i n i m a l residual disease. C y t o m e t r y 1994; 18:68-74. 81. B a k e r M A , T a u b R N , Carter W H . Immunotherapy for r e m i s s i o n maintenance in acute m y e l o b l a s t s l e u k e m i a . C a n c e r I m m u n o l o g y , Immunotherapy 1982; 13:85-8. . 82. F b o n K A , S m a i l e y R V , R i g g s C W , et a l . T h e role o f i m m u n o t h e r a p y in acute' myelogenous l e u k e m i a . A r c h i v e s o f Internal M e d i c i n e 1983; 143:1726-31. 83. F a v r e R, Sebahoun G , B a g a r r y - L i e g e y D , et al. M a i n t e n a n c e c h e m o i m m u n o t h e r a p y o f n o n l y m p h o b l a s t i c acute leukemias. Recent Results i n C a n c e r Research 1982; 80:56-63. 84. Laursen M L . Immunotherapy i n acute myelogeneous l e u k e m i a ( A M L ) — a tool f o r maintaining remission? M e d i c a l Hypotheses 1988; 26:221-5. 85. R o s e n b e r g S A , L o t z e M T . C a n c e r immunotherapy u s i n g interleukin-2 and interleukin-2-activated l y m p h o c y t e s . A n n u a l R e v i e w o f I m m u n o l o g y 1986; 4:681-709. 86. R o s e n b e r g S A , L o t z e M T , Y a n g J C , et a l . E x p e r i e n c e w i t h the use o f high-dose interleukin-2 in the treatment o f 652 cancer patients. A n n a l s o f Surgery 1989; 210:474-84; d i s c u s s i o n 484-5.  175  87. Pais R C , I n g r i m N B , G a r c i a M L , et a l . P h a r m a c o k i n e t i c s o f recombinant interleukin-2 i n c h i l d r e n w i t h malignancies: a Pediatric O n c o l o g y G r o u p study. Journal o f B i o l o g i c a l Response M o d i f i e r s 1990; 9:517-21. 88. H e s l o p H E , D u n c o m b e A S , Reittie J E , et a l . Interleukin 2 i n f u s i o n induces haemopoietic growth factors and m o d i f i e s m a r r o w regeneration after chemotherapy or autologous m a r r o w transplantation. B r i t i s h Journal o f H a e m a t o l o g y 1 9 9 1 ; 77:237-44. 89. R o b i n s o n N , B e n y u n e s M C , F e f e r A . Interleukin- 2 after autologous stem c e l l transplantation for hematologic m a l i g n a n c y : a phase I/n study. B o n e m a r r o w transplantation 1997; 19:435-442. 90. B o c c h i a M , W e n t w o r t h P A , S o u t h w o o d S, et al. S p e c i f i c b i n d i n g o f l e u k e m i a oncogene f u s i o n protein peptides to H L A class I m o l e c u l e s . B l o o d 1 9 9 5 ; 85:2680-4. 91. Barrett J , G u i m a r a e s A , C u l l i s J , et al. I m m u n o l o g i c a l characterization o f the tumor-specific bcr/abl j u n c t i o n o f P h i l a d e l p h i a c h r o m o s o m e positive c h r o n i c m y e l o i d l e u k e m i a . S t e m C e l l s 1 9 9 3 ; 11:104-8. 92. M c C u n e J M , K a n e s h i m a H , L i e b e r m a n M , et al. T h e scid-hu m o u s e : current status and potential applications. Current T o p i c s in M i c r o b i o l o g y & I m m u n o l o g y 1989; 152:18393. 93. M c C u n e J M . S C I D m i c e as i m m u n e system m o d e l s . Current O p i n i o n i n I m m u n o l o g y 1 9 9 1 ; 3:224-8. 94. D i c k JE, S i r a r d C , P f l u m i o F, et a l . M u r i n e m o d e l s o f n o r m a l and neoplastic h u m a n haematopoiesis. C a n c e r S u r v e y s 1992; 15:161-81. 95. L a p i d o t T , S i r a r d C , V o r m o o r J , et a l . A c e l l initiating h u m a n acute m y e l o i d l e u k a e m i a after transplantation into S C I D m i c e . Nature 1994; 367:645-8. 96. H e i s t e r k a m p N , Jenster G , ten H o e v e J , et a l . A c u t e l e u k a e m i a i n bcr/abl m i c e . Nature 1990; 344:251-253. 97.  transgenic  D a l e y G Q , V a n Etten R A , B a l t i m o r e D . Induction o f c h r o n i c myelogenous  l e u k e m i a in m i c e by the P 2 1 0 ^ 247:824-830.  c r /  ' ^ G e n e o f the P h i l a d e l p h i a c h r o m o s o m e . Science 1990; a  98. V o n c k e n J W , K a a r t i n e n V , Pattengale P K , et al. B C R / A B L P 2 1 0 and P 1 9 0 cause distinct l e u k e m i a in transgenic m i c e . B l o o d 1995; 86:4603-461 J. 99.  P e n n i n g e r J M , W e n T , T i m m s E, et a l . Spontaneous resistance to acute T- c e l l  leukaemias i n T C R V y l . l J y 4 C y 4 transgenic m i c e . N a t u r e 1995; 375:241-244. 100. A m a k a w a R, H a k e m A , K u n d i g T M , et a l . Impaired negative selection o f T cells i n H o d g k i n ' s Disease antigen C D 3 0 - deficient m i c e . C e l l 1996; 84:551-562. 101. H o l t s c h k e T , L o h l e r J , K a n n o Y , et a l . I m m u n o d e f i c i e n c y and c h r o n i c myelogenous leukemia-like syndrome in m i c e w i t h a targeted mutation o f the I C S B P gene. Cell 1996;87:307-17. 102. S z c z y l i k C , S k o r s k i T , N . C N , et al. S e l e c t i v e i n h i b i t i o n o f l e u k e m i a c e l l proliferation b y B C R - A B L antisense o l i g o n u c l e o t i d e s . S c i e n c e 1 9 9 1 ; 253:562-565.  176  103. M a r t i a t P, L e w a l l e P, Taj A S , et a l . R e t r o v i r a l l y transduced antisense sequences stably suppress P 2 1 0 B C R - A B L expression and i n h i b i t the proliferation o f B C R / A B L c o n t a i n i n g c e l l lines. B l o o d 1 9 9 3 ; 81:502-509. 104. L a n g e W , D a s k a l a k i s M , F i n k e J , et al. C o n i p a r i s o n o f different r i b o z y m e s for efficient and specific cleavage o f B C R / A B L related m R N A s . F E B S Letters 1994; 338:1758. 105. W a g n e r R W . T h e state o f the art in antisense research. Nature M e d i c i n e 1995; 1:1116-1118. 106. K m i e c E B . G e n o m i c targeting and genetic conversion in cancer therapy. Seminars i n O n c o l o g y 1996; 23:188-193. 107. H a n s e n R M , B o r d e n E C . C u r r e n t status o f interferons i n the treatment o f cancer. O n c o l o g y 1992; 6:19-24; d i s c u s s i o n 2 6 , 2 9 . 108. W e t z l e r M , Kantarjian H , K u r z r o c k R, et a l . Interferon-alpha therapy f o r c h r o n i c m y e l o g e n o u s l e u k e m i a . A m e r i c a n Journal o f M e d i c i n e 1995; 99:402-11. 109. L o w e n b e r g B, T o u w IP. H e m a t o p o i e t i c g r o w t h factors and their receptors in acute l e u k e m i a . B l o o d 1993; 81:281-292. 110. G i l l i o A P , G a b r i l o v e J L . C y t o k i n e treatment o f inherited bone m a r r o w failure s y n d r o m e s . B l o o d 1993; 8 1 : 1 6 6 9 - 1 6 7 4 . 111. D o n g F, B r y n e s R K , T i d o w N , et a l . M u t a t i o n s in the gene f o r the granulocyte colony- stimulating- factor receptor i n patients w i t h acute m y e l o i d l e u k e m i a preceded by severe congenital neutropenia. N e w E n g l . J M e d 1995; 333:487-493. 112. Naparstek E. G r a n u l o c y t e colony- stimulating factor, congenital neutropenia, and acute m y e l o i d l e u k e m i a . N e w E n g l . J M e d 1995; 333:516-518. 113. D o m b r e t H , Chastang C , F e n a u x P, et a l . A c o n t r o l l e d study o f r e c o m b i n a n t h u m a n granulocyte colony- stimulating factor in elderly patients after treatment for acute m y e l o g e n o u s l e u k e m i a . N E n g l . J M e d 1995; 3 3 2 : 1 6 7 8 - 1 6 8 3 . 114. Stone R M , B e r g D T , G e o r g e S L , et al. Granulocyte- macrophage c o l o n y stimulating factor after initial chemotherapy for patients at least 60 years o l d w i t h primary acute m y e l o g e n o u s l e u k e m i a . N e w E n g l . J M e d 1995; 332:1671-1677. 115. H a m b l i n TJ. D i s a p p o i n t m e n t s i n treating acute l e u k e m i a i n the elderly. N e w E n g l . J M e d 1995; 3 3 2 : 1 7 1 2 - 1 7 1 3 . 116. 21.  B o d e y G P . Therapy o f bacterial infections in acute l e u k e m i a . L e u k e m i a 1992; 6:17-  117. D e P a u w B E , D e r e s i n s k i S C , F e l d R, et a l . C e f t a z i d i m e c o m p a r e d w i t h p i p e r a c i l l i n and t ob ramy c i n for the e m p i r i c treatment o f fever in neutropenic patients w i t h cancer. A multicenter r a n d o m i z e d trial. T h e Intercontinental A n t i m i c r o b i a l Study G r o u p [see c o m m e n t s ] . A n n a l s o f Internal M e d i c i n e 1994; 120:834-44. 118. B o d e y G P , A n a i s s i e E J , E l t i n g L S , et a l . A n t i f u n g a l p r o p h y l a x i s d u r i n g r e m i s s i o n induction therapy for acute l e u k e m i a f l u c o n a z o l e versus intravenous amphotericin B. C a n c e r 1994; 7 3 : 2 0 9 9 - 1 0 6 .  177  119. B u c h n e r T , R o o s N . A n t i f u n g a l treatment strategy in l e u k e m i a patients. A n n a l s of H e m a t o l o g y 1992; 65:153-61. 120. K u r o s a w a M , O k a b e M , H a r a N , et a f R e v e r s a l effect o f itraconazole on a d r i a m y c i n and etoposide resistance in h u m a n l e u k e m i a cells. A n n a l s o f H e m a t o l o g y 1996; 72:17-21. 121. M c S h a r r y JJ. U s e s o f f l o w cytometry i n v i r o l o g y . C l i n i c a l M i c r o b i o l o g y R e v i e w s 1994;7:576-604. 122. Feusner J H , Hastings C A . Infections i n c h i l d r e n w i t h acute m y e l o g e n o u s l e u k e m i a . C o n c e p t s o f management and prevention. J P e d H e m a t o l / O n c o l 1995; 17:234-247. 123. G r e e n b a u m B H . D i f f e r e n c e s i n i m m u n o g l o b u l i n preparations for intravenous use: A c o m p a r i s o n o f six products. A m . J Pediatr. H e m a t o l . O n c o l 1 9 9 0 ; 12:490-496. 124. C a s p e r JT, S e d m a k G , H a r r i s R E , et a l . Intravenous i m m u n o g l o b u l i n : use i n pediatric bone m a r r o w transplantation. S e m . H e m a t o l 1992; 29:100-105. 125. R e a d M S , R e d d i c k R L , B o d e A P , et a l . Preservation o f hemostatic and structural properties o f rehydrated l y o p h i l i z e d platelets: potential f o r long- term storage o f dried platelets f o r transfusion. P r o c . N a t l . A c a d . S c i . U S A 1995; 9 2 : 3 9 7 - 4 0 1 . 126. W e n d l i n g F, M a r a s k o v s k y E, D e b i l i N , et al. c M p l l i g a n d is a h u m o r a l regulator o f m e g a k a r y o c y t o p o i e s i s . Nature 1994; 369:571-4. 127. V a i c k u s L, B r e i t m e y e r J B , S c h l o s s m a n R L , et al. Platelet transfusion and alternatives to transfusion i n patients w i t h m a l i g n a n c y . S t e m C e l l s 1995; 13:588-96. 128. B a t i u k T . Quantitating cyclosporine i m m u n o s u p p r e s s i o n : c o m p a r i n g calcineurin i n h i b i t i o n by t w o oral formulations, T h e C a n a d i a n Society f o r C l i n i c a l Investigation & T h e M e d i c a l Research C o u n c i l o f C a n a d a j o i n t program for c l i n i c a l scientists in training and M D - P h D students, M o n t r e a l , C a n a d a , 1995. C S C I & M R C . 129. F a y J W , W i n g a r d JR, A n t i n J H , et a l . F K 5 0 6 ( T a c r o l i m u s ) monotherapy f o r prevention o f graft-versus-host disease after histocompatible s i b l i n g allogenic bone m a r r o w transplantation. B l o o d 1996; 87:3514-9. 130. K o e h l e r M T , H o w r i e D , M i r r o J , et a l . F K 5 0 6 (tacrolimus) i n the treatment o f steroid-resistant acute graft-versus-host disease in c h i l d r e n u n d e r g o i n g bone m a r r o w .transplantation. B o n e M a r r o w Transplantation 1995; 15:895-9. 131. B l a z a r B R , T a y l o r P A , S n o v e r D C , et a l . M u r i n e recipients o f f u l l y m i s m a t c h e d donor m a r r o w are protected f r o m lethal graft-versus-host disease by the in v i v o administration o f r a p a m y c i n but develop an autoimmune-like syndrome. Journal o f I m m u n o l o g y 1 9 9 3 ; 151:5726-41. 132. A p p e r l e y JF, M a u r o F, G o l d m a n J M , et a l . B o n e m a r r o w transplantation f o r chronic m y e l o i d l e u k a e m i a in chronic phase: importance o f a graft- versus- l e u k a e m i a effect. B r . J H a e m a t o l 1988; 69:239-245. 133. Reisner Y , K a p o o r N , K i r k p a t r i c k D , et al. Transplantation for acute l e u k a e m i a w i t h H L A - A and B non- identical parental m a r r o w cells fractionated w i t h soya- bean agglutinin and sheep red b l o o d cells. L a n c e t 1 9 8 1 ; i i : 3 2 7 - 3 3 1 .  178  134. C a v a z z a n a - C a l v o M , B o r d i g o n i P, M i c h e l G , et al. A phase II trial o f partially incompatible bone m a r r o w transplantation for high- risk acute l y m p h o b l a s t i c l e u k a e m i a in c h i l d r e n : prevention o f graft rejection w i t h anti- L F A - 1 and anti- C D 2 antibodies Societe Francaise de G r e f f e de M o e l l e Osseuse. B r J H a e m a t o l 1996; 93:131-138. 135. Henslee- D o w n e y PJ, P a r r i s h R S , M a c D o n a l d JS, et a l . C o m b i n e d i n vitro and i n v i v o T l y m p h o c y t e depletion f o r the control o f graft- versus- host disease f o l l o w i n g haploidentical m a r r o w transplant. Transplantation 1996; 61:738-745. 136. K o e h l e r M , H u r w i t z C A , K r a n c e R A , et a l . X o m a Z y m e - C D 5 i m m u n o t o x i n i n conjunction w i t h partial T c e l l depletion for prevention o f graft rejection and graft-versushost disease after bone m a r r o w transplantation f r o m matched unrelated donors. B o n e M a r r o w Transplantation 1994; 13:571-5. 137. M a r t i n PJ, H a n s e n J A , Storb R, et a l . H u m a n m a r r o w transplantation: A n i m m u n o l o g i c a l perspective. In: D i x o n F J , A u s t e n K F , H o o d L E , U h r J W , eds. A d v a n c e s in I m m u n o l o g y . V o l . 4 0 . O r l a n d o , f l o r i d a : A c a d e m i c Press, Inc, 1987:379-423. 138. C h a m p l i n R. Graft-versus-leukemia without graft-versus-host disease: an e l u s i v e goal o f bone m a r r o w transplantation. S e m . H e m a t o l 1992; 29:46-52. 139. B l a i s e D , O l i v e D , M i c h a l l e t M , et al. Impairment o f leukaemia- free survival b y addition o f interleukin- 2- receptor antibody to standard graft- versus- host p r o p h y l a x i s . L a n c e t 1995; 3 4 5 : 1 1 4 4 - 1 1 4 6 . 140. A n t i n J H . Gradt-versus-leukemia: N o l o n g e r an e p i p h e n o m e n o n . B l o o d 1 9 9 3 ; 82:2273-2277. 141. A t k i n s o n K. C h r o n i c graft- versus- host disease. In: A t k i n s o n K, e d . C l i n i c a l bone m a r r o w transplantation: a reference textbook. C a m b r i d g e : C a m b r i d g e U n i v e r s i t y Press, 1994:312-324. 142. V o g e l s a n g S B , F a r m e r E R , H e s s A D , et al. T h a l i d o m i d e f o r the treatment o f c h r o n i c graft- versus- host- disease. N e w E n g l J M e d 1992; 3 2 6 : 1 0 5 5 - 1 0 5 8 . 143. W a n g W , M e a d o w s L R , den H a a n J M M , et a l . H u m a n H- Y : a male- s p c i f i c histocompatibility antigen derived f r o m the S M C Y protein. S c i e n c e 1995; 269:1588-1590. 144. G o u l m y E, S c h i p p e r R, P o o l J , et al. M i s m a t c h e s o f m i n o r h i s t o c o m p a t i b i l i t y antigens between H L A - i d e n t i c a l donors and recipients and the .development o f graft-versushost disease after bone m a r r o w transplantation [see c o m m e n t s ] . N e w E n g l a n d Journal o f Medicine 1996;334:281-5. 145. B e h a r E, C h a o N J , H i r a k i D D , et al. P o l y m o r p h i s m o f adhesion m o l e c u l e C D 3 1 and its role i n acute graft-versus-host disease [see c o m m e n t s ] . N e w E n g l a n d Journal o f M e d i c i n e 1996; 334:286-91. 146. K e r n a n N A , D u p o n t B. M i n o r h i s t o c o m p a t i b i l i t y antigens and m a r r o w transplantation. N e w E n g l a n d Journal o f M e d i c i n e 1996; 334:323-324. 147. W a l t e r s M C , Patience M , L e i s e n r i n g W , et a l . B o n e m a r r o w transplantation f o r s i c k l e c e l l disease. N e w E n g l . J M e d 1996; 335:369-376. 148. L u c a r e l l i G , G a l i m b e r t i M , P o l c h i P, et al. M a r r o w transplantation i n patients w i t h thalassemia responsive to i r o n chelation therapy. N e w E n g l J M e d 1 9 9 3 ; 3 2 9 : 8 4 0 .  179  149.  P i n k e l D . B o n e m a r r o w transplantation in c h i l d r e n . J . P e d 1 9 9 3 ; 122:331-341.  150. O' R e i l l y R J , M a c k i n n o n S, K w a k L W , et a l . A l l o g e n e i c m a r r o w transplants as adoptive immunotherapy: m o l e c u l a r and cellular interactions contributing to transferable l e u k e m i a resistance and v i r a l i m m u n i t y . H e m a t o l o g y 1995 1995:101-111. 151. Stuart R K . A u t o l o g o u s bone m a r r o w transplantation f o r l e u k e m i a . S e m in O n c o l 1993; 20:40-54. 152.  A r m i t a g e J O . B o n e m a r r o w transplantation. N e w E n g l . J . M e d 1994; 330:827-838.  153. G r i b b e n J G , F r e e d m a n A S , N e u b e r g D , et a l . I m m u n o l o g i c p u r g i n g o f m a r r o w assessed b y P C R before autologous bone m a r r o w transplantation f o r B- c e l l l y m p h o m a . New E n g l . J M e d 1 9 9 1 ; 3 2 5 : 1 5 2 5 . 154. B r e n n e r M K , R i l l D R , M o e n R C , et a l . G e n e - m a r k i n g to trace o r i g i n o f relapse after autologous bone-marrow transplantation. L a n c e t 1993; 341:85-6. 155. B r e n n e r M K , R i l l D R , M o e n R C , et a l . G e n e m a r k i n g and autologous bone m a r r o w transplantation. A n n a l s o f the N e w Y o r k A c a d e m y o f Sciences 1994; 716:204-14, 214-5, 225-7. 156. Y e a g e r A M , V o g e l s a n g G B , Jones R J , et al. Induction o f cutaneous graft-versushost disease by administration o f c y c l o s p o r i n e to patients u n d e r g o i n g autologous bone m a r r o w transplantation f o r acute m y e l o i d l e u k e m i a . B l o o d 1992; 79:3031-3035. 157. Kantarjian H M , D e i s s e r o t h A , K u r z r o c k R, et a l . C h r o n i c m y e l o g e n o u s l e u k e m i a : A concise update. B l o o d 1 9 9 3 ; 82:691-703. 158. C a r e l l a A M , F r a s s o n i F, N e g r i n R S . A u t o g r a f t i n g i n c h r o n i c m y e l o g e n o u s l e u k e m i a : new questions. L e u k e m i a 1995; 9:365-369. 159. D a l e y G Q , G o l d m a n J M . A u t o l o g o u s transplant f o r C M L revisited. E x p . H e m a t o l 1993;21:734-737. 160. G o l d m a n J M , H o y l e C . A u t o l o g o u s stem c e l l transplantation f o r c h r o n i c m y e l o i d l e u k e m i a . In: A t k i n s o n K, ed. C l i n i c a l bone m a r r o w transplantation: a reference textbook. C a m b r i d g e : C a m b r i d g e U n i v e r s i t y Press, 1994:124-128. 161. H u g h e s T P , M o r g a n G J , M a r t i a t P, et a l . D e t e c t i o n o f residual l e u k e m i a after bone m a r r o w transplant for chronic m y e l o i d l e u k e m i a : role o f polymerase chain reaction in p r e d i c t i n g relapse. B l o o d 1 9 9 1 ; 7 7 : 8 7 4 - 8 7 8 . " 162. T h o m p s o n J D , B r o d s k y I, Y u n i s JJ. M o l e c u l a r quantification o f residual disease in c h r o n i c myelogenous l e u k e m i a after bone m a r r o w transplantation. B l o o d 1992; 79:16291635. 163. M i y a m u r a K, T a h a r a T , T a n i m o t o M , et al. L o n g persistent ber- abl p o s i t i v e transcript detected b y polymerase c h a i n reaction after m a r r o w transplant f o r c h r o n i c myelogenous l e u k e m i a without c l i n i c a l relapse: a study o f 64 patients. B l o o d 1 9 9 3 ; 81:1089-1093. 164. G r i b b e n J G . Attainment o f m o l e c u l a r r e m i s s i o n : A w o r t h w h i l e goal ? J C l i n O n c o l 1994;12:1532-1534.  180  165. K l i n g e m a n n H G , D e a l H , R e i d D , et a l . D e s i g n and validation o f a c l i n i c a l l y applicable culture procedure for the generation of interleukin-2 activated natural k i l l e r cells in h u m a n bone m a r r o w autografts. E x p e r i m e n t a l H e m a t o l o g y 1993; 21:1263-70. 166. K l i n g e m a n n H G , N e e r u n j u n J , S c h w u l e r a U , et al. C u l t u r e o f n o r m a l and l e u k e m i c bone m a r r o w i n interleukin-2: analysis o f c e l l activation, c e l l proliferation, and c y t o k i n e p r o d u c t i o n . L e u k e m i a 1 9 9 3 ; 7:1389-93. 167. L o w r y P A , Tabbara I A . Peripheral hematopoietic stem cell transplantation: current concepts. E x p . hematol 1992; 2 0 : 9 3 7 - 9 4 2 . 168. A n d e r s o n K C . A u t o l o g o u s peripheral b l o o d progenitor c e l l transplantation. J . C l i n i c a l A p h e r e s i s 1995; 10:131-138. 169. L a s k y L C , B o s t r o m B, S m i t h J , et a l . C l i n i c a l c o l l e c t i o n and use o f peripheral b l o o d stem cells in pediatric patients. Transplantation 1989; 47:613-616. 170. S i e n a S, B r e g n i M , B r a n d o B, et a l . F l o w cytometry f o r c l i n i c a l estimation o f circulating hematopoietic progenitors for autologous transplantation in cancer patients. B l o o d 1 9 9 1 ; 77:400-. 171. B r u g g e r W , H e i m f e l d S, B e r e n s o n R J , et a l . R e c o n s t i t u t i o n o f hematopoiesis after high- dose chemotherapy by autologous progenitor cells generated ex v i v o . N e w E n g l . J M e d 1995; 333:283-287. 172. Janssen W E . M o b i l i z a t i o n of peripheral b l o o d stem cells for autologous transplantation. M e t h o d s , mechanisms, and role i n accelerating hematopoietic recovery. A n n a l s o f the N e w Y o r k A c a d e m y o f Sciences 1 9 9 5 ; 770:116-129. 173. S h p a l l E J , Jones R B . Release o f t u m o r c e l l s f r o m bone m a r r o w . B l o o d 1994; 83:623-625. 174. B r u g g e r W , B r o s s K J , Glatt M , et a l . M o b i l i z a t i o n o f tumor cells and hematopoietic progenitor cells into peripheral b l o o d o f patients w i t h s o l i d tumors. B l o o d 1994; 83:636640. 175. M a r t i n H , A t t a J , Z u m p e P, et al. P u r g i n g o f peripheral b l o o d stem cells yields B C R - A B L - negative autografts in patients w i t h B C R - A B L - positive acute l y m p h o b l a s t i c l e u k e m i a . E x p . H e m a t o l 1995; 2 3 : 1 6 1 2 - 1 6 1 8 . 176. ' L c m o l i R M , F o r t u n a A , M o t t a M R , et a l . C o n c o m i t a n t m o b i l i z a t i o n o f p l a s m a cells and hematopoietic progenitors into peripheral b l o o d o f multiple m y e l o m a patients: positive selection and transplantation o f enriched C D 3 4 + cells to remove cicrulating tumor cells. B l o o d 1996; 8 7 : 1 6 2 5 - 1 6 3 4 . 177. N i m g a o n k a r M , K e m p A , L a n c i a J , et al. A c o m b i n a t i o n of C D 3 4 selection and complement- mediated i m m u n o p u r g i n g (anti- C D 15 m o n o c l o n a l antibody) eliminates tumor cells w h i l e sparing n o r m a l progenitor cells. J . Hematotherapy 1996; 5:39-48. 178. T a n a k a J , K a s a i M , I m a m u r a M , et a l . C l i n i c a l application o f allogneic peripheral b l o o d stem cells transplantation. A n n a l s o f H e m a t o l o g y 1995; 71:265-269. 179. V o w e l s M R , T a n g R L . C o r d b l o o d and fetal tissue transplants. In: A t k i n s o n K, e d . C l i n i c a l bone m a r r o w transplantation: a reference textbook. C a m b r i d g e : C a m b r i d g e U n i v e r s i t y Press, 1994:637-642.  181  180. K u r t z b e r g J , L a u g h l i n M , G r a h a m , M L , et a l . Placental b l o o d as a source o f hematopoietic stem cells for transplantation into unrelated recipients. N e w E n g l J M e d 1996; 335:157.-166. 181. L a p o r t e J-P, G o r i n N - C , R u b i n s t e i n P, et a l . C o r d - b l o o d transplantation f r o m an unrelated donor in an adult w i t h c h r o n i c m y e l o g e n o u s l e u k e m i a . N e w E n g l J M e d 1996; 335:167-170. 182. G a l e R P . C o r d - b l o o d - c e l l trasnplantation— a real sleeper ? N e w E n g l . J M e d 1995; 3 3 2 : 3 9 2 . 183.  B i s h o p J M . M o l e c u l a r themes in oncogenesis. C e l l 1 9 9 1 ; 64:235-248.  184. W a t s o n J D , W i t k o w s k i J , G i l m a n M , et a l . O n c o g e n e s and antioncogenes. R e c o m b i n a n t D N A . N e w Y o r k : W . H . F r e e m a n and C o m p a n y , 1992:335-363. 185. G r o s s L. T h e role o f viruses i n the etiology o f cancer and l e u k e m i a in animals and in h u m a n s . P r o c . N a t l . A c a d . S c i . U S A 1 9 9 7 ; 9 4 : 4 2 3 7 - 4 2 3 8 . 186. S a w y e r s C L , D e n n y C T , W i t t e O N . L e u k e m i a and the disruption o f n o r m a l hematopoiesis. C e l l 1 9 9 1 ; 64:337-350. 187. H i d d e m a n n W , G r i e s i n g e r F. P r e c l i n i c a l aspects and therapeutic perspectives o f acute and chronic leukemias. Current O p i n i o n i n O n c o l o g y 1993; 5:13-25. 188. P o w i s G . A n t i c a n c e r drugs acting against s i g n a l i n g pathways. C u r r e n t O p i n i o n i n O n c o l o g y 1995; 7:554-9. 189. W i l k s A F . Protein tyrosine kinase growth factor receptors and their ligands i n development, differentiation, and cancer. A d v a n c e s i n C a n c e r R e s 1993; 60:43-73. 190. Imamoto A , So ri an o P. D i s r u p t i o n o f the c s k gene, e n c o d i n g a negative regulator o f Src f a m i l y tyrosine kinases, leads to neural tube defects and e m b r y o n i c lethality i n m i c e . Cell 1993;73:1117-1124. 191. K o n o p k a J B , W i t t e O N . Detection o f c-abl tyrosine kinase activity i n vitro permits direct c o m p a r i s o n o f n o r m a l and altered abl gene products. M o l . C e l l . B i 1985; 5:31163123. 192. . van Etten R A , Jackson P, B a l t i m o r e D . T h e mouse type I V c-abl gene product is a nuclear pro.tein, and activation o f transforming ability is associated w i t h c y t o p l a s m i c l o c a l i z a t i o n . CelVl989; 58:669-678.. " 193. W e l c h P J , W a n g J Y . A C-terminal protein-binding d o m a i n i n the retinoblastoma protein regulates nuclear c - A b l tyrosine kinase in the cell c y c l e . C e l l 1 9 9 3 ; 75:779-90. 194. T a u c h i T , B r o x m e y e r H E . B C R / A B L signal transduction. International Journal o f Hematology 1995;61:105-12. 195. L o w e S W , R u l e y H E , Jacks T , et a l . p53- dependent apoptosis modulates the c y t o t o x i c i t y o f anticancer agents. C e l l 1 9 9 3 ; 74:957-967. 196. M e t z T , H a r r i s A W , A d a m s J M . A b s e n c e o f p53 a l l o w s direct i m m o r t a l i z a t i o n o f hematopoietic cells b y the m y c and raf oncogenes. C e l l 1995; 82:29-36.  182  197. Ilaria R L , Jr., V a n Etten R A . T h e S H 2 d o m a i n o f P 2 1 0 B C R / A B L is not required for the transformation o f hematopoietic factor-dependent cells. B l o o d 1995; 86:3897-904. 198. A n d e r s o n S M , M l a d e n o v i c J . T h e B C R - A B L oncogene requires both kinase activity and src-homology 2 d o m a i n to induce c y t o k i n e secretion. B l o o d 1996; 87:238-44. 199. S h u a i K, H a l p e r n J , ten H o e v e J , et a l . C o n s t i t u t i v e activation o f S T A T 5 by the B C R - A B L oncogene i n c h r o n i c myelogenous l e u k e m i a . O n c o g e n e 1996; 13:247-54. 200. M o o r e M A S . C l i n i c a l implications o f positive and negative hematopietic stem c e l l regulators. B l o o d 1 9 9 1 ; 78:1-19. 201. G r a h a m G , J , W r i g h t E G , H e w i c k R, et a l . Identification and characterization o f an inhibitor o f hematopoietic stem c e l l proliferation. Nature 1990; 344:442-444. 202. C o o k D . T h e role o f M I P - l a i n i n f l a m m a t i o n and hematopoiesis. J L e u k o c y t e B i o l 1996; 59:61-66. 203.  E a v e s C J , C a s h m a n J D , W o l p e S D , et a l . U n r e s p o n s i v e n e s s o f p r i m i t i v e c h r o n i c  m y e l o i d l e u k e m i a cells to macrophage inflammatory protein- l a , an inhibitor o f p r i m i t i v e n o r m a l hematopoietic cells. P r o c . N a t l A c a d S c i U S A 1 9 9 3 ; 9 0 : 1 2 0 1 5 . 204. C a s h m a n J D , E a v e s A C , E a v e s C J . T h e tetrapeptide A c S D K P s p e c i f i c a l l y b l o c k s the c y c l i n g o f p r i m i t i v e n o r m a l but not leukemic progenitors i n long-term culture: evidence f o r an indirect m e c h a n i s m . B l o o d 1994; 84:1534-42. 205. G u i g o n M , B o n n e t D . Inhibitory peptides i n hematopoiesis. E x p H e m a t o l 1995; 23:477-481. 206. T u b i a n a M , C a r d e P, F r i n d e l E. W a y s o f m i n i m i s i n g hematopoietic damage induced b y radiation and cytostatic drugs—the possible role o f inhibitors. Radiotherapy & O n c o l o g y 1993; 29:1-17. 207. W i e r e n g a P K , K o n i n g s A W T . Goralatide ( A c S D K P ) selectively protects murine hematopoietic progenitors and stem cells against hyperthermic damage. E x p . H e m a t o l 1995; 24:246-252. 208. G e n e v a y M - C , M o r m o n t C , T h o m a s F, et a l . T h e synthetic tetrapeptide A c S D K P protects cells that reconstitute long- term bone m a r r o w stromal cultures f r o m the effects o f m a f o s f a m i d e ( A s t a Z 7654). E x p . H e m a t o l 1996; 24:77-81. 209. C o u t t o n C , G u i g o n M , B o h b o t A , et a l . Photoprotection o f n o r m a l h u m a n hematopoietic progenitors by the tetrapeptide N - A c S D K P . E x p e r i m e n t a l H e m a t o l o g y 1994; 22:1076-80. 210. G u i g o n M , L e m o i n e F, N a j m a n A . B o n e m a r r o w protection. B o n e M a r r o w Transplantation 1994; 13:93-95. 211. H u r l e y R W , M c C a r t h y J B , V e r f a i l l i e C M . D i r e c t contact w i t h bone m a r r o w stroma has negative regulatory effects on hematopoietic progenitors. B l o o d 1993; 82 (10 S u p p l . 1):21A.  183  212. G o r d o n M Y , D o w d i n g C R , R i l e y G P , et al. A l t e r e d adhesive interactions w i t h m a r r o w stroma o f hematopoietic progenitor cells i n chronic myelogenous l e u k a e m i a . N a t u r e 1984; 3 2 8 : 3 4 2 . 213. V e r f a i l l i e C M , H u r l e y R, L u n d e l l B I , et a l . Integrin-mediated regulation o f hematopoiesis: do B C R / A B L - i n d u c e d defects in integrin function underlie the abnormal circulation and proliferation o f C M L progenitors? A c t a H a e m a t o l o g i c a 1997; 97:40-52. 214. D o w d i n g C , G u o A P , O s t e r h o l z J , et al. Interferon-alpha overrides the deficient adhesion of chronic m y e l o i d l e u k e m i a p r i m i t i v e progenitor cells to bone m a r r o w stromal c e l l s . B l o o d 1 9 9 1 ; 78:499-505. 215. B h a t i a R, M c G l a v e P B , D e w a l d G W , et al. A b n o r m a l f u n c t i o n o f the bone m a r r o w microenvironment in chronic myelogenous l e u k e m i a : role o f malignant stromal macrophages. B l o o d 1995; 8 5 : 3 6 3 6 - 3 6 4 5 . 216. H a n k s S K , Polte T R . S i g n a l i n g throught f o c a l adhesion kinase. B i o e s s a y s 1 9 9 7 ; 19:137-145. 217. L i s t A F . M u l t i d r u g resistance: c l i n i c a l relevance i n acute leukemia. O n c o l o g y 1993; 7:23-28, 3 2 , 35-38. 218. M a r i e JP, Z i t t o u n R, S i k i c B I . M u l t i d r u g resistance (mdrX) gene e x p r e s s i o n i n adult acute leukemias: correlations w i t h treatment outcome and i n vitro drug sensitivity. B l o o d 1 9 9 1 ; 78:586-592. 219. L e m o l i R M . Characterization and selection o f benign stem cells in c h r o n i c m y e l o i d l e u k e m i a . H a e m a t o l o g i c a 1993; 78:393-400. 220. G r i b b e n J G , N a d l e r L M . P u r g i n g o f bone m a r r o w . In: D e V i t a V T , Jr, H e l l m a n S, R o s e n b e r g S A , eds. B i o l o g i c therapy o f cancer. P h i l a d e l p h i a : J . B . L i p p i n c o t t C o . , 1995:596-606. 221. O' B r i e n S G , G o l d m a n J M . Current approaches to hematopoietic stem- c e l l p u r g i n g i n chronic m y e l o i d l e u k e m i a . J Clin O n c o l 1995; 13:541-546. 222. R i z z o l i V , Carlo- S t e l l a C . S t e m c e l l p u r g i n g : an i n t r i g u i n g d i l e m m a . E x p H e m a t o l 1995;23:296-302. :  223. O n c o l i n k . P u r g e d bone m a r r o w p r i o r to autologous bone m a r r o w transplants decreased.relapse fate and treatment failure i n patients w i t h acute myelogenous l e u k e m i a . P e n n s y l v a n i a : U n i v e r s i t y o f P e n n s y l v a n i a , 1996. 224. N e g r i n R S , K u s n i e r z - G l a z C R , S t i l l B J , et al. Transplantation o f e n r i c h e d and purged peripheral b l o o d progenitor cells f r o m a single apheresis product i n patients w i t h non- H o d g k i n ' s l y m p h o m a . B l o o d 1 9 9 5 ; 8 5 : 3 3 3 4 - 3 3 4 1 . 225. B e a u j e a n F, H e r v e P. N e w approaches f o r bone m a r r o w p u r g i n g . T r a n s f u s . S c i 1992; 13:431-442. 226. G i d a l i J , S z a m o s v o l g y i S, F e h e r I, et al. S u r v i v a l and characteristics o f m u r i n e leukaemic and normal stem cells after hyperthermia: a murine m o d e l for h u m a n bone m a r r o w p u r g i n g . L e u k e m i a R e s 1 9 9 0 ; 14:453-457.  184  227. M o r i y a m a Y , H a s h i m o t o S, G o t o T , et a l . IN VITRO p u r g i n g o f c l o n o g e n i c l e u k e m i c cells f r o m h u m a n bone m a r r o w by heat: simulation experiments for autologous bone m a r r o w transplantation. L e u k . R e s 1 9 9 2 ; 16:973-977. 228.  Pui C H . C h i l d h o o d leukemias. N e w E n g l . J M e d 1995; 332:1618-1630.  229. L e b k o w s k i JS, S c h a i n L R , O k r o n g l y D , et al. R a p i d isolation o f h u m a n C D 3 4 hematopoietic stem cells- p u r g i n g o f h u m a n t u m o r cells. Transplantation 1992; 53:10111019. 230. S c h e f f o l d C , B r a n d t K, Johnston V , et a l . Potential o f autologous i m m u n o l o g i c effector cells for bone m a r r o w p u r g i n g i n patients w i t h c h r o n i c m y e l o g e n o u s l e u k e m i a . B o n e m a r r o w transplantation 1995; 15:33-39. 231. C h a n g J , Dexter T M . Long-term m a r r o w cultures: i n vitro p u r g i n g o f l e u k a e m i c cells. B a i l l i e r e s C l i n i c a l H a e m a t o l o g y 1 9 9 1 ; 4:775-88. 232. Barnett M J , E a v e s C J , P h i l l i p s G L , et a l . S u c c e s s f u l autografting i n c h r o n i c m y e l o i d l e u k e m i a after maintenance o f m a r r o w culture. B o n e m a r r o w transplant 1989; 4:345. 233. S h p a l l E J , Jones R B , B a s t R C , Jr., et a l . 4 - H y d r o p e r o x y c y c l o p h o s p h a m i d e p u r g i n g of breast cancer f r o m the m o n o n u c l e a r c e l l fraction o f bone m a r r o w i n patients r e c e i v i n g high-dose chemotherapy and autologous m a r r o w support: a phase I trial. Journal o f C l i n i c a l O n c o l o g y 1 9 9 1 ; 9:85-93. 234. M a k r y n i k o l a V , Kabral A , Bradstock K F . Effect of mafosfamide (ASTA-Z-7654) on the clonogenic cells in precursor-B acute l y m p h o b l a s t i c leukaemia: significance for ex v i v o p u r g i n g o f bone m a r r o w f o r autologous transplantation. B o n e M a r r o w Transplantation 1 9 9 1 ; 8:351-5. 235. W e i s s L, S l a v i n G , R e i c h S, et al. S u c c e s s f u l p u r g i n g o f m u r i n e p l a s m a c y t o m a by m a f o s f a m i d e ( A S T A - Z ) . B o n e M a r r o w Transplantation 1994; 13:27-30. 236. B l a a u w A , Spitzer G , D i c k e K, et a l . Potential drugs f o r e l i m i n a t i o n o f acute l y m p h a t i c l e u k e m i a cells f r o m autologous bone m a r r o w . E x p . H e m a t o l 1986; 14:683-688. 237. V o g l e r W R . B o n e m a r r o w p u r g i n g in acute l e u k e m i a w i t h a l k y l - l y s o p h o s p h o l i p i d s : a n e w f a m i l y of anticancer drugs. L e u k e m i a & L y m p h o m a 1994; 13:53-60. ,238. U.ckun F M , . K e r s e y JH, Y a l l e r a D A , et a l . A u t o l o g o u s bone marrow' transplantation i h high-risk remission T-lineage acute l y m p h o b l a s t i c l e u k e m i a u s i n g i m m u n o t o x i n s plus 4h y d r o p e r o x y c y c l o p h o s p h a m i d e f o r m a r r o w p u r g i n g . B l o o d 1990; 76:1723-1733. 239. U c k u n F M , K e r s e y J H , H a a k e R, et a l . A u t o l o g o u s bone m a r r o w transplantation i n high-risk remission B-lineage acute l y m p h o b l a s t i c l e u k e m i a using a c o c k t a i l o f three m o n o c l o n a l antibodies ( B A - 1 / C D 2 4 , B A - 2 / C D 9 , and B A - 3 / C D 1 0 ) plus c o m p l e m e n t and 4h y d r o p e r o x y c y c l o p h o s p h a m i d e f o r ex v i v o bone m a r r o w p u r g i n g . B l o o d 1992; 79:1094104. 240. S k o r s k i T , N i e b o r o w s k a - S k o r s k a M , B a r l e t t a C , et a l . H i g h l y efficient e l i m i n a t i o n o f P h i l a d e l p h i a leukemic cells by exposure to bcr/abl antisense oligodeoxynucleotides c o m b i n e d w i t h m a f o s f a m i d e . J . C l i n . Invest 1 9 9 3 ; 9 2 : 1 9 4 - 2 0 2 .  185  241. W u A G , J o s h i S S , C h a n W C , et a l . E f f e c t s o f B C R - A B L antisense oligonucleotides ( A S - O D N ) on human chronic m y e l o i d l e u k e m i c cells: A S - O D N as effective p u r g i n g agents. L e u k e m i a & L y m p h o m a 1 9 9 5 ; 20:67-76. 242. Seth P, B r i n k m a n n U , S c h w a r t z G N , et a l . A d e n o v i r u s - mediated gene transfer to h u m a n breast tumor cells: an approach for cancer gene therapy and bone m a r r o w p u r g i n g . C a n c e r R e s 1996; 56:1346-1351. 243. L a C a s s e E C , Saleh M T , Patterson B, et a l . Shiga- l i k e t o x i n purges h u m a n l y m p h o m a f r o m bone m a r r o w o f severe c o m b i n e d i m m u n o d e f i c i e n t m i c e . B l o o d 1996; 88:1561-1567. 244. C h a n C H , B l a z a r B R , E i d e C R , et al. A m u r i n e c y t o k i n e f u s i o n t o x i n s p e c i f i c a l l y targeting the murine granulocyte-macrophage colony-stimulating factor ( G M - C S F ) receptor on n o r m a l c o m m i t t e d bone m a r r o w progenitor cells and G M - C S F - d e p e n d e n t tumor cells. B l o o d 1 9 9 5 ; 86:2732-40. 245. K r a m e r R A , Z a k h e r J , K i m G . R o l e o f the glutathione redox c y c l e in acquired and de n o v o m u l t i d r u g resistance. S c i e n c e 1988; 2 4 1 : 6 9 4 - 6 9 7 . 246. L a n d e r H M . A n essential role for free radicals and d e r i v e d species in signal transduction. F A S E B Journal 1 9 9 7 ; 11:118-24. 247. H a l l i w e l l B. R e a c t i v e o x y g e n species i n l i v i n g systems: source, b i o c h e m i s t r y , and role i n h u m a n disease. A m . J . M e d 1 9 9 1 ; 91:14-23. 248.  C o c h r a n e C G . C e l l u l a r injury b y o x i d a n t s . A m . J . M e d 1 9 9 1 ; 91:23-30.  249. Pass H I . P h o t o d y n a m i c therapy in o n c o l o g y : m e c h a n i s m s and c l i n i c a l use. J . N a t l . C a n . Inst 1 9 9 3 ; 85:443-456. 250. B u t t k e T M , Sandstrom P A . O x i d a t i v e stress as a mediator o f apoptosis. I m m u n o l o g y T o d a y 1994; 7:7-10. 251. K a n e D J , Sarafian T A , A n t o n R, et a l . B c l - 2 i n h i b i t i o n o f neural death: decreased generation o f reactive o x y g e n species. S c i e n c e 1 9 9 3 ; 2 6 2 : 1 2 7 4 - 1 2 7 7 . 252. W i e d a u - P a z o s M , T r u d e l l JR, A l t e n b a c h C , et a l . E x p r e s s i o n o f bcl-2 inhibits cellular radical generation. Free R a d i c a l Research 1996; 24:205-12. 2 5 3 . . J a c o b s o n M D . Reactive o x y g e n species and p r o g r a m m e d c e l l death. Trends in B i o c h e m i c a l Sciences 1996; 21:83-6. 254. M i t c h e l l J B , C o o k J A , R u s s o A . B i o l o g i c a l basis o f phototherapy. In: M o r s t y n G , K a y e A H , eds. Phototherapy o f C a n c e r : H a r w o o d A c a d e m i c P u b l i s h e r s , 1990:1-22. 255. H e n d e r s o n B W , D o u g h e r t y TJ. H o w does p h o t o d y n a m i c therapy w o r k ? P h o t o c h e m & P h o t o b i o l 1992; 55:145-157. 256. S p i k e s J D . Photosensitization. In: S m i t h K C , e d . T h e S c i e n c e o f P h o t o b i o l o g y . N e w Y o r k : P l e n u m Press, 1989:79-110. 257. A l l i s o n B A , Pritchard P H , L e v y J G . E v i d e n c e f o r low- density l i p o p r o t e i n receptor- mediated uptake o f b e n z o p o r p h y r i n derivative. B r . J . C a n 1994; 69:833-839.  186  258. A l l i s o n B A , Pritchard P H , R i c h t e r A M , et al. T h e p l a s m a distribution o f b e n z o p o r p h y r i n derivative and the effects o f p l a s m a lipoproteins o n its b i o d i s t r i b u t i o n . P h o t o c h e m & P h o t o b i o l 1990; 52:501-507. 259. B o h m e r R M , M o r s t y n G . U p t a k e o f H e m a t o p o r p h y r i n derivative b y n o r m a l and malignant c e l l s : effcet o f serum, p H , temperature, and c e l l size. C a n c e r R e s 1985; 45:53285334. 260. G o m e r C J , F e r r a r i o A , H a y a s h i N , et a l . M o l e c u l a r , c e l l u l a r , and tissue responses f o l l o w i n g p h o t o d y n a m i c therapy. L a s e r s i n S u r g . & M e d 1988; 8:450-463. 261. Sieber F, G a f f n e y D K , Y a m a z a k i T , et al. Importance o f c e l l u l a r defense m e c h a n i s m s in the p h o t o d y n a m i c p u r g i n g o f autologous bone m a r r o w grafts, A d v a n c e s in bone m a r r o w p u r g i n g and p r o c e s s i n g : F o u r t h international s y m p o s i u m , 1994. W i l e y - L i s s , Inc. 262. A g a r w a l M L , C l a y M E , H a r v e y EJ, et al. P h o t o d y n a m i c therapy induces rapid c e l l death b y apoptosis i n L 5 1 7 8 Y mouse l y m p h o m a cells. C a n c e r Res 1 9 9 1 ; 51:5993-5996. 263. H e J , A g a r w a l M L , L a r k i n H E , et al. T h e i n d u c t i o n o f partial resistance to p h o t o d y n a m i c therapy by the protooncogene B C L - 2 . P h o t o c h e m i s t r y & P h o t o b i o l o g y 1996; 64:845-52. 264. R y t e r S W , G o m e r C J . N u c l e a r factor k a p p a B b i n d i n g activity i n mouse L 1 2 1 0 cells f o l l o w i n g photofrin Il-mediated photosensitization. P h o t o c h e m i s t r y & P h o t o b i o l o g y 1993; 58:753-6. 265. C u r r y P M , L e v y J G . Stress protein expression i n m u r i n e t u m o r c e l l s f o l l o w i n g p h o t o d y n a m i c therapy w i t h b e n z o p o r p h y r i n derivative. P h o t o c h e m & P h o t o b i o l 1993; 58:374-379. 266. L u n a M C , W o n g S, G o m e r C J . P h o t o d y n a m i c therapy mediated i n d u c t i o n o f early response genes. C a n c e r R e s e a r c h 1994; 54:1374-80. 267. T a o J-S, Sanghera JS, P e l e c h S L , et al. S t i m u l a t i o n o f stress- activated protein kinase and p38 H O G 1 kinase i n m u r i n e keratinocytes f o l l o w i n g p h o t o d y n a m i c therapy w i t h B e n z o p o r p h y r i n derivative. Journal o f B i o l . C h e m 1996; 2 7 1 : 2 7 1 0 7 - 1 5 . 268. Separovic D , H e J , O l e i n i c k N L . C e r a m i d e generation i n response to p h o t o d y n a m i c treatment o f L 5 1 7 8 Y mouse l y m p h o m a cells.,Cancer Res 1 9 9 7 ; 5 7 : 1 7 1 7 - 1 7 2 1 . 269.  L e v y J G . P h o t o d y n a m i c therapy. T I B T E C H 1995; 13:14-17.  270. 1994.  G r o s s w e i n e r L I . T h e S c i e n c e o f Phototherapy. V o l . 1. B o c a R a t o n : C R C Press,  271. M u l l e r PJ, W i l s o n B C . P h o t o d y n a m i c therapy for recurrent supratentorial g l i o m a s . Seminars i n S u r g i c a l O n c o l o g y 1995; 11:346-354. 272. Stables G l , A s h D V . P h o t o d y n a m i c therapy. C a n c e r Treatment R e v i e w s 1995; 21:311-323. 273. F i n g a r V H , W i e m a n T J , W i e h l e S A , et al. T h e role o f m i c r o v a s c u l a r damage i n p h o t o d y n a m i c therapy: the effect o f treatment on vessel constriction, permeability, and leukocyte adhesion. C a n c e r Res 1992; 52:4914-4921.  187  274. Ferrario A , G o m e r C J . E x p e r i m e n t s on laser mediated photosensitization: preclinical evaluation o f mono- 1- aspartyl c h l o r i n e6 ( N P e 6 ) . In: Chester A N , M a r t e l l u c c i J , S c h e g g i A M , eds. L a s e r S y s t e m s f o r P h o t o b i o l o g y and P h o t o m e d i c i n e . N e w Y o r k : P l e n u m Press, 1991:201-207. 275. M o r g a n A R , G a r b o G M , K e c k R W , et a l . N e w photosensitizers f o r p h o t o d y n a m i c therapy: c o m b i n e d effect o f metallopurpurin derivatives and light o n transplantable bladder tumors. C a n c e r R e s 1 9 8 8 ; 4 8 : 1 9 4 - 1 9 8 . 276. C h a n W S , M a r s h a l l J F , S v e n s e n R, et a l . P h o t o s e n s i t i z i n g activity o f phthalocyanine dyes screened against tissue culture cells. P h o t o c h e m . P h o t o b i o l 1987; 45:757-761. 277. R i c h t e r A M , K e l l y B, C h o w J , et a l . P r e l i m i n a r y studies o n a m o r e effective phototoxic agent than h e m a t o p o r p h y r i n . J N a t l . C a n c e r Inst 1987; 7 9 : 1 3 2 7 - 1 3 3 2 . 278. Richter A , Sternberg E, W a t e r f i e l d E, et al. Characterization o f b e n z o p o r p h y r i n derivative, a n e w photosensitizer. Proceedings o f S P I E 1988; 9 9 7 : 1 3 2 - 1 3 8 . 279. A v e l i n e B, H a s a n T , R e d m o n d R W . P h o t o p h y s i c a l and p h o t o s e n s i t i z i n g properties o f b e n z o p o r p h y r i n derivative m o n o a c i d r i n g A ( B P D - M A ) . P h o t o c h e m & P h o t o b i o l 1994; 59:328-355. 280.  R i c h t e r A M , Cerruti- S o l a S, Sternberg E D , et al. B i o d i s t r i b u t i o n o f tritiated  b e n z o p o r p h y r i n derivative (^H- B P D - M A ) , a n e w potent photosensitiser, i n n o r m a l a n d tumor- bearing m i c e . J . P h o t o c h e m . P h o t o b i o l . B 1 9 9 0 ; 5:231-244. 281. R i c h t e r A M , Y i p S, M e a d o w s H , et al. Photosensitising potencies o f the structural analogues o f b e n z o p o r p h y r i n derivative i n different b i o l o g i c a l test systems. J . C l i n . L a s e r M e d . & Surgery 1996; 14:335-341. 282.  R i c h t e r A M , Y i p S, W a t e r f i e l d E, et al. M o u s e S k i n photosensitization w i t h  b e n z o p o r p h y r i n derivatives and photofrin®: m a c r o s c o p i c and m i c r o s c o p i c evaluation. P h o t o c h e m & P h o t o b i o l 1 9 9 1 ; 53:281-286. 283. L o g a n P M , N e w t o n J , R i c h t e r A , et al. I m m u n o l o g i c a l effects o f p h o t o d y n a m i c therapy. S P I E . P h o t o d y n a m i c therapy: m e c h a n i s m s II 1990; 1203:153-158. 284. L e v y J G , C h a n A , Strong A . T h e c l i n i c a l status o f b e n z o p o r p h y r i n derivative. P r o c . S P I E 1996; 2 6 2 5 : 8 6 - 9 5 . 285. O b o c h i M O K , C a n a a n A J , Jain A K , et a l . Targeting activated l y m p h o c y t e s w i t h photodynamic therapy: susceptibility o f mitogen- stimulated splenic l y m p h o c y t e s to b e n z o p o r p h y r i n derivative ( B P D ) photosensitization. P h o t o c h e m . & P h o t o b i o l 1995; 62:169-175. 286. T a y l o r A , Gasparro F P . E x t r a c o r p o r e a l photochemotherapy f o r cutaneous T -ce ll l y m p h o m a and other diseases. S e m . i n H e m a t o l o g y 1992; 29:132-141. 287. N e y n d o r f f H C , Bartel D L , T u f a r o F, et al. D e v e l o p m e n t o f a m o d e l to demonstrate photosensitizer- mediated v i r a l inactivation i n b l o o d . T r a n s f u s i o n 1990; 30:485-490.  188  288. N o r t h J , C o o m b s R, J L. P h o t o i n a c t i v a t i o n o f H I V . by b e n z o p o r p h y r i n derivative. In: S p i n e l l i P, D a l Fante M , M a r c h e s i n i R, eds. P h o t o d y n a m i c therapy and b i o c h e m i c a l lasers: E l s e v i e r S c i e n c e P u b l i s h e r s , 1992:103-110. 289. O b o c h i M O K , Ratkay L G , L e v y J G . P r o l o n g e d s k i n allograft s u r v i v a l after p h o t o d y n a m i c therapy associated w i t h m o d i f i c a t i o n o f donor s k i n antigenicity. Transplantation 1997; 63:810-817. 290. M u l r o n e y C M , G l u c k S, H o A D . T h e use o f p h o t o d y n a m i c therapy i n bone m a r r o w p u r g i n g . Seminars in O n c o l o g y 1994; 21:24-7. 291. L e v y J G , D o w d i n g C , M i t c h e l D , et a l . Selective e l i m i n a t i o n o f malignant stem cells u s i n g photosensitizer f o l l o w e d by light treatment. S t e m cells 1995; 13:336-343. 292. J a m i e s o n C H M . B e n z o p o r p h y r i n derivative and the p h o t o d y n a m i c extracorporeal treatment o f l e u k e m i a . M i c r o b i o l o g y & I m m u n o l o g y . V a n c o u v e r : U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1992:309. 293. J a m i e s o n C H M , M c D o n a l d W N , L e v y J G . Preferential uptake o f b e n z o p o r p h y r i n derivative by l e u k e m i c versus n o r m a l cells. L e u k e m i a Res 1990; 14:209-219. 294. J a m i e s o n C , R i c h t e r A , L e v y J G . E f f i c a c y o f b e n z o p o r p h y r i n derivative, a photosensitizer, in selective destruction o f l e u k e m i a cells using a murine t u m o r m o d e l . E x p . H e m a t o l 1 9 9 3 ; 21:629-634. 295. K e a t i n g A , Jamieson C , H o r n b y A . P h o t o d y n a m i c e l i m i n a t i o n o f c l o n o g e n i c Ph-tc h r o n i c m y e l o i d l e u k e m i a cells. L e u k . L y m p h o m a 1 9 9 3 ; 11 (Suppl l ) : 2 6 5 - 2 6 9 . 296.  L e m o l i R M , Igarashi T , K n i z e w s k i M , et a l . Dye-mediated photolysis is capable o f  e l i m i n a t i n g drug-resistant ( M D R + ) t u m o r cells. B l o o d 1993; 81:793-800. 297. G l u c k S, Chadderton A , H o A D . T h e selective uptake o f B e n z o p o r p h y r i n D e r i v a t i v e mono- acid ring A results in differential c e l l k i l l o f multiple m y e l o m a cells in vitro. P h o t o c h e m & P h o t o b i o l 1996; 63:846-853. 298. S i n g e r C R J , L i n c h D C , B o w n S G , et a l . D i f f e r e n t i a l phthalocyanine photosensitization of acute m y e l o b l a s t s l e u k a e m i a progenitor cells: a potential p u r g i n g technique f o r autologous bone m a r r o w transplantation. B r . J . H a e m a t o l 1988; 68:417-422. 299. G u n t h e r W H H , Searle R,- Sieber F. Structure-activity relationships in the antiviral and antileukemic photopropeities o f M e r o c y a n i n e dves. S e m . in H e m a t o l o g y 1992, 29:8894. ' 300. M o r g a n J , M a c R o b e r t A J , G r a y s A G , et a l . U s e o f photosensitive, antibody directed liposomes to destroy target populations o f cells in bone m a r r o w : a potential p u r g i n g m e t h o d f o r autologous bone m a r r o w transplantation. B r . J . C a n c e r 1 9 9 2 ; 65:5864. 301. Sieber F, R a o S, R o w l e y S D , et a l . D y e - m e d i a t e d p h o t o l y s i s o f h u m a n neuroblastoma cells: implications for autologous bone m a r r o w transplantation. B l o o d 1986; 68:32-36.  189  302. Sieber F. P h o t o d y n a m i c therapy and bone m a r r o w transplantation. In: G o m e r C , ed. Future directions and applications o f p h o t o d y n a m i c therapy. V o l . IS 6. B e l l i n g h a m , W A : S P I E O p t i c a l E n g i n e e r i n g Press, 1990:209-218. 303. H e n d e r s o n B W , W a l d o w S M , Potter W R , „ e t a l . Interaction o f p h o t o d y n a m i c therapy and hyperthermia: tumor response and c e l l survival studies after treatment o f m i c e in v i v o . C a n c e r R e s e a r c h 1985; 45:6071-7. 304. M a L W , M o a n J , Steen H B , et al. A n t i - t u m o u r activity o f p h o t o d y n a m i c therapy in c o m b i n a t i o n w i t h m i t o m y c i n C i n nude m i c e w i t h h u m a n c o l o n adenocarcinoma. B r i t i s h Journal o f C a n c e r 1995; 71:950-6. 305. C i n c o t t a L, Szeto D , L a m p r o s E, et a l . B e n z o p h e n o t h i z i n e and B e n z o p o r p h y r i n derivative c o m b i n a t i o n phototherapy effectively eradicates large m u r i n e sarcomas. P h o t o c h e m & P h o t o b i o l 1996; 63:229-237. 306. G a n t c h e v T G , Brasseur N , v a n L i e r J E . C o m b i n a t i o n t o x i c i t y o f etoposide (VP-16) and photosensitisation w i t h a water-soluble a l u m i n i u m phthalocyanine i n K 5 6 2 human l e u k a e m i c cells. B r i t i s h Journal o f C a n c e r 1996; 74:1570-7. 307. C o w l e d P A , M a c k e n z i e L, F o r b e s IJ. P h a r m a c o l o g i c a l m o d u l a t i o n o f p h o t o d y n a m i c therapy w i t h hematoporphyrin derivative and light. C a n c e r R e s 1987; 47:971-974. 308. N a h a b e d i a n M Y , C o h e n R A , C o n t i n o M F , et al. C o m b i n a t i o n c y t o t o x i c chemotherapy w i t h cisplatin or d o x o r u b i c i n and p h o t o d y n a m i c therapy i n m u r i n e tumors. Journal o f the N a t i o n a l C a n c e r Institute 1988; 80:739-43. 309. W a l d o w S M , H e n d e r s o n B W , D o u g h e r t y T J . H y p e r t h e r m i c potentiation o f p h o t o d y n a m i c therapy e m p l o y i n g P h o t o f r i n I and II: c o m p a r i s o n o f results u s i n g three a n i m a l t u m o r m o d e l s . Lasers in Surgery & M e d i c i n e 1987; 7:12-22. 310. Schepartz S A . A n t i t u m o r screening procedures o f the N a t i o n a l C a n c e r Institute. Japanese Journal o f A n t i b i o t i c s 1977; 3 0 Suppl:35-40. 311. C o o p e r S, B r o x m e y e r H E . C l o n o g e n i c methods i n vitro f o r the enumeration o f granulocyte-macrophage progenitor cells ( C F U - G M ) i n h u m a n bone m a r r o w and mouse bone m a r r o w and spleen. J . T i s s . C u l t . M e t h 1 9 9 1 ; 13:77-82. 3 1 2 . . M o s m a n n T . R a p i d colorimetric assay f o r cellular g r o w t h and s u r v i v a l : application to proliferation and c y t o t o x i c i t y assays. J I m m u n o l o g i c a l M e t h o d s 1983; 65:55-63. :  313. M c H a l e A P , M c H a l e L. U s e o f a tetrazolium based c o l o r i m e t r i c assay i n assessing photoradiation therapy in vitro. C a n c e r Letter 1988; 41:315-321. 314. G a r l a n d J M . M u r i n e hematopoietic factor- dependent and stem c e l l lines. In: D o y l e A , G r i f f i t h s J B , N e w e l l D G , eds. C e l l & T i s s u e C u l t u r e : L a b o r a t o r y Procedures. V o l . 2. S a l i s b u r y : W i l e y , 1 9 9 3 : 2 1 B : 7.1-26. 315. M e s s n e r H A . M u l t i p o t e n t stem cells i n vitro. In: G o l d e D W , ed. H e m a t o p o i e s i s . N e w Y o r k : C h u r c h i l l L i v i n g s t o n e , 1984:73. 316. E y e r P, P o d h r a d s k y D . E v a l u a t i o n o f the m i c r o m e t h o d f o r determination o f glutathione u s i n g enzymatic c y c l i n g and E l l m a n ' s reagent. A n a l y t i c a l B i o c h e m i s t r y 1986; 153:57-66.  190  317. R i c h t e r A M , W a t e r f i e l d E, Jain J K , et a l . In vitro evaluation o f phototoxic properties o f f o u r structurally related b e n z o p o r p h y r i n derivatives. P h o t o c h e m . & P h o t o b i o l 1990; 5 2 : 4 9 5 - 5 0 0 . 318. M a L W , M o a n J , B e r g K, et al. Potentiation o f p h o t o d y n a m i c therapy b y m i t o m y c i n C i n cultured h u m a n c o l o n a d e n o c a r c i n o m a cells. R a d . Res 1993; 134:22-28. 319. K o s t r o n H , S w a r t z M R , M a r t u z a R L . P h o t o d y n a m i c therapy is potentiated b y C o 6 0 and intratumoral injection o f h e m a t o p o r p h y r i n derivative. Journal o f NeuroO n c o l o g y 1988; 6:185-91. 320. C h e n Q , C h e n H , S h a p i r o H , et a l . S e q u e n c i n g o f c o m b i n e d h y p e r t h e r m i a and p h o t o d y n a m i c therapy. R a d i a t i o n Research 1996; 146:293-7. 321. N e l s o n JS, L i a w L H L , L a h l u m R A , et a l . U s e o f m u l t i p l e photosensitizers and wavelengths during p h o t o d y n a m i c therapy: A new approach to enhance tumor eradication. J . N a t l . C a n . Inst 1 9 9 0 ; 82:868-873. 322. K r o s l G , K o r b e l i k M , K r o s l J , et al. Potentiation o f p h o t o d y n a m i c therapy-elicited antitumor response b y l o c a l i z e d treatment w i t h granulocyte-macrophage colony-stimulating factor. C a n c e r Research 1996; 56:3281-6. 323. B a a s P, van G e e l IP, Oppelaar H , et a l . E n h a n c e m e n t o f p h o t o d y n a m i c therapy b y m i t o m y c i n C : a p r e c l i n i c a l and c l i n i c a l study. B r i t i s h Journal o f C a n c e r 1996; 73:945-51. 324. B o o s e r D J , H o r t o b a g y i G N . A n t h r a c y c l i n e antibiotics i n cancer therapy. D r u g s 1994; 4 7 : 2 2 3 - 2 5 8 . 325. B o s e R, V e r h e i j M , H a i m o v i t z - F r i e d m a n A , et al. C e r a m i d e synthase mediates D a u n o r u b i c i n - i n d u c e d apoptosis: an alternative m e c h a n i s m s f o r generating death signals. C e l l 1995; 82:405-414. 326. T e p p e r C G , Jayadev S, L i u B , et a l . R o l e f o r ceramide as an endogenous mediator o f Fas- i n d u c e d c y t o t o x i c i t y . P r o c . N a t l . A c a d . S c i . U S A 1 9 9 5 ; 9 2 : 8 4 4 3 - 8 4 4 7 . 327.  W a n g C Y , M a y o M W , B a l d w i n J , A . S . T N F - and cancer therapy- i n d u c e d  apoptosis: potentiation b y i n h i b i t i o n o f N F - K p . S c i e n c e 1996; 2 7 4 : 7 8 4 - 7 8 7 . 328. L i p t o n S A . Janus faces o f N F - K B : neurodestruction versus neuroprotection. Nature M e d i c i n e 1997; 3:20-21. 329. D a s K C , W h i t e C W . A c t i v a t i o n o f N F - K B b y antineoplastic agents: role o f protein kinase C. J . B i o l . C h e m 1997; 272:14914-14920. 330. Jain R K . 1995 W h i t a k e r L e c t u r e : d e l i v e r y o f m o l e c u l e s , particles, and cells to s o l i d tumors. A n n a l s o f B i o m e d i c a l E n g i n e e r i n g 1996; 24:457-73. 331. O c h s n e r M . P h o t o p h y s i c a l and p h o t o b i o l o g i c a l processes i n the p h o t o d y n a m i c therapy o f tumours. J o f h o t o c h e m & P h o t o b i o l B: B i o l o g y 1 9 9 7 ; 39:1-18. 332. S h u l o k JR, W a d e M H , L i n C W . S u b c e l l u l a r l o c a l i z a t i o n o f h e m a t o p o r p h y r i n derivative i n bladder tumor cells in culture. P h o t o c h e m . & P h o t o b i o l 1990; 51:451-457.  191  333. G o m e r C J . P r e c l i n i c a l e x a m i n a t i o n o f first and second generation photosensitizers used i n p h o t o d y n a m i c therapy. P h o t o c h e m i s t r y & P h o t o b i o l o g y 1 9 9 1 ; 54:1093-107. 334. K e s s e l D , Jeffers R, F o w l k e s J B , et a l . E f f e c t s o f s o n o d y n a m i c and p h o t o d y n a m i c treatment o n cellular thiol levels. Journal o f Photochemistry & P h o t o b i o l o g y . B - B i o l o g y 1996; 32:103-6. 335. K e l l e y E E , Buettner G R , B u r n s C P . P r o d u c t i o n o f l i p i d - d e r i v e d free radicals i n L 1 2 1 0 murine l e u k e m i a cells is an early oxidative event in the p h o t o d y n a m i c action of Photofrin®. P h o t o c h e m . & P h o t o b i o l 1 9 9 7 ; 65:576-580. 336. B e l l n i e r D A . Potentiation o f p h o t o d y n a m i c therapy in m i c e w i t h recombinant h u m a n tumor necrosis factor-alpha. Journal o f Photochemistry & P h o t o b i o l o g y . B B i o l o g y 1 9 9 1 ; 8:203-10. 337. M y e r s R C , L a u B H , K u n i h i r a D Y , et a l . M o d u l a t i o n o f h e m a t o p o r p h y r i n derivative-sensitized phototherapy w i t h corynebacterium p a r v u m in murine transitional c e l l c a r c i n o m a . U r o l o g y 1989; 33:230-5. 338. C a s t r o D J , Saxton R E , H a g h i g h a t S, et a l . T h e synergistic effects o f rhodamine123 and merocyanine-540 laser dyes on h u m a n tumor c e l l lines: a n e w approach to laser phototherapy. O t o l a r y n g o l o g y - H e a d & N e c k Surgery 1993; 108:233-42. 339. C h e n Q , C h e n H , H e t z e l F W . T u m o r oxygenation changes post-photodynamic therapy. P h o t o c h e m i s t r y & P h o t o b i o l o g y 1996; 63:128-31. 340. M i y o s h i N , M a t s u m o t o N , H i s a z u m i H , et al. T h e effect o f h y p e r t h e r m i a on m u r i n e l e u k a e m i a cells in c o m b i n a t i o n w i t h p h o t o d y n a m i c therapy. International Journal o f H y p e r t h e r m i a 1988; 4:203-9. 341. M a L W , B e r g K, D a n i e l s e n H E , et al. E n h a n c e d antitumour effect o f p h o t o d y n a m i c therapy by m i c r o t u b u l e i n h i b i t o r s . C a n c e r Letters 1996; 109:129-39. 3 4 2 . . Y a m a z a k i T , Sieber F. T h e a l k y l - l y s o p h o s p h o l i p i d , E T - 1 8 - 0 C H 3 synergistically enhances the M e r o c y a n i n e 540-mediated photoinactivation of l e u k e m i a cells: implications f o r the extracorporeal p u r g i n g o f autologous hematopoietic stem cells. B o n e M a r r o w Transplantation 1997; 19:113-9. 343. Christensen T , F e r e n K , M o a n J , et al. P h o t o d y n a m i c effects o f h a e m a t o p o r p h y r i n derivative on s y n h r o n i z e d and asynchronous cells of different o r i g i n . B r . J . C a n c e r 1 9 8 1 : 44:717-724. ' 344. B a a s P, M i c h i e l s e n C , O p p e l a a r H , et al. Enhancement of interstitial p h o t o d y n a m i c therapy by m i t o m y c i n C and E 0 9 i n a mouse tumour m o d e l . International Journal o f C a n c e r 1994; 56:880-5. 345. V i g e v a n i A , W i l l i a m s o n M J . D o x o r u b i c i n . In: F l o r e y K, ed. A n a l y t i c a l profiles o f d r u g substances. N e w Y o r k : A c a d e m i c Press, 1980:246-270. 346. L a n k s K W , G a o JP, S h a r m a T . P h o t o d y n a m i c enhancement o f d o x o r u b i c i n c y t o t o x i c i t y . C a n c e r C h e m o t h e r . P h a r m a c o l 1994; 35:17-20. 347.  M e i s t e r A , A n d e r s o n M E . G l u t a t h i o n e . A n n . R e v . B i o c h e m 1 9 8 3 ; 52:711-760.  192  348. A r r i c k B A , Nathan C F . Glutathione m e t a b o l i s m as a determinant o f therapeutic e f f i c a c y : A r e v i e w . C a n c e r Res 1984; 44:4224-4232. 349. D u s r e L, M i m n a u g h E G , M y e r s C E , et al. Potentiation o f d o x o r u b i c i n c y t o t o x i c i t y b y buthionine s u l f o x i m i n e in multidrug-resistant h u m a n breast tumor cells. C a n c e r R e s 1989; 4 9 : 5 1 1 - 5 1 5 . 350. P e n d y a l a L, Perez R, W e i n s t e i n A , et a l . E f f e c t o f glutathione depletion on the cytotoxicity o f cisplatin and iproplatin i n a h u m a n m e l a n o m a c e l l line. C a n c e r Chemother. Pharmacol 1997;40:38-44. 351. L i n F, G e i g e r P G , G i r o t t i A W . Selenoperoxidase-mediated cytoprotection against merocyanine 540-sensitized photoperoxidation and p h o t o k i l l i n g o f l e u k e m i a cells. C a n c e r R e s e a r c h 1 9 9 2 ; 52:5282-90. 352. G o l d e D W , G a s s o n J C . H o r m o n e s that stimulate the g r o w t h o f b l o o d c e l l s . Scientific A m e r i c a n 1988; 259:62-71. 353. R e i s s m a n n K R . Studies on the m e c h a n i s m s o f erythropoietic stimulation i n parabiotic rats d u r i n g h y p o x i a . B l o o d 1 9 5 0 ; 5:372-376. 354. G o u g h N M , G o u g h L, M e t c a l f D , et al. M o l e c u l a r c l o n i n g o f c D N A e n c o d i n g a murine hematopoietic growth regulator: granulocyte- macrophage c o l o n y stimulating factor. Nature 1984; 3 0 9 : 7 6 3 - 7 6 6 . 355. G o l d e D W . H e m a t o p o i e t i c g r o w t h factors—an o v e r v i e w . International Journal o f Cell Cloning 1990;8:4-10. 356.  G o l d e D W . T h e stem c e l l . S c i . A m 1 9 9 1 ; 265:86-93.  357. D e m e t r i G D . H e m a t o p o i e t i c growth factors: current k n o w l e d g e , future prospects. Current p r o b l e m s i n cancer 1992; X V I : 179-259. 358. M e t c a l f D . T h e m o l e c u l a r control o f c e l l d i v i s i o n , differentiation c o m m i t m e n t and maturation in haematopoietic cells. Nature 1989; 339:27-30. 359. K a u s h a n s k y K. T h r o m b o p o i e t i n : b i o l o g i c a l and p r e c l i n i c a l properties. L e u k e m i a 1996; 10:S46-8. 360. P u i C H , B o y e t t J M , H u g h e s W T , et al. H u m a n granulocyte colony- s t i m u l a t i n g factor after i n d u c t i o n chemotherapv in children w i t h acute l y m p h o b l a s t i c l e u k e m i a . N e w E n g l . J M e d 1997; 3 3 6 : 1 7 8 1 - 1 7 8 7 . 361. H o e l z e r D . H e m a t o p o i e t i c g r o w t h factors- not whether, but w h e n and where. N e w E n g l . J M e d 1997; 336:1822-1824. 362. B r o x m e y e r H E . Suppressor c y t o k i n e s and regulation o f m y e l o p o i e s i s - b i o l o g y and possible c l i n i c a l uses. A m . J . Pediatr. H e m a t o l . O n c o l . 1 9 9 2 ; 14:22-30. 363. L e n f a n t M , W d z i e c z a k - B a k a l a J , Guittet E, et a l . Inhibitor o f hematopoietic pluripotent stem cell proliferation: purification and determination o f its structure. P r o c . Natl. A c a d . S c i . U S A 1989; 86:779-782. 364. V e i b y O P , L o C a s t r o S, B h a t n a g a r P, et a l . I n h i b i t i o n o f enriched stem cells i n v i v o and i n vitro b y the hemoregulatory peptide S K & F 1 0 8 6 3 6 . S t e m C e l l s 1996; 14:215-24.  193  365. B o n n e t D , Cesaire R, L e m o i n e F, e t a l . T h e tetrapeptide A c S D K P , an i n h i b i t o r o f the cell-cycle status f o r normal human hematopoietic progenitors, has no effect o n l e u k e m i c cells. E x p e r i m e n t a l H e m a t o l o g y 1992; 20:251-5. 366.  B o n n e t D , L e m o i n e F M , N a j m a n A , et a l . .Comparison o f the i n h i b i t o r y effect o f  A c S D K P , T N F - a , T G F - p \ and MIP-1 a on m a r r o w - p u r i f i e d C D 3 4 + progenitors. E x p Hematol 1995;23:551-556. 367. M o s e r M H , P a u k o v i t s W R . H a e m o p r o t e c t i o n against cytostatic drugs b y stem c e l l i n h i b i t i o n . T i P S 1 9 9 1 ; 12:304-310. 368. M a r s h a l l E. H a e m o p o i e t i c stem cell inhibition: potential for dose intensification. E u r o p e a n J C a n c e r 1 9 9 5 ; 31 A : 1586-1591. 369. M o r e b J , Z u c a l i JR, Z h a n g Y , et al. R o l e o f aldehyde dehydrogenase i n the protection o f hematopoietic progenitor cells f r o m 4 - h y d r o p e r o x y c y c l o p h o s p h a m i d e b y interleukin 1 /3 and t u m o r necrosis factor. C a n c e r R e s 1992; 52:1770-1774. 370. Eastgate J , M o r e b J , N i c k H S , et a l . A role f o r manganese superoxide dismutase in radioporotection o f hematopoietic stem cells b y interleukin-1. B l o o d 1993; 81:639-646. 371. E v a n s T . D e v e l o p m e n t a l b i o l o g y o f hematopoiesis. H e m a t o l o g y - O n c o l o g y C l i n i c s o f N o r t h A m e r i c a 1997; 11:1115-1147. 372. L e n f a n t M , G r i l l o n C , R i e g e r K J , et a l . F o r m a t i o n o f acetyl-Ser-Asp-Lys-Pro, a new regulator o f the hematopoietic system, through e n z y m a t i c processing o f t h y m o s i n beta 4. A n n a l s o f the N e w Y o r k A c a d e m y o f S c i e n c e s . V o l . 6 2 8 , 1991:115-25. 373. Pradelles P, Frobert Y , C r e m i n o n C , et al. D i s t r i b u t i o n o f a negative regulator o f haematopoietic stem c e l l proliferation ( A c S D K P ) and t h y m o s i n beta 4 i n mouse tissues. F E B S Letters 1 9 9 1 ; 289:171-5. 374. B o g d e n A E , Carde P, de Paillette E D , et a l . A m e l i o r a t i o n o f chemotherapy-induced toxicity b y cotreatment w i t h A c S D K P , a tetrapeptide inhibitor o f hematopoietic stem c e l l p r o l i f e r a t i o n . A n n a l s o f the N e w Y o r k A c a d e m y o f Sciences. V o l . 6 2 8 , 1991:126-39. 375. L i o z o n E, V o l k o v L, C o m t e L, et a l . A c S D K P serum concentrations vary d u r i n g chemotherapy in patients w i t h acute m y e l o i d leukaemia. B r i t i s h Journal o f H a e m a t o l o g y 1995; 89:917-20. 376. E z a n E, C a r d e P, L e K e r n e a u J , et al.' P h a r m c o k i n e t i c s in healthy volunteers and patients o f N A c - S D K P (seraspenide), a negative regulator o f hematopoiesis. D r u g M e t a b o l i s m & D i s p o s i t i o n 1994; 22:843-8. 377. Z u c a l i JR, M o r e b J , G i b b o n s W , et a l . R a d i o p r o t e c t i o n o f hematopoietic stem cells b y interleukin-1. E x p H e m a t o l 1994; 22:130-135. 378. D o u a y L, H u C , Giarratana M C , et a l . A m i f o s t i n e improves the a n t i l e u k e m i c therapeutic index o f m a f o s f a m i d e : i m p l i c a t i o n s f o r bone m a r r o w p u r g i n g . B l o o d 1995; 86:2849-55. 379. S h p a l l E J , S t e m m e r S M , H a m i L, et a l . A m i f o s t i n e ( W R - 2721) shortens the engraftment p e r i o d o f 4- H y d r o p e r o x y c y c l o p h o s p h a m i d e - p u r g e d bone m a r r o w i n breast  194  cancer patients r e c e i v i n g high- dose chemotherapy w i t h autologous bone m a r r o w support. B l o o d 1994; 8 3 : 3 1 3 2 - 3 1 3 7 . 380. R o b i n s o n S, L e n f a n t M , W d z i e c z a k - B a k a l a J , et al. T h e m e c h a n i s m o f action o f the tetrapeptide A c e t y l - N- Ser- A s p - L y s - P r o ( A c S D K P ) i n the c o n t r o l o f haematopoietic stem c e l l p r o l i f e r a t i o n . C e l l P r o l i f 1992; 25:623-632. 381. v a n der V i j g h W J , Peters G J . Protection o f n o r m a l tissues f r o m the c y t o t o x i c effects o f chemotherapy and radiation b y amifostine ( E t h y o l ) : p r e c l i n i c a l aspects. Seminars i n O n c o l o g y 1994; 21:2-7. 382. Patchen M L . A m i f o s t i n e plus granulocyte colony- stimulating factor therapy enhances recovery f r o m supralethal radiation exposures: p r e c l i n i c a l experience i n animals m o d e l s . E u r . J C a n c e r 1 9 9 5 ; 3 1 A S u p p l 1:S 17-21. 383. K y o i z u m i S, M c C u n e J M , N a m i k a w a R. D i r e c t evaluation o f radiation damage i n h u m a n hematopoietic progenitor cells i n v i v o . R a d i a t i o n Research 1994; 137:76-83. 384. T h o m a s E D , Storb R, C l i f t R A . B o n e - m a r r o w transplantation. N e w E n g l J M e d 1975; 2 9 2 : 8 3 2 - 8 4 3 , 895-902. 385. Testa N , H e n d r y J , M o l i n e u x G . L o n g - term bone m a r r o w damage after c y t o t o x i c treatment: stem cells and m i c r o e n v i r o n m e n t . In: Testa N , G a l e R, eds. H e m a t o p o i e s i s : long- term effects o f chemotheray and radiation. N e w Y o r k : M a r c e l D e k k e r , 1988:75. 386. G a r d n e r R V , A s t l e C M , H a r r i s o n D E . H e m a t o p o i e t i c precursor c e l l exhaustion i n a cause o f proliferative defect i n p r i m i t i v e hematopoietic stem cells ( P H S C ) after chemotherapy. E x p . H e m a t o l 1997; 25:495-501. 387. G u i g o n M , J . Y M , E n o u f J , et a l . Protection o f m i c e against lethal doses o f 1arabinofuranosyl- cytosine b y pluripotent stem c e l l inhibitors. C a n c e r R e r s 1982; 4 2 : 6 3 8 . 388. G u i g o n M , B o n n e t D , L e m o i n e F, et al. I n h i b i t i o n o f h u m a n bone m a r r o w progenitors b y the synthetic tetrapeptide A c S D K P . E x p . H e m a t o l 1990; 18:1112-1115. 389. B o n n e t D , L e m o i n e F M , K h o u r y E, et al. R e v e r s i b l e i n h i b i t o r y effects and absence o f t o x i c i t y o f the tetrapeptide acetyl- N- Ser- A s p - L y s - P r o ( A c S D K P ) i n h u m a n longterm bone m a r r o w culture. E x p . H e m a t o l 1992; 2 0 : 1 1 6 5 - 1 1 6 9 . 390. Z u c a l i J , M o r e b J , W e i n e r R. E f f e c t o f IL-1 and T N F - a l p h a o n early progenitor cells: i m p l i c a t i o n s f o r b o n e m a r r o w purging. B o n e M a r r o w Transplantation 1 9 9 1 ; 7:140. 391. Z u c a l i JR, M o r e b J , B a i n C . Protection o f cells capable o f reconstituting long-term bone m a r r o w stromal cultures f r o m 4 - h y d r o p e r o x y c y c l o p h o s p h a m i d e b y interleukin 1 and t u m o r necrosis factor. E x p . H e m a t o l 1992; 20:969-973. 392.  H a r r i s E D . R e g u l a t i o n o f antioxidant e n z y m e s . F A S E B J 1992; 6 : 2 6 7 5 .  393. M o r e l F, S z i l v a s s y SJ, T r a v i s M , et al. P r i m i t i v e hematopoietic cells i n m u r i n e bone m a r r o w express the C D 3 4 antigen. B l o o d 1996; 88:3774-3784. 394. N o v i t z k y N , M o h a m m e d R. Alterations i n the progenitor c e l l p o p u l a t i o n f o l l o w recovery f r o m m y e l o a b l a t i v e therapy and bone m a r r o w transplantation. E x p . H e m a t o l 1997; 25:471-477.  195  395. C o o p e r F R , C o o p e r M R . P r i n c i p l e s o f m e d i c a l o n c o l o g y . In: H o l l e b A I , F i n k D J , M u r p h y G P , eds. C l i n i c a l o n c o l o g y . A t l a n t a : T h e A m e r i c a n C a n c e r Society, 1991:47-69. 396. D e e g H J , S e i d e l K, H o n g D S , et a l . In v i v o radioprotective effect o f A c S D K P on canine m y e l o p o i e s i s . A n n . H e m a t o l 1 9 9 7 ; 7 4 : 1 1 7 - 1 2 2 . 397. W i e r e n g a P K , K o n i n g s A W T . G o r a l a t i d e ( A c S D K P ) selectively protects m u r i n e hematopoietic progenitors and stem cells against hyperthermic damage. E x p . H e m a t o l 1996; 2 4 : 2 4 6 - 2 5 2 . 398. G r i l l o n C , B o n n e t D , M a r y J Y , et a l . T h e tetrapeptide A c S e r A s p L y s P r o (Seraspenide), a hematopoietic i n h i b i t o r , m a y reduce the in vitro t o x i c i t y o f 3'-azido-3'd e o x y t h y m i d i n e to h u m a n hematopoietic progenitors. S t e m C e l l s 1993; 11:455-64. 399. M o n p e z a t JP, f r i n d e l E. Further studies o n the b i o l o g i c a s l activities o f the C F U - S inhibitory tetrapeptide A c S D K P . I. T h e precise point o f the c e l l c y c l e sensitive to A c S D K P . Studies on the effect o f A c S D K P on G M - C F C and on the possible i n v o l v e m e n t o f Tl y m p h o c y t e s i n A c S D K P response. E x p . H e m a t o l 1989; 17:1077-1080. 400. B e n - H u r E, Rosenthal I. P h o t o s e n s i t i z e d inactivation o f C h i n e s e hamster c e l l s b y phthalocyanines. P h o t o c h e m . & P h o t o b i o l 1 9 8 5 ; 4 2 : 1 2 9 - 1 3 3 . 401. F i e d o r o w i c z M , G a l i n d o JR, J u l l i a r d M , et al. E f f i c i e n t p h o t o d y n a m i c action o f v i c t o r i a blue B O against the h u m a n l e u k e m i c c e l l lines K- 562 and T F - 1. P h o t o c h e m . & P h o t o b i o l 1 9 9 3 ; 58:356-361. 402. G a n t c h e v T G , U r u m o v IJ, van l i e r JE. O n the relationship between rate o f uptake of P h o t o f r i n and cellular repsonses to p h o t o d y n a m i c treatment in vitro. C a n c e r b i o c h e m i s t r y B i o p h y s i c s 1994; 14:23-34. 403. S c h m i d t - E r f u r t h U , D i d d e n s H , B i r n g r u b e r R, et a l . P h o t o d y n a m i c targeting o f h u m a n retinoblastoma cells u s i n g covalent low-density lipoprotein conjugates. B r i t i s h Journal o f C a n c e r 1997; 75:54-61. 404. P h a r m i n g e n . T e c h n i c a l data sheet (catalog # 0 9 4 3 2 D ) . San D i e g o , C A : P h a r m i n g e n , 1996/97. 405. H u n t D W C , J i a n g H J , L e v y J G , et a l . Sens i ti vi ty o f activated m u r i n e peritoneal macrophages to p h o t o d y n a m i c k i l l i n g w i t h b e n z o p o r p h y r i n derivative. P h o t o c h e m & P h o t o b i 1 9 9 5 ; 61:417-421. 406. Sharkey. S M , W i l s o n B C , M o o r e h e a d R, et al. M i t o c h o n d r i a l alterations i n p h o t o d y n a m i c therapy-resistant cells. C a n c e r Research 1993; 53:4994-9. 407. W i l s o n B C , O l i v o M , S i n g h G . S u b c e l l u l a r l o c a l i z a t i o n o f Photofrin® and a m i n o l e v u l i n i c acid and p h o t o d y n a m i c cross- resistance in Vitro i n radiation- i n d u c e d f i b r o s a r c o m a cells sensitive or resistant to Photofrin- mediated p h o t o d y n a m i c therapt. Photochem. & Photobiol 1997; 65:166-176. 408. H u n t e r T , Pines J . C y c l i n s and C a n e r II: C y c l i n D and C D K inhibitors c o m e o f age. C e l l 1 9 9 4 ; 7 9 : 5 7 3 - 5 8 2 . 409.  H e i c h m a n K A , Roberts J M . R u l e s to replicate by. C e l l 1994; 79:557-562.  196  410. B e r g K, M o a n J . L y s o s o m e s and m i c r o t u b u l e s as targets f o r photochemotherapy o f cancer. P h o t o c h e m . & P h o t o b i o l 1997; 6 5 : 4 0 3 - 4 0 9 . 411. H e d l e y D W , C h o w S. E v a l u a t i o n o f methods for measuring cellular glutathione content u s i n g f l o w cytometry. C y t o m e t r y 1994; 15:349-358. 412. K e h r e r JP, L u n d L G . C e l l u l a r r e d u c i n g equivalents and o x i d a t i v e stress. F r e e R a d i c a l B i o l o g y & M e d i c i n e 1994; 17:65-75. 413. C h a t l a n i P T , N u u t i n e n PJ, T o d a N , et a l . Selective necrosis i n hamster pancreatic tumours u s i n g p h o t o d y n a m i c therapy w i t h phthalocyanine photosensitisation. B r . J S u r g 1992; 7 9 : 7 8 6 - 7 9 0 . 414. W e l l s W W , R o c q u e P A , X u D P , et a l . A s c o r b i c acid and c e l l s u r v i v a l o f a d r i a m y c i n resistant and sensitive M C F - 7 breast tumor cells. Free R a d i c a l B i o l o g y & M e d i c i n e 1995; 18:699-708. 415. G o m e r C J , L u n a M , Ferrario A , et a l . Increased transcription and translation o f heme oxygenase i n C h i n e s e hamster fibroblasts f o l l o w i n g p h o t o d y n a m i c stress or P h o t o f r i n II incubation. P h o t o c h e m i s t r y & P h o t o b i o l o g y 1 9 9 1 ; 53:275-9. 416. L i n F, G i r o t t i A W . Hyperresistance o f l e u k e m i a cells to p h o t o d y n a m i c inactivation after long-term exposure to h e m i n . C a n c e r Research 1996; 56:4636-43. 417. L e n a r d o M J , B a l t i m o r e D. N F - K B : A pleiotropic mediator o f i n d u c i b l e and tissuespecific gene control. C e l l 1989; 58:227-229. 418.  B o l a n d M P , Foster SJ, O ' N e i l l L A J . D a u n o r u b i c i n activates NFK(3 and induces  K(3- dependent gene expression in H L - 6 0 p r o m y e l o c y t i c and Jurkat T l y m p h o m a cells. Journal o f B i o l o g i c a l C h e m i s t r y 1997; 272:12952-12960. 419. B a l d w i n A S , Jr. T h e N F - k a p p a B and I k a p p a B proteins: n e w discoveries and insights. A n n u a l R e v i e w o f I m m u n o l o g y 1 9 9 6 ; 14:649-83. 420. Schreck R, R i e b e r P, B a e u e r l e P A . R e a c t i v e o x y g e n intermediates as apparently w i d e l y used messengers i n the activation o f the N F - k a p p a B transcription factor and H I V 1. E M B O Journal 1 9 9 1 ; 10:2247-58. 42.1. . Schreck R, M e i e r B, M a n n e l D N , et al. Dithiocarbamates as potent inhibitors o f nuclear factor kappa B activation in intact cells. Journal of E x p e r i m e n t a l M e d i c i n e 1992; 17-5:1181-94. 422. O s t h o f f - S c h u l z e K , Fiers W . O x y g e n radicals as second messengers. T r e n d s i n C e l l B i o l o g y 1991; 1:150. 423. L o s M , Schenk H , H e x e l K, et a l . IL-2 gene expression and N F - k a p p a B activation through C D 2 8 requires reactive o x y g e n production by 5-lipoxygenase. E M B O Journal 1995; 14:3731-40. 424.  Schieven G L , K i r i h a r a J M , M y e r s D E , et a l . Reactive o x y g e n intermediates activate  N F - K B in a tyrosine kinase-dependent m e c h a n i s m and in c o m b i n a t i o n w i t h vanadate activate the p 5 6 l k and p 5 9 f y tyrosine kinases in human l y m p h o c y t e s . B l o o d 1993; 82:1212-1220. c  n  197  425. A n d e r s o n M S , K a l y a n a r a m a n B, F e i x J B . E n h a n c e m e n t o f m e r o c y a n i n e 540mediated phototherapy b y salicylate. C a n c e r R e s 1 9 9 3 ; 53:806-809. 426. K o p p E, G h o s h S. I n h i b i t i o n o f N F - k a p p a B by s o d i u m salicyclate and aspirin. Science 1994;265:956-959. 427. P i e r c e J W , R e a d M A , D i n g H , et a l . Salicylates inhibit I k a p p a B-alpha p h o s p h o r y l a t i o n , endothelial-leukocyte adhesion m o l e c u l e expression, and neutrophil transmigration. Journal o f I m m u n o l o g y 1996; 156:3961-9. 428. T r a u l D L , A n d e r s o n G S , B i l i t z J M , et a l . Potentiation o f m e r o c y a n i n e 540mediated p h o t o d y n a m i c therapy by salicyclate and related drugs. P h o t o c h e m . & P h o t o b i o l 1995; 6 2 : 7 9 0 - 7 9 9 . 429. B h a t i a R, V e r f a i l l i e C M , M i l l e r JS, et al. A u t o l o g o u s transplantation therapy f o r c h r o n i c m y e l o g e n o u s l e u k e m i a . B l o o d 1997; 89:2623-2634.  198  

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