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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 PURGING USING BPD- M E D I A T E D P H O T O D Y N A M I C T H E R A P Y by Stephen Y i p B. Sc. The Un i ve rs i t y o f B r i t i sh C o l u m b i a 1990 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y , in T H E F A C U L T Y O F G R A D U A T E S T U D I E S D E P A R T M E N T O F M I C R O B I O L O G Y & I M M U N O L O G Y W e accept this thesis as con fo rming to the required standard T H E T J 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 September, 1998 © Stephen Y i p , 1998 In presenting this thesis in partial fulf i lment of the requirements for an advanced degree at The Univers i ty of B r i t i sh Co lumb ia , I agree that the L ib ra ry shall make it freely available for reference and study. I further agree that permission for extensive copy ing o f this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copy ing or publ icat ion of this thesis for f inancial gain shall not be a l lowed without my written permiss ion . Department c f M i c rob io logy & Immunology The Univers i ty of Br i t ish C o l u m b i a 6174 Univers i ty B l v d Vancouver , Canada V 6 T 1Z3 Date: 18 September 1998 A B S T R A C T Photodynamic therapy ( PDT) using the second generation photosensitiser benzoporphyrin derivative monoac id ring- A ( B P D , Verteporfin®) offers an attractive alternative to purging of contaminating neoplastic cells during autologous haematopoietic stem cel l transplantation. Enhancement of P D T cytotoxici ty was attempted using two independent approaches: combinat ion treatment wi th doxorubic in (Dox) and selective protection of normal haematopoietic cells using the tetrapeptide N - A c S D K P . The murine leukaemic cel l l ine L 1 2 1 0 was 45 x more susceptible to the sequenced combinat ion regimen of 1 h 2.5 u M D o x incubation fo l lowed by P D T mediated by 5.0 ng/ml B P D and 15 J /cm 2 red l ight (Dox-> P D T ) than normal murine haematopoietic cel ls. The significant enhancement in cytotoxicity was dependent on the concentration of B P D used as we l l as on the sequence of treatment. Spec i f ica l ly , it was observed wi th 5.0 ng/ml but not with 2.5 ng/ml B P D and only when D o x was used before P D T . The reverse sequence of PDT-> D o x and simultaneous D o x / P D T treatment were not associated with enhanced k i l l i ng . Interestingly, L 1 2 1 0 cells were much more susceptible to the combinat ion therapy than normal D B A / 2 haematopoietic progenitor cells wh ich offered interesting therapeutic impl icat ions. B P D uptake and cel lular G S H content, appeared not to be responsible for the potentiation of L 1 2 1 0 k i l l i ng in the Dox-> P D T sequence. Next , the potential of selective stem cel l protection in B P D - mediated P D T was investigated. Preincubation of D B A / 2 bone marrow cel ls wi th 100 n M N - A c S D K P for 1.5 h signif icantly protected resultant colony formation f rom P D T by a factor of 1.5- 2 over cel ls that were incubated with control peptides or with tissue culture med ium. Interestingly, L 1 2 1 0 cells were not protected by N - A c S D K P and the control peptides. However , N - A c S D K P -mediated photoprotection d id not appear to extend to earlier murine haematopoietic cells and stem cells as demonstrated by the long- term bone marrow culture ( L T B M C ) assay. The same f indings of differential photoprotection were also demonstrated in human haematopoietic cells. The mechanism of protection appeared to be mediated by inhibit ion of progression to S phase of the ce l l cyc le since depletion of cyc l ing D B A / 2 bone marrow cells with 50 p M cytosine arabinoside (ara- C ) resulted in cel ls more tolerant to subsequent P D T cytotoxicity. The above two approaches enhanced selective B P D - mediated P D T cytotoxicity and therefore maybe of merit in the c l in ica l use o f P D T in purging. ii A B S T R A C T II LIST OF T A B L E S X LIST OF FIGURES XII LIST OF ABBREVIATIONS X V A C K N O W L E D G E M E N T S XIX I N T R O D U C T I O N 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. Diagnost ics and Therapeutics .2 c. Support ive therapy 4 d. Bone marrow transplantation. 6 1.2 LEUKAEMIA : THE STATE OF T H E ART 1 0 a. C l i n i ca l advances 10 b. Diagnost ics and therapeutics 10 i. Ep idemio logy & aetiology 10 i i . N o v e l chemotherapeutics 11 i i i . Ant ibody- based diagnostics & therapeutics 13 iv. Ce l l- mediated immunotherapy 15 v. Mur ine models of human leukaemia 16 v i . Gene- based therapeutics 17 c. Support ive therapy 18 i. G- C S F and G M - C S F support for chemotherapy- induced neutropaenia 18 i i . Infection and haemostasis control 19 in 1.3 TRANSPLANTATIONS OF HAEMATOPOIETIC STEM C E L L S : CURRENT S T A T U S 20 a. Bone M a r r o w Transplantation ( B M T ) 21 i. A l logene ic bone marrow transplantation ( a l B M T ) 21 1. H L A matching 21 2. Prevention and the management of graft- versus- host disease ( G v H D ) 22 3. Identification of minor histocompatibi l i ty antigens ( m H C ) 23 4. Nove l indications for a l B M T in nonmalignant and malignant disorders 24 i i . Auto logous bone marrow transplantation ( a u B M T ) 25 1. a u B M T and chronic myelogenous leukaemia ( C M L ) 26 b. Peripheral b lood stem cel l transplantations 27 i. Auto logous transplantation of P B S C in cancer patients 28 c. U m b i l i c a l cord b lood transplantation 28 d. Future directions ' 29 1.4 EXPLOITABLE DIFFERENCES B E T W E E N N O R M A L AND L E U K A E M I C C E L L S 29 a. Leukaemogenesis 29 b. Genetic differences 30 c. Differences in intracellular s ignal l ing and ce l l death pathways 31 d. Response to extracellular regulatory signals 32 e. Ce l l surface markers, adhesion properties and the haematopoietic microenvironment33 f. Susceptibi l i ty to cytotoxic agents 33 1.5 PURGING IN AUTOLOGOUS S T E M C E L L TRANSPLANTATIONS 34 a. Introduction 34 b. Purg ing strategies 35 c. Combinat ion purging 36 d. Purg ing outside the traditional paradigm 37 1.6 PHOTODYNAMIC THERAPY (PDT) AND ITS R O L E IN PURGING 3 7 a. Reactive oxygen intermediates (ROIs) in b io logy 37 b. P D T : cytotoxic i ty and b io logica l responses 38 iv c. In v i vo uses of P D T 40 d. The Benzoporphyr in derivatives- second generation photosensitisers 40 e. E x v i vo uses of P D T 41 f. Bone marrow purging 42 g. C l i n i c a l trials 43 OBJECTIVES AND RATIONALE 4 3 EXPERIMENTAL PROCEDURES 46 2.1 EXPERIMENTAL REAGENTS 4 6 a. Benzoporphyr in derivative monoac id r ing A ( B P D , Verteporfin®) 46 b. Chemica l reagents 46 c. T issue culture reagents 46 d. Reagents for co lony assays and long- term marrow cultures 47 e. Fluorescence activated cel l sorting ( F A C S ) reagents 47 f. Synthetic peptides 48 2.2 LIGHT SOURCE ...4 8 2.3 EXPERIMENTAL ANIMALS 4 9 a. M i c e 49 2.4 C E L L PREPARATION 5 0 a. M o u s e bone marrow cells 50 b. Mouse leukaemic cel l l ines 50 c. Pr imary human haematopoietic cells 51 d. Human leukaemic cel l l ines 51 2.5 C Y T O T O X I C T R E A T M E N T OF C E L L S 51 a. P D T treatment of murine bone marrow cel ls and L I 210 cel ls for short term evaluations (combination experiments) 51 b. P D T treatment of normal and leukaemic murine haematopoietic cells preincubated wi th peptides (short- term co lony assay) 52 v c. P D T treatment of murine bone marrow cells for long- term bone marrow cultures 53 d. P D T treatment of human normal haematopoietic and leukaemic cells preincubated with peptides (short- term colony assay) 53 e. ara- C treatment of D B A / 2 bone marrow cells preincubated wi th peptides 53 f. P D T of D B A / 2 bone marrow cells preincubated with 50 u M ara- C 54 2.6 C Y T O T O X I C I T Y ASSAYS 5 4 a. A g a r co lony assay of treated murine bone marrow cel ls 54 b. C o l o n y assay o f L 1 2 1 0 cel ls 55 c. Short-term cytotoxici ty assay ( M T T assay) 55 d. One step long- term bone marrow culture ( L T B M C ) of murine haematopoietic cel ls56 e. Co lony assay of treated normal and leukaemic human haematopoietic cells 57 f. Co lony assay of the human leukaemic cel l l ine K 5 6 2 57 2.7 PHOTOMETRIC AND SPECTROSCOPIC ANALYSES 5 8 a. Spectrophotometric analysis 58 b. Spectrofluorimetric analysis of the interaction between 1BPD and D o x 58 2.8 C E L L ANALYSIS BY FLUORESCENCE A C T I V A T E D C E L L S SORTING (FACS) 58 2.9 FLUORESCENT MICROSCOPY 5 9 3.0 ANALYSIS OF INTRACELLULAR GLUTATHIONE (GSH) CONTENT 6 0 a. Tietze enzymatic assay 60 3.1 STATISTICAL ANALYSIS 60 a. Ana lys is o f co lony assay data f rom P D T / D o x combinat ion experiments 60 b. Ana lys is of co lony assay data f rom the inhibitory peptide pretreatment experiments61 CHAPTER 3: COMBINED T R E A T M E N T OF MURINE NORMAL HAEMATOPOIET IC AND LEUKAEMIC C E L L S WITH DOXORUBICIN AND PDT: CYTOTOXICITY STUDY 6 2 v i 3.1 ABSTRACT 62 3.2 INTRODUCTION 63 3.3 RESULTS 65 Single agent cytotoxicity 65 Combinat ion experiments with B P D - mediated P D T and D o x : simultaneous and sequenced treatments of D B A / 2 haematopoietic progenitor cells 72 Combinat ion experiments with B P D - mediated P D T and D o x : simultaneous and sequenced treatments of L 1 2 1 0 leukaemic cells 72 Differential susceptibility of L 1 2 1 0 cells and D B A / 2 haematopoietic progenitors to Dox-> P D T sequenced combination treatment 77 Statistical analysis of combination treatments 77 DISCUSSION 84 CHAPTER 4: COMBINED T R E A T M E N T OF MURINE NORMAL HAEMATOPOIET IC AND LEUKAEMIC C E L L S WITH DOXORUBICIN AND PDT: MECHANISTIC STUDY 87 4.1 A B S T R A C T 87 4.2 INTRODUCTION 88 4.3 RESULTS 9 0 Photophysical properties o f B P D and D o x 90 Intracellular localisations of B P D and D o x in the human leukaemic cel l l ine K 5 6 2 . . . 97 The role o f excitation wavelength (A,ex) has on the photobleaching o f B P D in the presence o f D o x 101 Uptake of B P D by L 1 2 1 0 cells as affected by the presence of D o x in different combinat ion regimens 107 Deplet ion of cel lular glutathione (GSH ) in L 1 2 1 0 cells by preincubation with D o x . 111 4.4 DISCUSSION 113 v i i CHAPTER 5: SELECTIVE PREPROTECTION OF NORMAL HAEMATOPOIETIC COMMITTED PROGENITIOR CELLS FROM PDT WITH N-ACSDKP: CYTOTOXICITY STUDY 118 5 .1 A B S T R A C T 1 1 8 5 . 2 I N T R O D U C T I O N 1 1 9 5 . 3 R E S U L T S 1 2 1 Photoprotective effect of N - A c S D K P on D B A / 2 haematopoietic progenitors 121 Absence of N - A c S D K P mediated photoprotection of L 1 2 1 0 cel ls 121 Statistical analyses of 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 cel ls 133 Selective photoprotection of normal human bone marrow cells but not leukaemic cells f rom P D T by N - A c S D K P 138 D I S C U S S I O N 1 4 1 CHAPTER 6: PREPROTECTION OF NORMAL HAEMATOPOIETIC CELLS WITH N-ACSDKP: MECHANISTIC STUDY.... 144 6 .1 A B S T R A C T 1 4 4 6 . 2 I N T R O D U C T I O N 1 4 5 6 . 3 R E S U L T S 1 4 8 B P D uptake by D B A / 2 bone marrow cells preincubated with 100 n M N - A c S D K P . 148 Inhibit ion of the proliferative activity o f murine C F U - G M progenitors by 100 n M N-A c S D K P . .152 Ce l l cycle inhibit ion of D B A / 2 haematopoietic progenitor cells mediated by 100 n M N-A c S D K P 152 Glutathione level in D B A / 2 bone marrow cel ls treated with 100 n M N - A c S D K P . . .157 v i i i 6 . 4 D I S C U S S I O N 1 5 9 C H A P T E R 7: D I S C U S S I O N 1 6 4 P D T C O M B I N A T I O N T H E R A P Y 1 6 4 N - A C S D K P - M E D I A T E D P H O T O P R O T E C T I O N F R O M B P D 1 6 5 F A C T O R S A F F E C T I N G P D T C Y T O T O X I C I T Y 1 6 7 Th io l s , a- tocopherol , and haeme oxygenase 167 N F - K B and its role in P D T cytotoxic i ty 167 Summary 169 R E F E R E N C E S 1 7 1 ix LIST O F T A B L E S Table 2.1 Table 3.1a Table 3.1b Table 3.2a Table 3.2b Table 5.1a Table 5.1b Table 5.2a Title Reagents used for F A C S analysis Bonferron i (all- pairwise) multiple comparison test . o f clonogenicity data of L 1 2 1 0 leukaemic cells: significance of B P D doses Page 48 T w o way analysis o f variance ( A N O V A ) of cytotoxici ty data f rom 80 D B A / 2 haematopoietic progenitor cells treated wi th the three different drug combinations invo lv ing D o x and B P D - mediated P D T : signif icance of treatment sequence and B P D dose Statistical analyses o f data f rom the different D o x / P D T 81 combinations f rom D B A / 2 haematopoietic cells: Bonfer ron i (all-pairwise) mult iple comparison testings o f B P D concentrations and treatment sequences T w o way analysis of variance ( A N O V A ) of cytotoxici ty data f rom 82 L I 2 1 0 leukaemic cells treated with the three different drug combinations invo lv ing D o x and B P D - mediated P D T : signif icance of treatment sequence and B P D dose Statistical analyses o f data f rom the different Dox/ P D T 83 combinations f rom L I 2 1 0 leukaemic cells: Bonfer ron i multiple-comparison testings of B P D concentrations and treatment sequences Bonferron i (all- pairwise) multiple compar ison test of 129 clonogenicity data of D B A / 2 haematopoietic progenitors: s ignif icance of B P D doses Bonferron i (all- pairwise) multiple comparison test of 130 clonogenicity data of D B A / 2 haematopoietic progenitors: signif icance of peptides 131 Table 5.2b Bonferron i (all- pairwise) multiple compar ison test of 132 clonogenicity data o f L 1 2 1 0 leukaemic cells: signif icance of peptides Table 5.3 Suspension cel l numbers o f weekly harvests o f long term bone 135 marrow culture of D B A / 2 haematopoietic cells preincubated in the presence or absence of 100 n M N - A c S D K P fo l lowed by B P D -mediated P D T Table 5.4 Numbers of C F U - G M progenitor colonies generated f rom 138 x nonadherent cells harvested weekly f rom long term bone marrow culture of D B A / 2 haematopoietic cells preincubated in the presence or absence of 100 n M N - A c S D K P fo l l owed by B P D -mediated P D T Table 6.1 B P D uptake in the C D 3 4 expressing subpopulat ion o f D B A / 2 151 bone marrow cells x i LIST OF FIGURES Title Page F igure 3.1 The effect of B P D - mediated P D T on the clonogenicity of normal 66-7 D B A / 2 haematopoietic progenitors and the • leukaemic cell l ine L 1 2 1 0 F igure 3.2 Cytotoxic i ty of B P D in the absence or presence of 15 J / cm 2 red 68 light irradiation on L 1 2 1 0 cells as determined by the M T T cytotoxicity assay F igure 3.3 The effect of D o x and 15 J / cm 2 red light on the clonogenicity o f 69-D B A / 2 haematopoietic progenitor cells and L 1 2 1 0 leukaemic cells 70 F igure 3.4 Cytotoxic i ty o f D o x in the absence or presence o f 15 J / cm 2 red 71 light irradiation on L 1 2 1 0 cells as determined by the M T T cytotoxicity assay F igure 3.5 Surv ival fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 73 cells to combinations of 5 ng/ml B P D and 2.5 u M D o x in conjunct ion with 15 J /cm 2 o f red l ight exposure F igure 3.6 Surv iva l fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 74 cells to combinations o f 5 ng/ml B P D and 1.25 u M D o x in conjunct ion with 15 J /cm 2 o f red l ight exposure F igure 3.7 Surv iva l fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 75 cells to combinations of 2.5 ng/ml B P D and 2.5 u M D o x in conjunct ion with 15 J /cm 2 o f red l ight exposure F igure 3.8 Surv iva l fraction of D B A / 2 haematopoietic progenitors and L 1 2 1 0 76 cells to combinations of 2.5 ng/ml B P D and 1.25 u M D o x in conjunct ion with 15 J / cm 2 o f red l ight exposure F igure 3.9 Differential k i l l ing o f L 1 2 1 0 cells over D B A / 2 progenitors in the 79 different P D T / D o x combinations F igure 4.1 Chemica l structure o f the benzoporphyr in derivative monoac id 92 ring- A ( B P D , Verteporfin®) F igure 4.2 Chemica l structure of Doxorub i c in (Dox) 93 F igure 4.3 Absorpt ion spectra of 1 pg/ml B P D and 10 u M D o x in 10 % HI- 94 F C S / P B S F igure 4.4a Fluorescence emiss ion spectra o f B P D in the presence of D o x 95 (550- 700 nm) xn F igure 4.4b Fluorescence emission profi le o f B P D in the presence o f D o x 96 (650- 700 nm) F igure 4.5 Fluorescence micrograph of K 5 6 2 leukaemic cells incubated with 98 B P D F igure 4.6 Fluorescence micrograph of K 5 6 2 leukaemic cells incubated with 99 D o x F igure 4.7 Fluorescence micrograph of K 5 6 2 leukaemic cells incubated with 100 B P D and D o x F igure 4.8 Photobleaching o f 10 ng/ml B P D in the presence o f different 103 concentrations of D o x (A,ex = 440 nm) F igure 4.9 Summary o f B P D photobleaching in the presence of different 104 concentrations of D o x (440 n m excitation) F igure 4.10 -phe effect o f X e x on B P D photobleaching in the presence and ^ absence of. 5 u M D o x F igure 4.11 L igh t transmittance of 1 m M D o x in 10 % HI- F C S / P B S 106 F igure 4.12a Uptake o f B P D by L 1 2 1 0 cells as determined by 108 spectrofluorimetric measurements of cel l lysates F igure 4.12b Uptake o f D o x by L 1 2 1 0 cells as determined by 109 spectrofluorimetric measurement of cel l lysates F igure 4.13 Uptake of B P D by L 1 2 1 0 cells when incubated simultaneously 110 ( BPD/ Dox ) or after D o x (Dox-> B P D ) F igure 4.14 Cel lu lar glutathione levels in L 1 2 1 0 cells after 1 h incubation with 112 different doses of D o x F igure 5.1 Selective cytoprotective effects of N - A c S D K P on D B A / 2 122-haematopoietic progenitor cells subjected to B P D - mediated P D T 3 F igure 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 , 124 and medium on the clonogenicity o f D B A / 2 haematopoietic progenitor cells F igure 5.3 Absence of cytoprotective effects of N - A c S D K P on L 1 2 1 0 125-leukaemic cel l l ine subjected to B P D - mediated P D T 6 F igure 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 , 127 x i i i and medium on the clonogenicity of L 1 2 1 0 leukaemic cells F igure 5.5 One step long- term bone marrow culture ( L T B M C ) o f D B A / 2 136 bone marrow cel ls F igure 5.6 Compar ison of week 1 long- term bone marrow culture ( L T B M C ) 137 harvests of nonadherent cel ls f rom samples preincubated wi th 100 n M N - A c S D K P or med ium fo l lowed by P D T F igure 5.7 Cytoprotection conferred by 100 n M of N - A c S D K P against 140 subsequent B P D - mediated P D T was specific for normal human bone marrow cells F igure 6. la-c Representative F A C S histograms o f B P D uptake in control and 149-N - A c S D K P - treated murine bone marrow mononuclear cells 150 ( B M M N C ) F igure 6.2 Effect of 1.5 h preincubation with 100 n M N - A c S D K P had on the 154 cyc l ing status of C F U - G M f rom D B A / 2 mice F igure 6.3 Effect of 5.0 h preincubation wi th 100 n M N - A c S D K P had on the 155 cyc l ing status of C F U - G M f rom D B A / 2 mice F igure 6.4 Correlation between cell cyc l ing and susceptibil ity to B P D - 156 mediated P D T in D B A / 2 C F U - G M progenitors F igure 6.5 Cel lu lar glutathione content in D B A / 2 bone marrow cells 158 incubated w i th 0, 10, or 100 n M N - A c S D K P xiv LIST OF ABBREVIATIONS 4- H C 4- hydroperoxycyc lophosphamide 8- M O P 8- methoxypsoralen A C T H Adreno- corticotrophic hormone a l B M T Al logene ic one marrow transplantation A L D H A ldehyde dehydrogenase A L L Acute l ymphoid leukaemia A l S P c Sulphonated a lumin ium phthalocyanine A M L Acute myelo id leukaemia A N O V A Ana lys is of variance A P L Acute promyelocyt ic leukaemia A r a - C Cytosine arabinoside A T G Ant i thymocyte g lobul in A T R A A l l - trans retinoic acid a u B M T Auto logous bone marrow transplantation B C G Bac i l lus Calmette- Guer in B M M N C s Bone marrow mononuclear cells B M P Bone marrow purging B M T Bone marrow transplantation B P D Benzoporphyr in derivative monoac id ring- A B S A Bov ine serum a lbumin B S O Buthionine su lphoximine B u Busulphan C F U - G M Co lony fo rming unit- granulocyte & macrophage C F U - L Co lony forming unit- leukaemic cel l C M L Chronic mye lo id leukaemia C M V Cytomegalov i rus C S F Co lony stimulating factor C T L Cytotoxic T lymphocyte C W Cont inuous wavelength C Y Cyc lophosphamide XV C Y A Cyc lospor in A D H E Dihaematoporphyrin ether D I C Disseminated intravascular coagulation D M E M Du lbecco ' s modi f ied Eagle ' s med ium D T N B 5,5'- dithiobis- (2- nitrobenzoic acid) D o x Doxorub i c in hydrochloride E P O Erythropoietin E t N B S 5- ethylamino- 9- diethylaminobenzo[a] phenothiazinium chloride F A B French- Amer ican- Br i t i sh classif ication F A C S Fluorescence activated ce l l sorting F C S Foetal calf serum F I T C Fluorescein isothiocyanate G- C S F Granulocyte- colony stimulating factor G M - C S F Granulocyte & macrophage- colony stimulating factor G S H Glutathione G v H D Graft- versus- host- disease G v L Graft- versus- leukaemia HI- F C S Heat- inactivated- foetal calf serum H L A Human leukocyte antigen HOC1 Hypoch lorous acid H P C Haematopoietic progenitor cel l H P D Haematoporphyrin derivative H S C Haematopoietic stem cel l H U C Human umbi l i ca l cord b lood cells I C S B P Interferon consensus b inding sequence protein I F N a Interferon a M D M Iscove's M o d i f i e d Du lbecco 's M e d i u m L A K Lymphok ine activated k i l ler L D L L o w density l ipoprotein L D L r L o w density l ipoprotein receptor L T B M C Long- term bone marrow culture x v i M C 5 4 0 Merocyanine 540 M D R Mu l t id rug resistance M E S N A Sod ium 2- mercaptoethanesulfonate M F I M e a n fluorescence intensity M H C Major histocompatibil ity complex m H C minor histocompatibil ity complex M IP- l a Macrophage inflammatory protein- l a M L R M i x e d lymphocyte reaction M M C M i tomyc in C M n S O D Manganese superoxide dismutase M R D M i n i m a l residual disease M T T 3- [4,5- Dimethy l th iazol- 2- yl]- 2,5- diphenyltetrazol ium bromide M U D Matched unrelated donor N - A c S D K E N-Acetyl- Ser- Asp- Lys- A s p N - A c S D K P N-Acetyl- Ser- Asp- Lys- Pro N A C N-Acetyl- cysteine N C I National Cancer Institute N E M N- ethylmaleimide N F - K P Nuc lear factor- K(3 O D N Oligodeoxynucleotide P B S Phosphate buffered saline P B S C Peripheral b lood stem cel l P B S C T Peripheral b lood stem cel l transplantation P C R Polymerase chain reaction P D T Photodynamic therapy P D T C Pyrrol idone derivative of dithiocarbamate p E E D C K Py roG lu- G lu- Asp- Cys- L y s P H A - L C M phytohaemaggultinin stimulated- human leucocyte conditioned medium P H S C Pluripotent haematopoietic stem cel l P H ' Phi ladelphia chromosome xv i i P M T Photomult ipl ier tube P T K Protein tyrosine kinase P W M - S C C M Pokeweed mitogen- spleen cel l condit ioned med ium qRT- P C R Quantitative reverse transcriptase- polymerase chain reaction R A P A Rapamycin R B Retinoblastoma protein R B C Red b lood cel l R F L P Restrict ion fragment length po lymorph ism R h l 2 3 Rhodamine 123 rhIL- 2 Recombinant human interleukin- 2 RO Is Reactive oxygen species RT- P C R Reverse transcriptase- polymerase chain reaction S C A Sickle cel l anaemia S C I D Severe combined immunodef ic iency S D K P Ser- Asp- Lys- Pro S E M Standard error of the mean SLT- 1 Shiga- l ike toxin 1 S O D Superoxide dismutase s O D N Phosphorothioate- modi f i ed ol igodeoxynucleot ide T B I Total body irradiation T C D T cel l depletion T G F - P Transforming growth factor- (3 T I L Tumour infi ltrating lymphocyte T N F a Tumour necrosis factor a T N F r Tumour necrosis factor receptor T P O Thrombopoiet in xv i i i A C K N O W L E D G E M E N T S I wou ld l ike to thank the members of m y thesis committee: Drs . Noe l Buskard , D a v i d Do lph in , and M i k e G o l d . I wou ld also w ish to acknowledge the advice and support prov ided by Dr . An thony C h o w , in his capacity as the director of the U B C M.D.- Ph .D . program. I wou ld l ike to thank Dr . Ju l ia L e v y and L u c y . A n d lastly, P iggy Y i p , in his capacity as my fa i thful dog for the past 10 years. B a B a Ra. . . x i x B l o o d is the originat ing cause o f a l l men's diseases. The Ta lmud Baba Nathra, III. 58a I N T R O D U C T I O N 1.1 LEUKAEMIA : HISTORY a. Cl inica l observations The history of leukaemia in the past two centuries says as much about the disease itself as about the scientific advancements and paradigm shifts in the f ie ld of medicine. S i r W i l l i a m Osier, in his seminal work The Principles and Practice of Medicine, described the three components of disease as manifestations, pathogenesis, and cause. True appreciation of an il lness and therefore formulat ion of rational treatments require understandings of all three of the above. Unfortunately, the extent o f our medica l knowledge does not a l low us to fu l ly comprehend their combined signif icance. W e are often left with the most obvious facet of the disease tr i logy, the manifestations, wh ich was never more true than in Osier's days. The manifestations of a disease belong to the domain of signs, symptoms, and the treatments designed to ameliorate them. Symptomatic treatments were universal during Osier's t ime, some of which are st i l l in use today. In 1893, when the first edit ion of the textbook was published, physicians already had an idea that leukaemia constituted a distinct entity of the systemic disorders. 1 The first documented case of leukaemia was reported in 1827 by the French physic ian A l f r e d Ve lpeau. "The b lood was thick, l ike gruel... resembl ing in consistency and co lo r the yeast of red wine... One might have asked i f it were not rather laudable pus, m ixed with blackish co lor ing matter, than b l ood . " 1 Ten years later, gross observations were complemented wi th rudimentary microscopic examination of the b lood f rom leukaemic patients. Donne concluded that the proliferation of "mucous ce l l s " was responsible for the patient's hepatosplenomegaly. A t that t ime, there was still great controversy concerning the aetiology of leukaemia. M a n y prominent physicians bel ieved that the disease was a severe fo rm o f general pyaemia and therefore was suppurative in nature. V i r c h o w correctly defined leukaemia as a distinct disease with "an increase in the number of colorless b lood corpuscles to the extent that the red color of b lood turns into reddish, ye l low ish , or greenish wh i t e " and was associated with the f indings of hepatosplenomegaly and lymphadenopathy. H e also attempted to classify leukaemia into two categories: the splenic and lymphatic f o r m . 1 In 1854, V o g e l described in detail the signs and symptoms of leukaemia wh ich inc luded lethargy, diarrhoea, pal lor, hepatosplenomegaly, and lymphadenopathy in some cases. S igni f icant ly , V o g e l questioned whether the leukaemic b lood prof i le was caused by enlargements of the spleen, l iver, and lymphat ics or the other way around. In the late 1800's, W o o d w a r d introduced the anil ine dyes into diagnostic haematology. Ehr l i ch later exploited the differential staining abil ity of these dyes to identify the different b lood cel ls in both normal subjects and leukaemic patients; his most significant contribution to the f ie ld was the demonstration that leukaemia cou ld be diagnosed by simple b lood smears. Thus entered S i r W i l l i a m Osier's textbook in 1893. The manifestations of leukaemia were fa ir ly we l l understood and physicians were starting to focus their attention on the pathogenesis and aetiology of the disease. However , leukaemic treatments were sti l l symptomatic in nature. In fact, many were pall iative and inc luded " f resh air, good diet, and abstention f rom mental worry and care. . . " . 2 b. Diagnostics and Therapeutics The 20th century has brought significant progress in both the diagnosis and treatment of leukaemia. One important landmark was the discovery of the Phi ladelphia chromosome (PH' ) by N o w e l l and Hunger ford in I 960 . 3 P H ' is a product of the t(9; 22)(q34; q21) rearrangement in chronic myelogenous leukaemia ( C M L ) . Th is discovery not only prov ided a unique and reliable marker for C M L , it also paved the path for further discoveries in the molecular pathogenesis and aetiology o f this f o rm of leukaemia. Other 2 chromosomal abnormalities were soon l inked to different forms o f cancers; i ronical ly , the majority of chromosomal translocations are found to be associated with leukaemias. 4 Great strides were made in elucidating the cause of leukaemia f rom studies in the viral transmission of leukaemia in animals and f rom epidemiological studies of the patients exposed to radiation and chemicals. A t the same t ime, development of novel chemotherapy was being conducted at the Nat ional Cancer Institute (NCI ) under C . Go rdon Zubrod's d i rect ion. 1 The systemic nature of leukaemia precluded the effective use (except during oncological emergency or in local control) of surgery and radiation treatments; therefore, chemotherapy by default became the therapy of choice. Ear ly use of chemical compounds in the control of leukaemic growth was often empir ica l in nature. In 1907, Osier mentioned the use of potassium arsenic by his contemporaries in the management of leukaemia; the results, however, were predictably unpredictable. Eh r l i ch , pessimist ic in regard to the future of chemotherapy in cancer management, displayed a sign wi th the l ine f rom Dante's Inferno; " Abandon all hope all ye who enter here," prominently at the entrance to his laboratory. Pess imism concerning the success of leukaemia chemotherapy also was shared by W o g l o m half a century later. "Those who have not been trained in chemistry or medicine.. . may not realize how dif f icult the problem of cancer treatment really is. It is almost-not quite, but almost- as hard as f ind ing some agent that w i l l d issolve away the left ear, say, yet leave the right ear unharmed: So slight is the difference between the cancer cel l and its normal ancestor." 5 Research into alkylat ing agents such as busulphan and cyc lophosphamide fo l lowed rapidly after the init ial discovery of the use o f nitrogen mustard in leukaemia control . Busu lphan, developed by S i r A lexander Haddow, was found to be extremely myelosuppressive and was therefore useful in the control of excessive neutrophils in chronic phase C M L . 6 Ear ly observations of the b io logica l activities of corticosteroids had lead to the subsequent use of adreno- corticotrophic hormone ( A C T H ) and cortisone on patients wi th lymphocyt ic leukaemia. A t the same time, development of the different antimetabolites was underway. Fo l i c acid plays an important role in haematopoiesis; its deficiency leads to retarded haematopoiesis and its abundance accelerates the growth of leukaemic c e l l s . 7 ' 8 The therapeutic potential of antimetabolites was realised init ia l ly with aminopterin and later with 3 methotrexate, both fo l i c ac id antagonists. In 1948, H i tch ings and E l i o n , who later won the Nobe l pr ize for medic ine and phys io logy, developed the purine analogue 6- mercaptopurine which was subsequently used for the maintenance of patients wi th acute lymphocyt ic leukaemia ( A L L ) . 9 C l i n i ca l introduction of the pyr imid ine analogue cytosine arabinoside (Ara- C ) in 1963 signif ied a milestone in acute myelogenous leukaemia ( A M L ) management. 1 0 C o m m o n l y used in conjunction with anthracycline antibiotics such as daunorubicin or more recently idarubic in, Ara- C has been the mainstay of induct ion therapy for a variety o f A M L s . The next b ig breakthrough in therapeutics was the discovery of the v inca alkaloid vincristine and its extraordinary eff icacy in paediatric A L L when used in combinat ion with p r e d n i s o n e . 1 1 ' 1 2 D iscovery of the anthracycline antibiotics and the podophyl lotoxins further complemented an already potent arsenal o f drugs. Real isat ion of the potential of combinat ion chemotherapy in the early 1950's was due pr imar i ly to understanding o f cel lu lar m e t a b o l i s m . 1 3 - 1 4 Encourag ing results f rom experiments that used drugs to effect sequential and concurrent b lock ing of b iochemical pathways lead to the initiation of more ambitious c l in ica l t r i a l s . 1 4 The rationale for combinat ion therapy has changed somewhat since then and drugs wi th different mechanisms of action have been used together to increase the potency of treatment, decrease the combined adverse effects as we l l as the possibi l i ty of developing mult idrug r e s i s t ance . 1 5 ' 1 6 Current treatment protocols often cal l for combinat ions o f different chemotherapeutic agents at different stages of the disease so as to tailor the pharmacological profi les of the drugs to the unique growth dynamic and peculiarity o f the leukaemia. c. S u p p o r t i v e therapy Signif icant improvement in the prognosis of leukaemic patients in the period between 1900- 1970 was also due to the introduction of various anci l lary therapies. Advances in transfusion science a l lowed cl inic ians to provide "custom" haematological supports in the fo rm of packed red b lood cel l or platelet transfusions. Ear ly recognit ion of the relationship between thrombocytopaenia and bleeding diathesis in patients had lead to the c l in ica l introduction of platelet transfusions, init ia l ly as whole b lood and later as platelet concentrates. 1 7 Improvement in both purif icat ion and storage techniques also contributed to 4 the universal use o f platelet transfusions in leukaemic patients suffering f rom thrombocytopaenia. However , early recognit ion of the l ink between thrombocytopaenia and bleeding diathesis d id not lead to increased use o f platelet replacements. The delay was main ly due to technical inexperience in the handl ing of platelets ex vivo. In addit ion, scepticism concerning the merits of the procedure also contributed to delays in its implementation in the c l in ics. Paradoxica l ly , increased alio- transfusions of platelets lead to immunolog ica l l y mediated- "transfusion refractor iness" . 1 8 Th is problem was later ameliorated with the use of partially matched donors. Ironical ly, adequate control of haemostasis by platelet transfusions accentuated the problem of infections related to the under ly ing disease or chemotherapy- induced neutropaenia. The role of neutrophils in nonspecif ic immuni ty was clearly established in the 1800's. Correct ion o f neutropaenia not only decreases the incidence of life- threatening infect ions, it also permits the use of more aggressive chemotherapy. Strumia, in 1934, attempted to reinfuse leukocytes into a patient wi th unmitigated infection. H e described the miraculous effects that " leukocyte c ream" , a dubious mixture o f whole cel l and ce l l debris, had on the pat ient . 1 9 Later, " su rp lus " neutrophils f rom chronic phase C M L patients were used as supplements in neutropaenic patients, a rather adventurous fo rm of replacement therapy . 2 0 Increased use o f neutrophil transfusions exposed the recipient to the prob lem o f cytomegalovirus ( C M V ) transmission, even f rom asymptomatic donors with latent C M V in f ec t i ons . 2 1 - 2 2 Development of C M V infections in leukaemia patients is extremely problematic because of suppressed immuni ty caused by the disease and the cytotoxic treatments. The appropriate use of ant imicrobial therapy later supplanted the use of neutrophil transfusions in patients. Gera ld Bodey was an early advocate of the appropriate yet aggressive use of antibiotics in neutropaenic pat ients. 2 3 The emerging problem wi th fatal fungal infections in leukaemic patients mirrored that o f bacterial infection. A g a i n , the pr incipal culprit was persistence of the neutropaenic state initiated by the leukaemia wh i ch was sustained by the ever more aggressive myelosuppressive chemotherapy regimens. A retrospective review conducted by Baker in 1962 demonstrated the increased incidence of fatal fungal infections and concluded that "some factor of modern therapy is responsible for the fa ta l i t i es " . 2 4 L ibera l and invaluable use o f indwe l l ing catheters such as the H i c k m a n l ine also contributed to the emergence of systemic infections. Amphoter i c in B was the drug of choice f rom a smal l arsenal of antifungal agents. Unfortunately, the side effects were 5 horrendous and patients often compla ined of the "bake and shake" effects of "amphote r r ib l e " . 2 5 d. Bone marrow transplantation Appreciat ion of the curative potential of B M T in humans began not long after the dawn of the atomic age. Fear of the consequences of radiation exposures; inadvertently f rom c iv i l i an accidents or deliberately and more sinisterly, as a result of mi l i tary confl icts, perpetuated early research in human B M T . 2 6 In 1922 Fabricious- M o e l l e r demonstrated, albeit obl iquely, the principles o f B M T . He showed that shielding of the legs during lethal irradiation protected guinea pigs f rom subsequent bone marrow ap las i a . 2 7 Jacobson and colleagues repeated similar experiments in mice with splenic shie lding; in addit ion, they observed that intraperitoneal injections of splenocytes also rescued the irradiated an ima l s . 2 8 Meanwh i l e , Mor r i son and Samwick successfully treated a patient aff l icted with idiopathic aplastic anaemia with sternal injections o f donated bone marrow. They were, however, mistaken in their interpretation of the result in that they thought the donated marrow contained humoral factors that promoted the maturation of the diseased host haematopoietic compartments . 2 9 Recogni t ion o f a stable populat ion of haematopoietic cel ls of donor or ig in or ch imer ism posttransplantation and its role in the protection of the lethally irradiated host further strengthened the theoretical foundation of B M T . 3 0 Add i t iona l animal experiments also helped to define the relatively novel concepts of graft- versus- host disease ( G v H D ) , also known as secondary or runt disease, and immune t o l e r a n c e . 3 1 - 3 3 However , it was not unti l much later that the human equivalents of the mouse major histocompatibi l i ty antigens H- 2 were identif ied and the concept of human leucocyte antigen ( H L A ) matching was introduced into c l in ica l marrow t ransp lanta t ion . 3 4 - 3 6 In a landmark paper publ ished in 1957, E. Dona ld Thomas and colleagues demonstrated the feasibi l i ty o f c l in ica l marrow transplantation in leukaemia and more importantly, they showed evidence of transient marrow engraftment in a pat ient . 3 7 W i t h great prescience, the authors foresaw the use of B M T as a form of salvage regimen in h igh dose radiotherapy, 6 "In selected patients with disseminated neoplasia it may be advantageous to use total- body radiation in large dosage and to cover the resultant aplasia by marrow transplantation"; they also predicted the increasing demand for this novel fo rm of medical technology r iva l l ing that of kidney transplantations. Pr ior to 1968, al logeneic B M T s were performed without the benefits of H L A matching and the results were dismal . The rare cases o f success were later ascribed to fortuitous H L A matching between the host and the s ib l ing d o n o r . 2 6 In 1968, a patient with congenital severe combined immunodef ic iency disease ( SC ID) received marrow f rom a H L A - matched s ib l ing, thus heralding in a new era in c l in ica l marrow transplantation, one that is based on the rational selection of donors . 3 8 T o compensate for the paucity of genotypical ly H L A - matched s ib l ing donors, partly as a result of the l imi ted size of the Western fami ly and also a consequence of the enormous po lymorph ism o f the H L A haplotypes - different matching criteria were used. Comp lex algorithms were developed for the identif ication of marrow donors, starting f rom the best to the least compatible match i.e. f rom a monozygot ic twin donor to a H L A - matched unrelated donor ( M U D ) , respect ive ly . 3 9 Ut i l i sat ion of less r igorously matched marrow donors necessitated the use of more eff icacious immunosuppressive agents. Introduction of cyc lospor in A and later, a more effective cyclosporin A and methotrexate combinat ion signif icantly decreased the incidence of G v H D and hence improved the outcomes of al logeneic B M T s . 4 0 - 4 1 Development of new condit ioning regimens not only reflected the paradigm shifts in B M T , but also the newly acquired knowledge in immunob io logy and haematology. Or ig ina l ly intended for the rescue of v ict ims of accidental radiation exposure, B M T was later adapted as a fo rm of experimental therapy for endstage leukaemic patients. Subsequently, people with congenital or acquired def ic iency of the haematopoietic system also benefited f rom B M T . The purpose of the precondit ioning regimen is to achieve adequate immunosuppression of the host to effect graft acceptance, to " re l i eve " the marrow microenvironment of diseased host haematopoietic cel ls so that the donor cells can "take up residence"; and, in the case of a host with mal ignancy, to destroy a signif icant number of residual cancer c e l l s . 4 2 The need for unique precondit ioning regimens was evident f rom the different types of disease indicated above. Fo r example, patients with Fanconi 's anaemia, a form of congenital aplastic aplasia, are exceptionally sensitive to a lkylat ing agents therefore 7 necessitating a less severe round of pretransplant cond i t i on ing . 4 3 The funct ional status of the host's immune system, in addit ion to the degree of donor- host disparity, also determines the intensity of the regimen. Obv ious ly , a foetal host is different f rom an adult host wi th congenital immunodef ic iency and is quite different f rom a host with an intact immune system, most l ikely a leukaemic patient. Ear ly B M T recipients, wi th their restricted range of disease indications, were often precondit ioned with radiation (mostly unintentional) or h igh doses of chemotherapeutic agents. Later, wi th al logeneic B M T assuming a more "proact ive" role in the overall management of diseases such as congenital immunodef ic iency, novel and often more effective condit ioning regimens were developed. S ingle dose total body irradiation (TBI ) was or iginal ly designed to suppress the immune system and ablate the host marrow; later, fractionated T B I was used to reduce extramedullary t o x i c i t y . 4 4 Cyc lophosphamide ( C Y ) , a nitrogen mustard derivative, and antithymocyte g lobul in ( A T G ) are both potent immunosuppressants that do not appreciably ablate the marrow; therefore, regimens that util ise C Y or A T G were successful only in patients wi th intrinsic stem cel l dysfunct ion i.e. severe marrow aplasia. A transplant candidate suffering f rom T cel l- mediated aplastic anaemia wou ld require a regimen that both suppresses host immunity and ablates residual endogenous haematopoiesis. S ince busulphan (Bu) has the capabil ity to damage cel ls that reside in the G 0 /G, phases of the cel l cyc le , i.e. the quiescent stem cel l compartment, B u along wi th T B I became the rational choice for marrow ab l a t i on . 4 2 In 1968, Santos and Haghshenass out l ined a new regimen busulphan- cyc lophosphamide ( BuCy ) , wh i ch was subsequently used for the pretransplant condi t ion ing of various types of d i seases . 4 5 - 4 6 In the same year, B a ch and col leagues performed a successful allogeneic B M T on a paediatric patient suffering f rom Wiskott-A l d r i c h syndrome; donor marrow was obtained f rom a K L A - identical s i b l i n g . 4 7 Shortly thereafter, the first detailed study of allogeneic B M T in acute leukaemia was performed by the Fred Hutchinson group; H L A - matched marrows were infused into end stage leukaemic patients condit ioned with C Y and T B I fo l lowed by a two week anti- G v H D regimen of methotrexate 4 8 The history of leukaemia mirrors the progress of medic ine in general; it evo lved f rom an area of empir ica l observations and speculations to more substantive (and often more objective) investigations. Leukaemic therapy also improved f rom the early use of 8 alkylat ing agents to the development and incorporation of B M T into the overall -management plan of the disease. 9 1.2 LEUKAEMIA : THE STATE OF THE ART a. C l i n i c a l advances Recent developments in the f ie ld of leukaemic research have not only signif icantly contributed to the understanding of the disease but also, more importantly, have led to improvements in both the prolongation and the quality of l ife of the leukaemic patient. b. Diagnost ics and therapeutics i. Epidemiology & aetiology Ep idemio log ica l studies of ch i ldhood leukaemia in geographical regions exposed to high levels of radioactive fallout f rom Chernoby l further illustrated the correlation between foetal radiation exposure and an increased incidence of l eukaemia . 4 9 However , the contribution of previous paternal radiation exposure, wh ich implicates mutations in the male germ cel l compartment, to the increased incidence of ch i ldhood leukaemia is stil l cont rovers ia l . 5 0 F r om retrospective studies o f the occurrence of A L L in Br i t i sh chi ldren that were relocated during the Second W o r l d War , M e l Greaves proposed that common chi ldhood infections promote leukaemogenesis of haematopoietic precursor cells that have received mutations (first hit but silent ?) in utero.5*'52 The novel fus ion gene products BCR- ABL and PML- RARa are impl icated in the init iat ion and maintenance of chronic myelogenous leukaemia ( C M L ) and acute promyelocyt ic leukaemia ( A P L ) , respectively. In fact, countless chromosomal abnormalities have been attributed to the different types o f leukaemia 4 Unfortunately, the role of many o f these genomic events in the actual init iation and progression of leukaemia is not certain. S ince leukaemia, and cancer in general, results f rom mult iple genetic and sometimes epigenetic events, f ind ing the proverbial " smok ing g u n " has proven to be diff icult . One must appreciate the combined role of proto-oncogenes, tumour suppressor genes, and the environment in leukaemia pathogenesis. 10 However , there is little doubt that definit ive identif ication of an aberrant genetic event in the initiation and maintenance of leukaemia provides a unique target for molecular therapy. Technical advances in molecular diagnostics permit the sensitive detection of genetic changes in leukaemic cel ls, wh ich provide cl inicians with invaluable leukaemia- specif ic markers for the purpose of detecting m in ima l residual d isease. 5 3 Di f ferent mutagenic agents leave behind unique basepair alterations in the p53 tumour suppressor gene wh ich afford the investigator with clues as to the identity the aetiologic agent, thereby prov id ing an important tool in the emerging f ie ld of molecular ep idemio logy . 5 4 One has to realise that societal and pol icy changes often come as a result of the posit ive identif ication of leukaemogenic agents, especial ly in the face of " irrefutable scientif ic proofs" . Therefore, the improved understanding of the molecular events that underlie the initiation and maintenance of leukaemia w i l l not only translate into improved diagnostics and therapeutics in the c l i n i c , but also the overal l wel lness of soc ie ty . 5 5 //. Novel chemotherapeutics The search for novel chemotherapeutic agents diverges into two paths: identif ication of naturally occurr ing antineoplastic agents and the chemical synthesis of potentially useful cytotoxic compounds. The majority of the anticancer drugs in current c l in ica l use are naturally derived and are usually chemical ly modi f ied to achieve reasonable pharmacokinetic and pharmacodynamic characteristics. Late ly , signif icant developments in the f ie ld of combinatorial libraries have created large databases of potentially useful pharmacological agents. 5 6 > 5 7 A foreseeable use of this technology in leukaemia therapy wou ld be the production of a large number of compounds wh i ch cou ld potentially interact with the signal transduction machinery of the leukaemic ce l l . Meydan and colleagues have shown that the tyrphostin analogue A G - 4 9 0 , synthesised using traditional chemica l techniques, inhibited the growth of lymphoblast ic leukaemic cells in vitro and in vivo. Importantly, the authors showed that the compound selectively inhibited the J A K 2 protein tyrosine kinase, a downstream component of the cytokine receptor superfamily s ignal l ing cascade; J A K 2 is shown to be elevated in relapsing lymphoblast ic leukaemic cel ls, thus prov id ing a rational molecular target for future invest igat ions. 5 8 Several groups are 11 work ing on the development of other compounds wh ich cou ld inhibit the tyrosine kinase activity of the pathognomonic P210 BCR- ABL fus ion protein in C M L . Recent ly , D ruker and colleagues showed that the 2- phenylaminopyr imidine- based compound C G P 57148 selectively inhibited the growth of leukaemic cells f rom C M L patients with no deleterious effects on the colony formation of normal haematopoietic c e l l s . 5 9 Research into the myr iad signal transduction pathways of b lood cells w i l l hopeful ly identify more potential targets for pharmacological intervention. Screening of naturally derived compounds sti l l remains the backbone of chemotherapeutic drug discovery. Treatment of hairy cel l leukaemia was signif icantly improved wi th the discovery of the new purine analogues such as 2'- deoxycoformyc in , chlorodeoxyadenosine, and f ludarab ine . 6 0 Deve lopment of chemotherapy protocols based on all- trans retinoic acid ( A T R A ) , in combinat ion with traditional induction therapy, remains one of the major achievements of medical science in the late 20 t h century and illustrates that mutual ly beneficial cross talk exists between science and med i c ine . 6 1 L eo Sachs init ia l ly pioneered the theory that growth of some leukaemic clones cou ld be successfully control led with differentiation- induc ing chemica l compounds or cy tok ines . 6 2 ' 6 3 Later, Bre i tman and colleagues demonstrated that retinoic acid ( R A ) could induce the A M L cel l l ine H L - 60 to differentiate in vitro.64 Subsequently, A M L M 3 or acute promyelocyt ic leukaemia ( A P L ) became the obvious therapeutic candidate for R A - mediated differentiation therapy because of the cumulative in vitro data and the therapeutic urgency in its management. The use of oral A T R A successful ly control led life- threatening episodes of haemorrhagic diathesis of A P L by forc ing the differentiation of the leukaemic promyelocytes into endstage cells of the granulocyte lineage; c l inic ians also found that a " 3 + 7 " regimen of cytosine arabinoside /anthracycline fo l low ing A T R A therapy was successful in maintaining the remission. Apparent ly , leukaemic promyelocytes contain vast amounts of tissue factor whose release leads to v ic ious cycles of c lott ing and thrombolys is , a condit ion known c l in ica l l y as disseminated intravascular coagulopathy or D I C . Subsequent molecular studies demonstrated that the canonical t(15; 17) translocation of A P L results in the fusion of the retinoic acid receptor a gene (rara) wi th the pml gene. Ectopic cel lular expression of the fusion protein PML- RARa and inappropriate dimerisation with the normal b ind ing partners o f PAR expla ined partly the dysregulated growth of A P L cel ls ; however, the exact mechanisms of enforced differentiation of the 12 leukaemic cells by h igh dose A T R A is sti l l not c lear . 6 1 Reports f rom C h i n a suggested arsenic trioxide (As203) is effective in treating A P L patients refractory to A T R A and conventional chemotherapeutic regimens; in addit ion, Chen and colleagues recently demonstrated that As203 promotes apoptotic cel l death in A P L cells v i a the downregulation of the antiapoptotic protein BCL- 2 as we l l as modulat ion of the intracellular local isation of PML- RARa and PML.65 Recent developments in the area of tumour angiogenesis have lead to the discovery of posit ive and negative regulators of angiogenesis, as we l l as the appreciation of angiogenesis in tumour growth and metastasis . 6 6 Leukaemia has tradit ionally been v iewed as " b l o o d tumour" in wh ich angiogenesis is irrelevant; however, many o f the haematopoietic growth factors are angiogenic factors and vice ve rsa . 6 7 F o l kman and colleagues recently showed increased microvessel density in leukaemic bone marrow with the capil laries surrounded, by cords of leukaemic cel ls, an observation common ly made in so l id tumours; in addit ion, the levels of urinary basic f ibroblastic growth factor, a potent inducer of angiogenesis, were elevated in al l the leukaemic subjects studied ( A L L ) 6 8 Th is discovery has lead to c l in ica l trials of the use of angiostatin, a potent angiogenesis inhibitor derived f rom proteolytic cleavage o f f ibr inogen, on paediatric A L L patients who have fai led init ial therapy. iii. Antibody- based diagnostics & therapeutics The concept of eradication of neoplasm by the specif ic targeting of cancer cells first evolved from Ehr l i ch 's "mag ic bul le t " theor)' o f immune recognition. Initial opt imism in antibody- directed immunotherapy of leukaemia was tempered by various scientif ic and technical p r o b l e m s . 6 9 ' 7 0 The majority o f the problems associated wi th immunotherapy is due to the paucity of genuine leukaemia- specif ic antigens and subsequently the compromised choice of the target antigens. In addit ion, therapeutically effective eradication of min ima l residual disease requires the expression of the target antigen on leukaemic stem cel ls. Fortunately, judic ious selection of the disease and target antigen can sometimes circumvent the above problems. No rma l expression of the B cel l developmental antigens C D 19 and C D 2 0 are tightly regulated; both are not expressed on haematopoietic stem cells 13 and plasma cel ls. However , B ce l l l ymphoma and B cel l precursor leukaemia express abundant levels of the above two antigens, thus offer ing unique targets for immunotherapy without signif icantly affecting haematopoiesis and antibody secretion. K a m i n s k i and colleagues demonstrated the eff icacy of 1 3 II- an t i B l (CD20 ) radioimmunoconjugates on B ce l l l ymphoma patients refractory to chemotherapy in a recent t r i a l . 7 1 Another approach, using immunoconjugates of the tyrosine kinase inhibitor genistein- ant iB4 ( C D 19), was successful ly used by U c k u n ' s group in abrogating the growth o f lethal doses o f human B ce l l precursor leukaemia, wh ich constitutes 7 5 % of all adult A L L s , in a SCTD mouse m o d e l . 7 2 U s i n g radioimmunoconjugates against the panhaematopoietic antigen C D 4 5 , a group at the Fred Hutchinson Cancer Research Centre was able to deplete B M T recipients of host lymphohaematopoietic cells for the purpose of pretransplant condit ioning whi le sparing the radiosensitive organs such as the l u n g s . 7 3 - 7 4 Identif ication of therapeutically useful monoclonal antibodies and the production of humanised mouse antibody w i l l s ignif icantly improve the uti l i ty of in vivo immunotherapy 7 5 Monoc lona l antibodies have also been used therapeutically in the ex vivo manipulat ion of autologous or al logeneic bone marrow grafts for the purpose of leukaemic ce l l purging or the removal of G v H D -mediat ing donor T lymphocytes, respect i ve ly . 7 6 Regardless, widespread use of immunoconjugates in leukaemia treatment is far f rom reality. Development of antibody- based diagnostics has made more of an impact in the overal l c l in ica l management of leukaemia, much more so than the antibody- based therapeutics discussed above. Haematopoietic maturation is accompanied by the appearance and disappearance of developmentally- restricted cel l surface antigens. Leukaemogenesis f rom normal haematopoietic precursor cells means that leukaemic cells wou ld share many of the lineage antigens present on normal cel ls, hence the.diff iculty in the specif ic targeting of leukaemic cells in therapeutics. Immunophenotyping of peripheral b lood and bone marrow aspirates with panels of antibodies against lineage- specif ic antigens has dramatically improved the specificity of traditional morphologic and cytochemical diagnosis; for example, the definit ive diagnosis of F A B M 7 acute megakaryoblastic leukaemia using monoclonal antibodies that recognise the platelet antigens glycoprotein Ib and l i b- I l i a . 7 7 ' 7 8 In addit ion, antibody- based diagnostics also provide informat ion with significant prognostic implicat ions such as the identif ication of biphenotypic leukaemia, wh ich coexpresses both mye lo id and l ympho id markers and may require more aggressive 14 treatment. 7 9 Improved detection of leukaemic cel ls with f low cytometry also permits the sensitive detection of m in ima l residual disease during and posttherapy, wh ich hopeful ly should translate into better c l in ica l management of the pat ient . 8 0 iv. Cell- mediated immunotherapy Bac i l lus Calmette- Guer in ( B C G ) and other adjuvants such as Corynebacterium Parvum have been used experimental ly as immunomodulatory agents for leukaemia, unfortunately with m ixed results due to the nonspecif ic nature of the el ic ited immune r e s p o n s e . 8 1 - 8 3 Several groups were actively invo lved in therapeutic trials in wh i ch patients were vaccinated with allogeneic leukaemic cells (l ive or irradiated), often coadministered w i th adjuvants . 8 4 However , realisations of the immense potential of cel lular immuni ty in leukaemia therapy came only after publ icat ion of results f rom cl in ical trials conducted by Steven Rosenberg's group at the N C I . Lymphocy tes isolated f rom the draining l ymph nodes or biopsy samples of patients wi th so l id tumours were stimulated in vitro wi th recombinant human interleukin- 2 (rhIL- 2). Reinject ion of these activated lymphokine-activated k i l ler ( L A K ) cells or tumour- inf i l trat ing lymphocytes (TILs) , often fo l lowed with in vivo administration of rhIL- 2, was found to have tremendous therapeutic effects on selected patient p o p u l a t i o n s . 8 5 , 8 6 Deve lopment of c l in ica l protocols that take advantage of the above discoveries in leukaemia patients first came about in trials studying the eff icacy of in vivo administration of rhIL- 2 . 8 7 " 8 9 Surface M H C I presentation of antigenic peptides consist ing of the fus ion region of the translocation product on leukaemic cel ls, for example BCR- ABL in C M L , presents the possibi l i ty of ce l l mediated anti leukaemia response in the patient. Presence of fusion peptide- specif ic cytotoxic T lymphocytes ( C T L s ) has been demonstrated; however, the c l in ica l s igni f icance o f these cel ls remains amb iguous . 9 0 - 9 1 Investigators soon realised that active and adoptive immunotherapy could be incorporated into B M T s for various malignancies. Hand l i ng of the autologous or al logeneic graft ex vivo offers unique opportunities for the generation of a leukaemia- specif ic immune response in vitro as a fo rm of bone marrow purging in a u B M T and in v i vo to eradicate min ima l residual 15 disease in both a l B M T and a u B M T patients postgraft reinfusion. These forms of immune manipulat ion w i l l be discussed in a later section. v. Murine models of human leukaemia Bas ic immunolog ica l research pr incipal ly focuses on the development and effector funct ion of members o f the l ympho id compartment. However , one can easily appreciate that information garnered f rom the study of the immune system can be appl ied to leukaemia research (and not just restricted to l ympho id leukaemia). One example is the development of the S O D - hu mode l o f human lymphopoies is by M c C u n e ' s t e a m . 9 2 ' 9 3 Th is concept was later extended to the study o f human haematopoietic development in a murine host. The SC ID- hu system is an invaluable tool for the study of human haematopoiesis because the available in vitro assays can only detect progenitor cells with l imi ted proliferating and replating potentials; the murine system also offers an opportunity to test the in vivo effects of various cytokines and therapeutic protocols on human b lood ce l l deve lopment . 9 4 The same system later a l lowed for the identif ication o f a rare populat ion of human mye lo id leukaemic precursor cells that behave s imi lar ly in the immunocompromised murine hosts as in the or iginal human pat ients. 9 5 The above examples illustrate the importance of the murine system for the study of human haematopoiesis; in addit ion, phylogenetic closeness between the two species means the sharing of genes, developmental pathways, and signal l ing pathways. Genetic alterations in transgenic mice and gene knockout mice provide numerous clues to the normal and abnormal funct ioning o f the human haematopoietic system. Express ion (increased or ectopic) or deletion of genes impl icated in human leukaemias in the mouse w i l l often recapitulate the human phenotype. Neoplast ic development in ber- abl transgenic mice and in mice reconstituted with haematopoietic stem cells infected with ber- abl carrying retroviruses partially recapitulated the human p h e n o t y p e . 9 6 - 9 8 However , the occasional unexpected results serve to remind the investigators of the complex i ty of the b io logica l system and the fact that cancer often results f rom polygenetic cooperativity. Serendipitous discovery by Tak M a k ' s group o f natural leukaemia resistance in the T c R V y l . U y 4 C Y 4 transgenic mice w i l l certainly spur additional 16 research into the role of the y8 T ce l l population in immune su rve i l l ance . " In a separate study, Tak M a k ' s group deleted the mouse equivalent of the gene coding for the human Hodgk in ' s Disease antigen C D 3 0 ; the resulting impairment in lymphocyte cel l death signal l ing and thymic deletion offers an intr iguing gl impse into the normal regulatory role of this protein in the organism, and possib ly its role in H o d g k i n ' s D i s e a s e . 1 0 0 Recent ly , Hol tschke and colleagues engineered a knockout mouse strain that faithful ly recapitulates the human equivalent of C M L . 1 0 1 M i c e without functional interferon consensus b ind ing sequence protein (ICSBP"'") develop acute leukaemia after an init ial prodromal period characterised by benign neutrophil ia. Numerous other examples also serve to illustrate the importance of the mouse model for the study of human leukaemia, either in its pathogenesis or in the search for different treatment modalit ies - in vivo Veritas. vi. Gene- based therapeutics A s mentioned in an earlier section, identification of leukaemia- specif ic genetic changes in patients offers unique targets for molecular therapy. Encourag ing results in the development of antisense technology with ol igodeoxynucleot ides (ODNs ) , retroviral vectors, or r ibozymes serve to propel the disc ip l ine forward. Examples include the specif ic eradication of ber- abt m R N A and the growth of ber- abt cells with junction- region specif ic antisense O D N s . 1 0 2 S imi la r results were obtained wi th 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 lear ly , one can appreciate the potential applications o f this technology in leukaemia therapy, either in bone marrow purging in vitro or the eradication of min ima l residual disease in vivo. Recent technical advances in the design of nuclease- resistant nucleotides and of cationic l iposome delivery systems enable greater latitude in antisense- based the rapy . 1 0 5 The current system is restricted to the inhibit ion of m R N A transcription without affecting the source of the corrupted genetic code; however, the future o f gene- specif ic therapeutics appears to lie with catalytic O D N s or r ibozymes with the capabil ity to effect site- specific D N A hence permanent c o r r e c t i o n s . 1 0 6 17 c. S u p p o r t i v e therapy i. G- CSF and GM- CSF support for chemotherapy- induced neutropaenia Haematotrophic cytokines, particularly the colony stimulating factors (CSFs ) in their current capacity are not considered to be true therapeutic agents. A notable exception is the successful application of a- interferon ( I FNa ) for the treatment of chronic phase C M L and hairy cel l l e u k a e m i a . 1 0 7 ' 1 0 8 C S F s essentially play an anci l lary role in leukaemia management; principal ly in accelerating normal haematopoietic recovery postradiochemotherapy to reduce the incidence o f opportunistic infections, thereby fu l f i l l i ng the same function as Strumia 's " leukocyte c ream" more than f ifty years ago. Ut i l isat ion of C S F s in leukaemia management poses the theoretical d i l emma of the unintentional stimulation of leukaemic growth. In vitro studies have shown that many leukaemic cells upregulate the expression o f both C S F s and their corresponding receptors . 1 0 9 C S F s are mitogenic for both pr imary leukaemic cel ls and cel l l ines in vitro and in vivo:95 In addit ion, pro longed use of granulocyte- C S F (G- C S F ) in patients with severe congenital neutropaenia (Kostmann 's syndrome) may augment an already increased r isk of A M L . 1 1 0 A recent study revealed mutations in the domain responsible for the transmission of maturation signals in the G- C S F receptor gene and is impl icated in the pathogenesis of congenital neutropaenia and A M L , as we l l as the possible contributory role o f therapeutic G- C S F in l eukaemogenes is . 1 1 1 Nevertheless, at the present time the c l in ica l benefits of exogenous G- C S F in congenital neutropaenia outweigh the elevated risk of •subsequent-AML deve lopment , 1 1 2 , Results f rom two recent multicentre, double- b l ind , randomised, placebo-control led c l in ica l trials questioned the eff icacy of G- C S F and G M - C S F in elderly A M L p a t i e n t s . 1 1 3 ' 1 1 4 Bo th studies showed that use of either cytokine d id not lead to st imulation of leukaemic relapse; however, despite improvements in objective parameters such as shortening of the neutropaenic per iod, overal l survival was not enhanced. Treatment-related failures (i.e. intolerance to chemotherapy) and possibly the presence of a bio logica l ly distinct form of A M L remain the major problems that plague A M L management in the e l d e r l y . 1 1 5 Unfortunately, 6 0 % of A M L s occur in patients 60 years and older. Age-18 related intolerance to chemotherapy also extends to the intense preparative regimen for a l B M T and the subsequent episodes of G v H D ; therefore, most centres refuse to perform a l B M T on patients 55 years and older. One can clearly appreciate the urgency in identifying new agents that cou ld protect the patient f rom the severity of leukaemia treatments. ii. Infection and haemostasis control Ant ib iot ics are often used prophylact ical ly or empir ica l ly to manage episodes of pyrex ia of unknown or ig in in neutropaenic p a t i e n t s . 1 1 6 - 1 1 7 Funga l infections have proven to be more recalcitrant to therapy; however, introduction of the less toxic f luconazole and l iposomal amphoteric in B should improve somewhat the c l in i ca l e x i g e n c y . 1 1 8 - 1 1 9 Itraconazole, another antifungal agent, was discovered to have the abil i ty to reverse the mult idrug resistance phenotype, possibly v ia steric interference o f the membrane glycoprotein p u m p . 1 2 0 Carefu l selection o f b lood product donors contribute to the reduction of v ira l transmission; in addit ion, application of f low cytometry improved the rapidity of viral diagnosis and therefore t reatment . 1 2 1 Nevertheless, infect ion sti l l remains one of the thorniest problems in the management of leukaemic pa t i en ts . 1 2 2 L im i t ed availabil ity of effective antifungal and antiviral agents has created significant interest in the use of intravenous gamma globul in as prophylaxis against infections, especial ly in the B M T s e t t i n g . 1 2 3 - 1 2 4 Recent advances in platelet storage techniques should enable the prolonged and safe storage of p la te le ts . 1 2 5 However , the most important development in haemostatic research was the successful identif ication and c lon ing of the human platelet growth factor or thrombopoietin ( T P O ) . 1 2 6 P rec l in ica l research has demonstrated that T P O , often in combinat ion with other cytokines, has signif icant mitogenic effects on megakaryocytes and the resultant platelet counts; clearly desirable in patients receiving myelosuppressive chemothe rapy . 1 2 7 Understanding of leukaemia and the c l in ica l management o f the disease has come a long way since Osier 's era. However , except for selected diseases such as paediatric A L L or A P L , overall survival of the leukaemic patient is st i l l fa ir ly d ismal . Technologica l advances in molecular diagnosis does not necessarily translate into better outcome. 19 Nowadays , c l in ic ians have knowledge of the exact location of the chromosomal translocation or the type of basepair alteration. Unfortunately, the majority of treatments are st i l l archaic invo lv ing various cytotoxic substances with little selectivity; not much different f rom Osier 's prescription o f "c lean air and absence of wor r y " for leukaemic patients. The next hurdle in disease management w i l l be the translation of knowledge and sk i l l gained in molecular diagnosis to molecular therapeutics - i.e. targeting and the corrections o f the genetic lesions. 1.3 TRANSPLANTATIONS OF HAEMATOPOIETIC STEM CELLS: CURRENT STATUS Intravenous infusion of haematopoietic progenitor and stem cel ls ( H P C s & H S C s ) into a patient with damaged or defective bone marrow is already an established fo rm of treatment for several benign and malignant haematological conditions. Ce l l s can be harvested f rom the marrow cavities o f the donor, hence the term bone marrow transplantation ( B M T ) ; in peripheral b lood stem cel l transplantation ( P B S C T ) , c irculat ing stem and progenitor cells are harvested f rom the patient (autologous) or an al logeneic donor through apheresis, an extracorporeal process wh i ch phys ica l ly separates b lood into its cel lular components. Placental and umbi l i ca l cord b lood represents a third source of stem cells wi th unique properties which appear to permit greater margins in donor: recipient H L A disparity. The fo l low ing section w i l f f o c u s o n recent;advances-in stem ce l l transplantation. 20 a. Bone Marrow Transplantation (BMT) i. Allogeneic bone marrow transplantation (alBMT) In brief, successful engraftment of allogeneic haematopoietic cells in a foreign host requires a modus vivendi between the graft and the host. Th is is aided by the careful selection of donors based on the matching of major and even minor histocompatibi l i ty antigen loc i as we l l as the effective posttransplantation management of the patient in the fo rm of adequate immunosuppression wi th vigi lant infection control and c l in ica l precautions. 1. H L A matching L imi ta t ion in the size of the opt imal marrow donor poo l i.e. human leukocyte 'antigen or H L A matched sibl ings drives the search for other sources o f stem cel ls. Less desirable donors include H L A matched unrelated donors ( M U D s ) and related donors with partial H L A matches. The science of H L A matching has progressed f rom phenotyping through the use of the m ixed lymphocyte reaction ( M L R ) and serology to the more precise method of genotyping using the polymerase chain reaction ( PCR ) . Accurate matching o f donors and recipients should decrease the incidence of graft rejection and of graft- versus-host disease ( G v H D ) ; however, the paucity o f potential wel l- matched donors often necessitates the use of marrows f rom the suboptimal donor pools mentioned above. D N A typing o f - H L A genes using analytical techniques such as D N A restriction fragment length po lymorph ism ( R F L P ) , P C R - sequence- specif ic ol igonucleot ide probe typ ing, and P C R ' f ingerprint ing' played significant roles in improv ing the outcome o f M U D transplantations by opt imis ing the H L A matching between unrelated donors and rec ip ients . 3 9 Creat ion of the national and global registries of volunteer bone marrow donors facilitates the search for potential M U D donors. Nonetheless, G v H D and graft rejection w i l l remain a fact of l i fe in a l B M T regardless of the improvements made in H L A matching technology s imply because of the enormous po lymorph ism of the H L A loc i and consequently the scarcity of perfectly matched donors. Therefore, improvement of the outcome of a l B M T requires developments 21 of novel immunosuppressive regimens and patient management techniques such as the effective control of infections, in addition to better H L A matching technology. 2. Prevention and the management of graft- versus- host disease ( G v H D ) Cyc lospor in A ( C Y A ) , the venerable antirejection drug, revolutionised the management of graft/ host H L A discordance in B M T . However , C Y A is nephrotoxic and requires frequent drug level monitor ing. The novel microemuls ion formulat ion of C Y A appears to improve the pharmacokinetics of the drug in patients, therefore a lower drug dose is required to maintain the same level o f immunosuppress ion . 1 2 8 Several ongoing a l B M T trials are being conducted to compare the eff icacy of C Y A with the macrol ide antibiotic F K 5 0 6 , an immunosuppressive agent w i th a relatively successful history in l iver t r ansp lan ta t ions . 1 2 9 - 1 3 0 Rapamyc in ( R A P A ) , another naturally der ived macrol ide- based immunosuppressant, binds to the same molecular target as F K 5 0 6 yet mediates different downstream signal l ing pathways. Pre l iminary murine studies indicated the potential of R A P A in M H C - mismatched B M T ; however, this agent has several unique immunolog ica l effects wh i ch warrant addit ional studies pr ior to any c l in ica l testing on pa t ien ts . 1 3 1 Ex vivo T cel l depletion ( T C D ) using a - C D 3 ( O K T 3 ) or a - C D 5 2 (Campath l ) monoc lona l antibodies is an effective method of preventing acute G v H D by removing donor T lymphocytes. However , this often comes with the price of increased incidences of leukaemia relapse and graft f a i l u r e . 1 3 2 - 1 3 3 The expression of other T cel l- specif ic antigens are also exploi ted in antibody-, mediated ex vivo and in vivo T C D ; in fact, several trials have combined both modalities of T C D in the hope of improv ing the c l in ica l outcome, especial ly of M U D transplantations. Monoc l ona l antibodies specif ic for the T c R afj chain, L F A 1 , C D 2 , and C D 5 have all been used s ingly or in different combinat ions in T C D . 1 3 4 -1 3 6 Ex vivo T C D is typical ly accompl ished by antibody- complement mediated lysis of the target cel ls whi le in vivo T C D is often carried out by antibody- toxin conjugates. Nonetheless, the c l in ic ian must realise that T cel ls mediate proper engraftment of the donor stem cel ls, immune surveil lance of leukaemic relapses (GvL ) as we l l as viral- associated lymphoprol i ferat ive disorders, and unfortunately also G v H D . 1 3 7 The graft- versus-22 leukaemia effect (GvL ) is mediated by donor T cel ls wh ich recognise residual leukaemic cells in the patient. Studies have demonstrated that G v L is responsible for the lower rates of leukaemic relapses in patients receiv ing a l B M T ; in addit ion, reinfusions of donor- derived "buffy- coat" containing lymphocytes have been shown to suppress leukaemic relapse post-a l B M T in C M L pat ients . 1 3 8 . Therefore, overzealous and injudicious use of T C D w i l l affect not only the f inal outcome of the graft but ult imately the welfare of the pa t ient . 1 3 9 Better def init ion of the T cel l subset(s) responsible for G v H D should help in the selective removal of this populat ion without affecting the o the r s . 1 4 0 Chron i c G v H D , a different fo rm of graft/host conf l ic t than acute G v H D , can sti l l occur despite pharmacological control of the latter; chronic G v H D manifests itself as a mult isystem disorder wh ich resembles the col lagen vascular d i seases . 1 4 1 Prolongat ion of the survival o f the transplantation recipient highlights the problems associated with this disorder. Management of chronic G v H D with thal idomide offers an attractive alternative to the traditional regimen of prednisolone and azathiaprine because of the former 's effectiveness in treating skin chronic G v H D without m y e l o t o x i c i t y . 1 4 2 3. Identification of minor histocompatibi l i ty antigens ( m H C ) The minor histocompatibi l i ty antigens ( m H C ) denote transplantation antigens which are not encoded by the major histocompatibi l i ty antigen loc i ( H L A in the human and H- 2 in the mouse). Po lymorphisms of these genes in the populat ion contribute to their roles as allorgactive, antigens in organ transplantations. The most-obvious m H C is the sex- l inked male antigen H- Y , wh ich elicits alloreactive T cel l responses in male patients receiving M H C - matched female g ra f t s . 1 4 3 S ince only H L A matching is performed in c l in ica l a l B M T , G v H D often can result f rom mismatches in the m H C even in transplantations between H L A - identical individuals. Recently, two reports highl ighted the importance of the non- sex- l inked m H C in c l in ica l a l B M T s . G o u l m y and colleagues demonstrated the importance of H A - 1 matching in H L A - matched a l B M T . 1 4 4 Interestingly, the authors showed that mismatches in H A - 1 contributes to G v H D only in donors and recipients positive for H L A - A 2 ; therefore, a complex interplay exists between m H C (simple "protein 23 antigens" presented by the M H C ) and the M H C (alloantigens, protein antigens, and b inding partners of 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 complementary D N A of the C D 3 1 ( P E C A M - 1) adhesion molecule revealed a single po lymorph ism in normal subjects; further investigation in a l B M T patients who received bone marrow cells f rom H L A - identical s ib l ing donors revealed a posit ive correlation between donor: recipient C D 3 1 po lymorph ism and the inc idence o f G v H D . 1 4 5 Unfortunately, the insuff ic iency o f H L A - matched donors precludes the routine matching of m H C in actual c l in ica l practice. However , as indicated in an accompanying editorial, m H C matching may be beneficial in situations where more than one H L A -identical donor is available or when the degree o f m H C disparity can be used to tailor the extent o f the immunoprophy lax is for the pa t i en t . 1 4 6 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 of a l B M T in paediatric patients symptomatic for s ickle cel ls anaemia ( S C A ) . 1 4 7 The authors showed that a l B M T was able to halt the various symptoms of S C A and to abol ish the need for R B C transfusions. S im i l a r l y , patients suffering f rom another benign haematological disorder, |3-thalassaemia, also benefited f rom a l B M T . 1 4 8 A l B M T , when used responsibly, is able to improve the qual ity of l i fe in patients suffering f rom various forms of benign haemato logica l 'd isorders . 1 4 9 Th is however w i l l increase the demand for an already scarce resource and w i l l probably provide an impetus for ut i l i s ing novel sources of stem cells such as umbi l i ca l cord b lood and mobi l i sed peripheral b lood stem cel ls. F ina l l y , several groups are actively pursuing the use of a l B M T as a form of adoptive immunotherapy; infusion of donor leukocytes for leukaemic relapse control post- a l B M T and the transfer of tumour antigen specif ic immuni ty f rom an immunised al logeneic donor are two such e x a m p l e s . 1 5 0 24 ii. Autologous bone marrow transplantation (auBMT) "wa i t ing for a M U D is l ike wait ing for Godot"- Sometimes, a patient becomes an unwit t ing player in Beckett ' s play. [Golstone, 1993 #610] Auto logous B M T ( a u B M T ) offers the only hope for elderly patients or without H L A - compatible donors; elderly patients are especially susceptible to the deleterious effects of G v H D associated wi th a l B M T and therefore most centres do not per form the procedure in patients more than 50 years o l d . 1 5 1 - 1 5 2 Theoret ical ly , bone marrow harvest should be performed when the probabi l i ty o f neoplastic cel l contamination is at its lowest i.e. dur ing remiss ion. Th is per iod usual ly fo l l ows successful intensive chemotherapy, i.e. at first complete remission postinduction therapy in A M L and during chronic phase in C M L . The patient then receives high- dose chemotherapy and radiation therapy fo l lowed by reinfusion of the harvested marrow cel ls. The lack of G v H D in a u B M T unfortunately means the absence of the G v L effect; therefore, the higher rate of leukaemic relapses essentially balances out the lower incidence o f B M T procedure- related mortal ity (i.e. G v H D ) in a u B M T pa t i en ts . 1 5 2 In addit ion, re infusion o f contaminating tumour cel ls also increase the chance of relapse. T w o independent studies, indirectly and direct ly, demonstrated the contributory role of unpurged autografts in subsequent relapses. The tumour- specif ic translocation t(14; 18), wh i ch jo ins together the bcl- 2 gene wi th the immunoglobu l in heavy chain promoter, is common to a majority of fo l l i cu lar non-Hodgk in ' s l ymphoma ( N H L ) and 3 0 % o f B- ce l l N H L and al lows the use of P C R to detect rare l ymphoma cel ls. P C R posit iv i ty of the reinfused purged autograft correlated wi th the incidence o f subsequent l ymphoma re l apse . 1 5 3 Brenner and colleagues unambiguous ly and directly demonstrated, v i a ex vivo retroviral mark ing of the harvested product, that neoplastic cells in the reinfused graft contribute to subsequent relapses in A M L and neuroblastoma p a t i e n t s . 1 5 4 ' 1 5 5 However , this does not discount the presence o f endogenous neoplastic cel ls that have surv ived the condit ioning regimen. Several groups have tried to improve the odds of a u B M T by the administration of l ow dose cyc lospor in A , wh ich appears to disrupt immune tolerance and thereby provokes a fo rm of G v L but also G v H D in the pa t i en t . 1 5 6 25 1. a u B M T and chronic myelogenous leukaemia ( C M L ) A l B M T is currently the sole curative regimen for chronic myelogenous leukaemia ( C M L ) possibly because of the role o f G v L . 1 4 0 C M L can strike at all ages yet the median age of incidence is 50 years, wh i ch renders most patients unsuitable for a l B M T for reasons discussed a b o v e . 1 5 7 Therefore, intensive chemotherapy coupled wi th autologous bone marrow rescue becomes an alternative regimen for those patients in which a l B M T is not i n d i c a t e d . 1 5 8 ' 1 5 9 Contaminat ion o f the harvested marrow by C M L cel ls and the absence o f G v L effects combine to increase the incidence of relapse in patients receiv ing unpurged autologous marrow g r a f t s . 1 6 0 Presence of the unique molecular lesion ber- abl in C M L cells a l lows cl inic ians to assess the extent of min ima l residual disease ( M R D ) in the patient's marrow and circulat ion using sensitive reverse transcriptase- polymerase chain reaction (RT- P C R ) , wh ich can detect a single ber- abl+ ce l l in 10 5 to 10 6 normal c e l l s . 1 6 1 Newer quantitative RT- P C R (qRT- P C R ) methodology based on the competit ive ampli f icat ion of exogenous competitor templates permits the quantitation of in vivo M R D or the degree of autologous graft contamination as wel l as the eff icacy of the graft purging procedure. Serial monitor ing of the patient posttransplantation using qRT- P C R facilitates the early detection o f r e l apse . 1 6 2 However , M i y a m u r a and colleagues have shown that bcr-abl P C R posit iv ity post- a l B M T does not necessary correlate with c l in ica l re l apses . 1 6 3 Therefore, c l in ica l remission may exist as a state of equi l ibr ium between the residual leukaemic cel ls and the immune s y s t e m . 1 6 4 Regardless, P C R technology a l lows for the rapid evaluation of ex vivo purg ing eff icacy and is invaluable for the compar ison of the different purging systems. Without significant improvements in the management of G v H D and'the development of other forms of therapies, a u B M T w i l l remain a feasible alternative in leukaemia treatment. Development of novel ex vivo techniques such as leukaemic ce l l purging and immune modulat ion w i l l make a u B M T a more viable option than a l B M T . 1 6 5 ' 166 26 b. Peripheral blood stem cell transplantations Peripheral b lood stem cel l ( P BSC ) transplantation can be performed in either autologous or allogeneic setting. Advantages and problems associated with a l B M T and a u B M T are also applicable respectively to al logeneic and autologous P B S C grafts. However , transplantation of P B S C s in general has several advantages over B M T - the former procedure does not require general anaesthesia and P B S C grafts mediate faster haematological recovery than marrow grafts, possibly because of an abundance of commit ted progenitor cells in the c i r c u l a t i o n . 1 6 7 ' 1 6 8 The procedure is especially useful in paediatric patients because of the risks associated with bone marrow harvests in c h i l d r e n . 1 6 9 The harvesting of P B S C can be accompl ished by leukapheresis, an extracorporeal procedure in which the pe r son ' s b lood is circulated through a b lood ce l l separator such as the Fenwal l 3000CS and cel ls wi th in a certain range of specif ic density (mainly mononuclear cells) are isolated v ia centrifugal elutriation. Lymphocytes and monocytes are col lected along with the committed progenitor and stem cel ls, wh ich can be selected posit ively using monoclonal antibody specif ic for the haematopoietic stem cel l antigen C D 3 4 . M o s t of the experience in P B S C transplantation has been wi th autologous grafts; however, al logeneic P B S C transplantation is gaining more acceptance s imply because of the relative ease of the harvesting procedure. " M o b i l i s a t i o n " is necessary to increase the number of stem and progenitor cells in the circulation prior to the actual harvests. Recent advances in the functional characterisation of P B S C and the development of novel mobi l isat ion and stem cel l expansion protocols have established P B S C transplantation as an integral part of the standard treatment regimen in oncology. Fo r example, f low cytometric analysis , 0 f i C D 3 4 + cel ls in the P B S C harvests enables direct .and rapid enumeration of progenitor and stem cel ls wh i ch provides an index of the reconstitutive potential o f the harvested product without resorting to the labour- intensive and time- consuming b io logica l assays ( C F U - G M assays, delta assays, and long- term culture- init iat ing cel l assays ) . 1 7 0 Furthermore, Brugger and colleagues recently showed that ex vivo expansion of posit ively selected C D 3 4 + cells f rom a single session o f leukapheresis was sufficient to effect rapid and sustained haematopoietic engraf tment . 1 7 1 The above f indings coupled with enhanced design of the ce l l separator and better 27 haemodynamic support dur ing leukapheresis signif icantly 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 wi th the autologous marrow such as inadequate specimen size due to marrow f ibrosis (from pelv ic irradiation or disease process) and tumour contamination of the marrow space. To obtain enough P B S C , mobi l isat ion of the oncology patient can be accompl ished by myeloablative chemotherapy in conjunction with the administration of recombinant haematopoietic growth factors such as G- C S F . F o r obvious reasons, on ly growth factors are used for al logeneic donor mobi l isat ion. Th is procedure increases the number of P B S C to a level wh ich is practical for harvesting; usually three leukapheresis sessions are required to obtain the 2 X 1 0 6 C D 3 4 + P B S C / k g of recipient needed for adequate haematological engra f tment . 1 7 2 Cl in i c ians , however, must be vigi lant in deal ing with mobi l i sed autologous harvests f rom these patients because the operation also increases the circulatory tumour l o a d . 1 7 3 - 1 7 4 Therefore, additional steps such as C D 3 4 + posit ive selection coupled wi th tumour cel l purging are necessary in autologous P B S C transplantations invo l v ing leukaemic patients; whereas C D 3 4 + posit ive selection alone wou ld suffice for patients wi th so l id tumours because of the absence of C D 3 4 expression on these tumour c e l l s . 1 7 5 " 1 7 7 In al logeneic P B S C transplantation, posit ive selection of C D 3 4 + c e l l s also reduces the possibi l i ty o f reinfusion of donor lymphocytes , wh i ch can contribute to G v H D . 1 7 8 c. Umbilical cord blood transplantation Human umbi l i ca l cord b lood ( H U C ) , normal ly discarded after del ivery of the foetus, is an unique source o f haematopoietic stem and progenitor cel ls for transplantation. M o s t studies have focused on H U C transplantations in the paediatric setting because of the fear of insufficient cel ls for adult recipients, even though H U C contains approximately the same concentration of stem cel ls as adult bone marrow but only at a typical harvest volume 28 of 100 m l . 1 7 9 The unique developmental state or immunolog ica l naivete o f the H U C s endows them with the capabil ity to engraft across disparate H L A barriers without resulting in s ignif icant G v H D . 1 8 0 H U C s also appear to be unusual ly robust, poss ib ly due to their developmental status, in their abil ity to effect haematopoietic engraftment; therefore adult patients may benefit f rom H U C transplantat ions. 1 8 1 Standardisation o f H U C col lect ion, storage, and characterisation should increase the use of this fo rm o f stem cel l t ransp lanta t ion . 1 8 2 d. Future directions The raison d ' etre of stem cel l rescue in leukaemia therapy is the reconstitution of the haematopoietic system damaged by the high dose chemotherapeutic agents and radiation treatment needed to eradicate leukaemic cells f rom the host. The relative nonselectivity of the current generation of treatments results in "co l la te ra l " damage to normal tissues such as cells of the haematopoietic system and other rapidly d iv id ing cells. Development of more specific antineoplastic agents such as gene- based or s ignal l ing pathway specif ic antagonists w i l l make this particular indicat ion redundant . 1 5 2 In the future, stem cel l transplantation probably w i l l be used for the correction o f intrinsic genetic defects or in customised modif icat ions to the host immune system. >>.k -1.4 EXPLOITABLE DIFFERENCES BETWEEN NORMAL AND LEUKAEMIC CELLS a. Leukaemogenesis Oncogenesis in cells of haematopoietic or ig in is often a multistep event and involves the acquisit ion of oncogenic changes as we l l as the loss or inactivation of normal 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, especial ly those be longing to the fami ly o f retroviridae, can cause leukaemia in a variety of mammal ian hosts. Recently, 29 Gross speculated that dormant oncogenic viruses may play a even bigger role in animal leukaemogenesis than previously t hough t . 1 8 5 Some oncogenic events are so potent that no other genetic changes are necessary for leukaemic transformation; however, successful evolution of the leukaemic clone in the host often needs more than one genetic " h i t " in the guise of additional oncogenic changes or abrogation of normal growth c o n t r o l . 1 8 6 B iochemica l and physiological differences between normal and leukaemic cells fo rm the basis of antileukaemic therapy; however, the differences can be quite subtle since leukaemogenesis arises f rom normal haematopoietic precursors. The therapeutic w indow o f opportunity between normal and malignant cells relies on the differential sensitivity to treatment which results f rom a range of d i s s im i l a r i t i e s . 1 8 7 The fo l l ow ing is a br ief description of selected leukaemia- specif ic changes resulting f rom the ber- abl translocation in C M L , and their implicat ions in therapy. b. Genetic differences Gene- based differences fo rm the foundation of subsequent deviations in signal transduction, adhesion properties, and response to extracellular st imul i in leukaemic cel ls 5 5 Acqu is i t ion of transforming oncogenes for example, through point mutations and chromosomal translocations, in conjunct ion wi th the loss of tumour suppressor genes conceive the script for the ensuing leukaemic transformation. These changes, wh i ch are absent in normal cel ls, are responsible for the init iation as we l l as maintenance of the leukaemic phenotype. Ment ioned previously, ber- abl and pml- raraaxe respectively unique to C M L and A P L ; both translocation events result in a plethora of changes wh i ch endow the cells with growth autonomy. Absence of oncogenic changes in normal haematopoietic cells therefore al lows specific targeting of the leukaemic clone. Complementary binding of specif ic antisense ol igodeoxynucleotides ( O D N s ) or r ibozymes can target leukaemic- specif ic sequences at the m R N A l e v e l . 1 0 5 S z czy l i k and colleagues have demonstrated the specif ic ity of the ber- abl junction- region antisense O D N in the inhibi t ion of ber- abl+ cells by both RT- P C R as we l l as co lony a s says . 1 0 2 Therefore, identification of genetic aberrations wh i ch are pathognomonic for leukaemia remains important in not only the diagnosis, but also the treatment of the disease. 30 c. Differences in intracellular signalling and cell death pathways Signal transduction is paramount in the conveyance of extracellular information to the nucleus or executive centre of the ce l l ; it is also important in the regulation of cel lular development. Aberrant signal transduction events can lead to dysregulated growth or oncogenesis, therefore such pathways have been deemed appropriate targets in cancer the rapy . 1 8 8 Tyros ine phosphorylat ion, wh i ch consists of less than 2 % of total cel lu lar protein phosphorylat ion, plays an extremely important role in the mediation of cel lular s ignal l ing. Dysregulat ion of the expression and regulation of protein tyrosine kinases ( P T K ) can result in catastrophic changes in cel ls and the o r g a n i s m . 1 8 9 For example, genetic knockout of the CSK tyrosine kinase in mice , wh i ch negatively regulates the SRC P T K , results in substantial upregulation of SRC kinase activity. A phenotype of embryonic lethality wi th signif icant neural tube defects was el ic i ted f rom the CSK knockout m i c e . 1 9 0 The BCR- ABL fus ion protein in C M L has s ignif icant ly upregulated tyrosine kinase activity in compar ison to the wi ld- type ABL protein in normal haematopoietic c e l l s . 1 9 1 In addit ion, BCR- ABL local ises to the cy top lasm whereas w i ld- type ABL expression is restricted to the nucleus where it interacts with growth regulatory proteins such as the ret inoblastoma p r o t e i n . 1 9 2 ' 1 9 3 . S ince tyrosine kinase act ivat ion of BCR- ABL appears to be crucial in the initiation and maintenance of the leukaemic phenotype in C M L , signal transduction events mediated by BCR- ABL therefore become logica l targets for therapeutic in te rvent ions . 1 9 4 Several examples of the successful use o f tyrosine kinase inhibitors have been ment ioned p r e v i o u s l y . 5 8 - 5 9 Transformed cel ls, in addition to changes wh i ch promote prol iferation and growth autonomy, also are deficient in regulatory pathways resulting in resistance to normal homeostatic controls. Fo r example, loss of the wi ld- type p53 phenotype results in the abrogation of intrinsic senescence control and sensitivity to apoptosis- inducing anticancer a g e n t s . 1 9 5 - 1 9 6 Restorat ion of the w i ld- type p53 phenotype, v i a retrovirus- or adenovirus-mediated gene transfer, can therefore increase sensitivity to chemotherapeutic agents or effect cel lular differentiation. 31 d. Response to extracellular regulatory signals Leukaemic cells exhibit heightened responses to cytokine stimulation or may even become independent of survival or mitogenic factors. BCR- ABL endows cytokine independence to the interleukin- 3 (IL- 3) dependent cel l l ine F D C P - 1 by substituting for normal signal transduction events mediated by IL- 3 b ind ing to its receptor; in addit ion, BCR- ABL, v i a its Src- homology 2 domain (SH2) , induces the secretion of IL- 3 to effect paracrine s t i m u l a t i o n . 1 9 7 ' 1 9 8 Furthermore, Shuai and colleagues have demonstrated that S T A T 5 , a cytoplasmic protein wh ich mediates G M - C S F s ignal l ing, is constitutively activated by BCR- ABL in the absence of G M - C S F . 1 9 9 Therefore, BCR- A B L , through its exaggerated P T K activity and the presence of various adapter or b ind ing regions such as the S H 2 and S H 3 domains, can substitute for mitogenic and survival cytokines wh i ch normal ly maintain homeostasis. A t the other extreme, evidence has shown that leukaemic cells do not respond to the growth inhibitory effects of a fami ly of cytokines loosely termed haematopoietic or stem cel l i n h i b i t o r s . 2 0 0 One such molecule, the macrophage inf lammatory protein- l a (MIP-l a ) , is a member of the chemokine fami ly but was or ig inal ly characterised as an inhibitor of haematopoietic stem ce l l p r o l i f e r a t i o n . 2 0 1 - 2 0 2 Eaves and colleagues reported that C M L cells are unresponsive to the regulatory effects of MIP- l a . 2 0 3 Another example is the lack of response to the tetrapeptide N-Acetyl- Ser- Asp- Lys- Pro ( N - A c S D K P ) by C M L but not by normal haematopoietic c e l l s . 2 0 4 N - A c S D K P and py roG lu- G lu- Asp- Cys- L y s ( p E E D C K ) belong to a new class of low molecular weight haematopoietic inhibitors wh ich b lock cel l cyc le progression f rom the quiescent or G 0 phase; normal ly , quiescent cells are not susceptible to the cytotoxic effects of cycle- specific chemotherapeutic agents and to ionis ing r ad i a t i on . 2 0 5 Differential sensitivity to the above molecules can be exploi ted in therapeutics. Selective cycle inhibit ion of normal cells by A c S D K P protects them f rom chemotherapy, hyperthermia, radiat ion, as we l l as P D T t r ea tmen t s . 2 0 6 " 2 0 9 Select ive inhibit ion of normal haematopoietic ce l l cyc l ing by N - A c S D K P , p E E D C K , and MIP- l a should permit dose intensification in the in vivo treatment of leukaemia 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 in the bone marrow. Th is is control led by the expression and activity of mult ip le adhesion molecules and their counter receptors. Engagement of selected receptors on the surface of progenitor cel ls and stromal l igands results in the transduction of growth inhibi tory s i g n a l s . 2 1 1 Gordon and colleagues first demonstrated that C M L progenitor cells exhibit attenuated adhesion to the marrow stroma, even though C M L progenitors express s imi lar numbers of integrin adhesion receptors as than normal c o u n t e r p a r t s . 2 1 2 ' 2 1 3 Subsequent studies showed that treatment wi th interferon- a ( IFN- a) restores normal (31 integrin- dependent adhesion in C M L progenitor cel ls , poss ib ly v i a mediat ion by M IP- l a . 2 1 4 ' 2 1 5 Reestablishment o f stromal contact and transduction of growth inhibi tory signals by IFN-a may explain its c l in ica l effects in C M L management . 1 0 8 S ince P T K s are impl icated in the mediation of integrin s ignal l ing, abnormality in P T K activation cou ld therefore affect normal cel lular responses to integrin- mediated ce l l adhes i on . 2 1 6 In a recent review article, Ver fa i l l i e and colleagues speculated the BCR- ABL fusion protein in C M L interfered with integrin- mediated signal transduction events and brings about abnormal adhesion, t raf f icking and as we l l as prol i ferat ion observed in C M L . 2 1 3 f. Susceptibility to cytotoxic agents In theory, the sum of the above differences should lead to a comfortable therapeutic w indow of opportunity in leukaemia treatment. Unfortunately, most practical experience in therapy does not give credence to the scientif ic evidence. In addit ion, most neoplastic cells have evolved sophisticated protective mechanisms that confer resistance to various radiochemotherapeutic regimens. Fo r example, expression of the P I 7 0 A T P - dependent membrane glycoprotein pump, encoded by the mdrl gene, is s ignif icant ly increased in cells wh ich express the mult idrug resistance ( M D R ) phenotype . 2 1 7 In one study o f adult acute leukaemias, heightened mdrl expression was documented in 5 0 % of leukaemic relapses but only 1 9 % of newly diagnosed leukaemia; in addit ion, an inverse correlation was made 33 between the complete remission rate and the amount of mdrl e x p r e s s i o n . 2 1 8 Successful treatment of leukaemia w i l l therefore depend on the application of newly acquired knowledge in the molecular b io logy of leukaemia to the c l i n i c . 2 1 9 7.5 PURGING IN AUTOLOGOUS STEM CELL TRANSPLANTATIONS a. I n t r o d u c t i o n Bone marrow purging ( B M P ) is deemed beneficial in certain situations invo lv ing the reinfusion of autologous marrow cells because of contamination of the harvested product by neoplastic c e l l s . 2 2 0 Purg ing assumes that exploitable differences exist between normal and leukaemic cells such that selectivity can be ensured in the removal or destruction of the undesirable populat ion. C l i n i ca l l y effective B M P presents a chal lenging d i l emma because the c l in ic ian must achieve a balance between sufficient eradication of the leukaemic cells with preservation of enough of the normal cells for haematopoietic reconstitution. Overzealous purging w i l l lead to eventual graft failure or a delay in engraftment; on the other hand, inadequate purging may contribute to leukaemic relapses in the patient. B M P has been studied extensively in C M L autografts and provides an excellent model system for the study o f the different purging strategies because of the avai labi l i ty of ber- abT ce l l lines and the sensitivity of the P C R detection a s s a y . 2 2 1 - 2 2 2 Accumula t ing evidence also points to the effectiveness of B M P in A M L patients receiving autografts. A report f rom a recent conference showed signif icant survival advantage in patients receiv ing purged compared to unpurged autografts and led the coauthor of the report, E l izabeth j . Shpal l to declare "These results suggest that autotransplantation of acute myelogenous leukaemia should use purged bone marrow for restoration of b lood p r o d u c t i o n " . 2 2 3 Las t l y , purging is also being considered for autologous P B S C transplantations in order to reduce the level of tumour cel l contamination o f the mobi l i sed leukapheresed p r o d u c t . 2 2 4 34 b. Purging strategies The four chief modalit ies of purg ing; phys ica l , immuno log ica l , b io log ica l , and pharmacological , are used singly or in combinations to effect c l in ica l ly beneficial r e s u l t s . 2 2 2 ' 2 2 5 Dif ferences in b iophys ica l characteristics are exploi ted in phys ica l purging. Examples include counterf low elutriation, density gradient separation, f i l trat ion, and heating. Fo r example, clusters o f neuroblastoma cel ls can be removed f rom the autograft v ia f i ltration through a 40pm mult i layer mesh fi lter wh i ch results in the e l iminat ion of 0.5 log of the cancer cells. Several groups have discovered that leukaemic ce l l l ines are much more susceptible to hyperthermia than normal haematopoietic c e l l s . 2 2 6 - 2 2 7 Surface expression of differentiation markers permit recognit ion of cel ls by antibodies. However , as mentioned earlier, leukaemic and normal haematopoietic cells share many of the same markers since the former populat ion derives f rom neoplastic transformation of the la t ter . 2 2 8 For example, many A M L cells and essentially al l A M L stem cells express the haematopoietic stem cel l antigen C D 3 4 ; therefore, procedures which involve only the positive selection of C D 3 4 + cells are applicable solely for so l id nonhaematopoietic tumours, wh i ch do not express C D 3 4 . Nevertheless, several commerc ia l ventures have been started to take advantage of C D 3 4 positive selection technology in the ex vivo manipulat ion of stem cel ls. C D 3 4 - specif ic monoc lona l antibodies conjugated to a sol id- phase support, either in the fo rm o f ferromagnetic beads or the surface of tissue culture f lasks, are in various stages o f c l in ica l trials for stem cel l puri f icat ion and such approaches afford promise for condit ions l ike neuroblastoma and breast c ance r . 2 2 9 Posit ive selection of C D 3 4 + cells f rom a leukaemic sample w i l l therefore require additional steps such as negative selection or pharmacological purging to eradicate C D 3 4 - expressing leukaemic cel ls. Negat ive selection based on the expression of mature . lineage markers provides greater latitude in purging since normal committed progenitor cells and haematopoietic stem cel ls do not express such markers. Fo r example, presence of C D 2 , 3, 5, 7, and C D 1 l a on leukaemic cel ls targets them for destruction by immunotoxins and radioisotope immunoconjugates, without signif icant reduction in the v iabi l i ty and c lonogenic i ty of normal haematopoietic c e l l s . 6 9 - 7 0 B i o l og i ca l purging operates on the basis that normal and leukaemic cells behave differently in culture due to unique expression profi les of haematopoietic growth factors, their receptors, as we l l as adhesion molecules. Scheffo ld and associates showed that C M L - specif ic cytokine- induced k i l ler cel ls can be 35 generated f rom the peripheral circulation of C M L patients; furthermore, they showed that these C D 5 6 + cel ls can be used as a form of bone marrow purg ing in C M L . 2 3 0 G rowth kinetics in response to cytokines also differentiate normal cel ls f rom their malignant counterpart, as mentioned in a previous section. In vitro culture of bone marrow cel ls harvested f rom selected C M L patients resulted in the gradual disappearance of Philadelphia-chromosome posit ive leukaemic cells with a concomitant expansion of normal haematopoietic c e l l s . 2 3 1 ' 2 3 2 Establ ished chemotherapeutic agents such as 4-hydroperoxycyc lophosphamide (4- H C ) and its congenor mafosfamide (or A S T A - Z®) are both analogues o f the commonly used alkylat ing agent cyc lophosphamide; the two former drugs have been mod i f i ed such that in vivo hepatic activation is not required. A S T A - Z® and 4- H C remain the two most common l y used pharmacologica l purging a g e n t s . 2 3 3 - 2 3 5 In addit ion, other chemotherapeutic agents are also being considered for ex vivo p u r g i n g . 2 3 6 A l k y l - lysophosphol ip ids, analogoues of the platelet- activating factors, also appear to be promis ing candidates in various purging app l i ca t i ons . 2 3 7 c. Combination purging A s shown in c l in ica l pharmacology, combinat ion therapy in cancer management is often superior to the single drug approach. Therefore, research in purging wh i ch combines the different modal i t ies, such as immunolog ica l and pharmacologica l purging, or different drugs, such as mafosfamide with etoposide, are being act ively p u r s u e d . 2 2 5 The ex vivo nature of the treatment affords the c l in ic ian with greater latitude of graft manipulation without much fear of systemic toxic i ty ; the singular goal of purging is to. effect maximal-log reduction of the contaminating neoplastic cells whi le preserving an adequate number of normal haematopoietic cells for rapid yet durable engraftment. Other examples of combinat ion purging include combinations of 4- H C or A S T A - Z® wi th antibody-mediated- complement lysis, immunotoxins, and ber- abl junct ion specif ic antisense o l igodeoxynuc leot ide ( O D N ) . 2 3 8 " 2 4 0 36 d. Purging outside the traditional paradigm N o v e l purging modalit ies are constantly being developed to exploit the preferential k i l l i ng of leukaemic cel ls. Gene- based strategies are used to target leukaemia- specif ic oncogenes such as the ber- abl translocation sequence in C M L . Some examples include the ber- abl junct ion region- specif ic antisense O D N and retroviral expression v e c t o r . 1 0 3 - 2 4 1 Enforced reexpression of the w i ld- type tumour suppressor gene p53 can lead to.a reversion of the tumour phenotype or even apoptosis in cells wi th mutated or deleted p53 sequence. Seth and colleagues used an adenoviral vector in a human breast cancer purging mode l to effect the transfer of the w i ld- type p53 tumour suppressor gene, wh i ch induced apoptosis preferentially in the breast cancer c e l l s . 2 4 2 Often, aberrant signal transduction events are implicated in the initiation and maintenance of the leukaemic phenotype; therefore, specif ic targeting of s ignal l ing pathways can lead to the selective destruction of the cel ls dependent on their survival . N o v e l compounds wh ich disrupt the s ignal l ing pathways of neoplastic cel ls or promote apoptosis w i l l increasingly f i nd their way into p u r g i n g . 5 8 - 5 9 - 7 2 LaCasse and col leagues described the successful purg ing o f the human B- cel l l ymphoma cel l l ine Daud i with the protein synthesis inhibitor Shiga- l ike toxin 1 (SLT- 1), wh i ch binds to C D 7 7 , a ce l l surface g l y co l i p id 2 4 3 Restr icted expression of C D 7 7 to a subset of activated B cells and l ymphoma cells means that normal haematopoietic stem and precursor cells are spared f rom SLT- 1- mediated k i l l i ng . A s imi lar strategy util ises a fusion G M - C S F / diphtheria toxin wh ich selectively targets and destroys G M -C S F dependent leukaemic cells (as we l l as normal granulocyte- macrophage progenitors) in vitro.244 The above examples illustrate some of the novel methods of purging wh ich show promise for future c l in ica l success. 1.6 PHOTODYNAMIC THERAPY (PDT) AND ITS ROLE IN PURGING a. Reactive oxygen intermediates (ROIs) in biology Molecu la r oxygen is essential for the survival o f the vast majority of terrestrial b io log ica l organisms. It, however, can behave deleteriously i f not for the elaborate protective mechanisms that are evolutionari ly conserved in the different species. Reactive oxygen intermediates (ROIs) such as superoxide anions (02~) and hydrogen peroxide 37 ( H 2 0 2 ) are generated during oxidative respiration and are normal ly deactivated by superoxide dismutase ( SOD) and catalase, respectively. The fami ly of glutathione (GSH)-dependent enzymes are extremely important in the detoxif ication o f a variety of RO Is and l i p i d p e r o x i d e s . 2 4 5 In addition to their role as byproducts of mitochondria l respiration, RO I s are also impl icated in a myr iad of intracellular and intercellular signal transduction pathways. 2 4 6 Act ivated phagocytic cells produce 0 2 " that is bactericidal for intracellular pathogens. Phagocytes f rom patients suffering f rom chronic granulomatous disease are defective in the generation of 0 2 , wh i ch predisposes these patients to persistent and severe infections. Neutrophi ls contain the enzyme myeloperoxidase, wh ich catalyses the oxidat ion of chloride ion by H 2 0 2 i n t o the h igh ly reactive hypochlorous acid (HOC1). Therefore, the body util ises RO I s for various aspects of nonspecif ic immune de f ence . 2 4 7 Ox idat ive injury to cells can occur at the different levels of cel lular constituencies. L i p i d peroxidat ion, oxidative damage to the thiol groups of structural proteins as we l l as enzymes, and direct damage to D N A are observed in cel ls exposed to oxidat ive s t r e s s . 2 4 8 ' 2 4 9 Increasing evidence f rom different experimental systems also demonstrate the role of RO I s in the induct ion of apoptotic ce l l d e a t h . 2 5 0 In addit ion, some studies have shown that the antiapoptotic protein BCL- 2 has antioxidative capabil ity wh ich can inhibit the formation and actions of R O I s . 2 5 1 - 2 5 2 However , contradictory f indings also suggest that RO I s are not necessary for the induction of apoptosis and that BCL- 2 retains its antiapoptotic abil ity even in anoxic c o n d i t i o n s . 2 5 3 In summary, RO I s are generated during normal cel lular metabol ism and are also impl icated in the various signal transduction pathways. Phagocytes use RO I s as part of the nonspecif ic defence against pathogens.-All cel lular components are potential•targets^of oxidative damage; in addit ion, oxidative stress can effect its cytotoxic i ty v i a apoptosis. b. P D T : cytotoxicity and biological responses Photodynamic therapy ( PDT) exploits the biophysica l characteristics of a group of photoreactive compounds that, when activated by l ight of the appropriate wavelengths, enter into a higher energy state and w i l l subsequently excite either cel lular biomolecules (type I photosensitised reaction) or ground state oxygen (type II photosensitised reaction); 38 singlet oxygen ( '0 2 ) , wh ich is extremely reactive, is generated as a result of the type II r e a c t i o n . 2 5 4 - 2 5 5 The relatively short l i fe span o f ' 0 2 (4 |is) in aqueous solution means that the sites of oxygen- dependent photodamage are dictated by the init ial localisation of the photosens i t i se r . 2 5 6 Bo th apoptosis and necrosis have been observed after P D T and this appears to be dependent on the type and the dose of the photosensitiser. S ince these agents are inert unti l activated by light, the specif icity of the P D T cytotoxic response can be fine-tuned by manipulat ing the time and the site of photoirradiation. Neoplast ic tissues and other rapidly prol i ferat ing tissues such as angiogenical ly stimulated endothelial cells readily take up the photosensitising compound, one of the mechanisms is mediated by the upregulation of l ow density l ipoprotein ( L D L ) receptors . 2 5 7 Var ious porphyrin- based photosensitisers associate wi th p lasma l ipoproteins; therefore, upregulation of the L D L receptors ( L D L r ) w i l l increase the accumulation of the photosensitiser- L D L complex, and hence subsequent P D T c y to tox i c i t y . 2 5 8 Other factors such as the local mi l i eu of the cel l and the chemical characteristics of the compound also determine the uptake profi le and susceptibil ity to P D T . 2 5 9 Oxidat ive damage to cel lular components results in various changes to the ce l ls , tissues, and the organism 2 6 0 . The degree of P D T cytotoxicity is dependent on the amount and activity of the various cel lular protective enzymes mentioned p r e v i ous l y . 2 6 1 Recent ly , much research has been devoted to the role of apoptosis in P D T cytotoxic i ty , wh i ch appears to be mediated by phospholipase-C in L 5 1 7 8 Y l ymphoma c e l l s . 2 6 2 He and col leagues showed that overexpression o f antiapoptotic protein BCL- 2 in the Chinese hamster ovary cel l l ine effected protection f rom PDT- mediated c y to tox i c i t y . 2 6 3 Several established stress signal transduction pathways are also initiated by P D T ; increased expression o f specif ic heat shock proteins and various early response:genes'as we l l as the activation o f the N F - K{3, J N K / S A P K pathways as we l l as increased product ion of ceramide are observed post- P D T . 2 6 4 - 2 6 8 These pathways may be invo lved either as a normal protective response to PDT- mediated stress or as part of the cel l death pathways initiated by P D T . Numerous photosensitising compounds w i th different b iophysica l and b iochemica l characteristics have been proposed for P D T . T w o members of the porphyr in fami l y , Photofrin® (porf imer sodium) and the benzoporphyr in- derivatives, are in current c l in i ca l u s e . 2 6 9 Other photosensitisers such as sulphonated a lumin ium phthalocyanine (A l SPc ) and Merocyan in 540 ( M C 540) being considered as we l l . The "personal i ty " of each 39 photosensitiser, wh i ch includes hydrophobic i ty , ext inct ion coeff ic ient, absorption spectra, and singlet oxygen y ie ld , is determined by the unique chemical structure o f the compound. These factors, in conjunction wi th the b io logica l attributes of the ce l l (in vitro and in vivo P D T ) and the organism (in vivo P D T ) determine the overal l effectiveness of P D T . 2 7 0 c. In vivo uses of PDT Access ib i l i ty of the sk in to photoirradiation and the high occurrence of cutaneous tumours combined to provide an ideal indicat ion for P D T . In addit ion, P D T has shown promise in the treatment of tumours of the bra in, lung, oesophagus, and o f the b ladder . 2 4 9 -271,272 Damage to the tumour- associated vasculature by P D T also contributes to the destruction of the t u m o u r . 2 7 3 However , l imi ted photopenetration remains one of the major impediments in the application of in vivo P D T for the treatment of large tumours; this l imitat ion was evident in the first generation haematoporphyrin derivative- based photosensitiser Photofrin® (porf imer sodium) with an activation wavelength at 630nm, wh ich coincides wi th tissue absorption. d. The Benzoporphyrin derivatives- second generation photosensitisers T o further improve the eff icacy of P D T , various second generation photosensitising compounds with absorption peaks between 650- and 700 nm are under investigation. Some of these include the benzoporphyr in- derivatives, phthalocyanines, purpurins, and ch lor in C f . 274-276 The benzoporphyr in derivatives absorb max ima l l y at approximately 690 nm, a wavelength with at least twice the •tissue penetration o f Photofrin® . 2 7 7 > 2 7 8 One analogue 1 in current c l in ica l testing, benzoporphyrin- derivative monoac id r ing A (BPD- M A or B P D ) , possesses rather h igh singlet and triplet oxygen quantum yields wh i ch contribute to its potential as an efficient photosens i t i ser . 2 7 9 In addit ion, B P D has a pharmacokinet ic prof i le wh ich favours tumour accumulat ion and therefore optimal tumour- to- tissue ratios in D B A / 2 mice wi th the implanted rhabdomyosarcoma tumour M l . 2 8 0 - 2 8 1 E ven though B P D persists for up to 72 h in the mouse sk in , photosensit ivity only lasts for 24 h postinjection possibly as a result of inactivation rather than clearance of the photosens i t i ser . 2 8 2 Furthermore, Logan and colleagues have shown in vivo P D T mediated by B P D increased 4 0 the immunogenic i ty of M l tumour in the murine h o s t . 2 8 3 In summary, the advantages of B P D make it an effective and ideal photosensitiser for in vivo app l i c a t i ons . 2 8 4 e. Ex vivo uses of PDT The ex vivo use of P D T , pr inc ipa l ly in the manipulat ion of b lood products, bypasses the many constraints of in vivo P D T ; namely, interference o f photoactivation by the tissues and b lood surrounding the target site, and concerns wi th achiev ing a therapeutic concentration of the photosensitiser in the target without el ic i t ing significant sk in photosensitivity and other side effects. M a n y of the techniques developed for precl in ical in vitro studies of P D T can be adapted into c l in ica l ex vivo applications. In addit ion, whole b lood can be separated into its different components prior to P D T . The concentration and purif icat ion of leukocytes through apheresis eliminates most of the red b lood cel ls, wh ich may interfere with both the uptake of the photosensitiser and subsequent l ight activation. Essential ly , ex vivo P D T simpl i f ies the pharmacokinetics and pharmacodynamics by restricting the potential target populations. Fo r example, leukapheresis of a patient with autoimmune disease w i l l y ie ld both activated (proliferating) and nonactivated (quiescent) lymphocytes; the former populat ion has been shown to be more susceptible to P D T because of its prol iferative s ta te . 2 8 5 Gasparro 's group has completed several p romis ing c l in ica l trials on the extracorporeal U V A irradiation of b lood cells pretreated with 8-methoxypsoralen (8- M O P ) , for the management of cutaneous T- cel l l ymphoma and several other autoimmune d i seases . 2 8 6 Photophoresis usual ly takes place two hours after the ingestion o f 8- M O P wi th m i n i m u m plasma drug level at 50 ng/ml. A special ly designed photophoresis unit is used to separate the patient's b lood into different components -fo l lowed by photoactivation of the leukocytes in recirculating clear plastic tubings sandwiched between banks of U V A lamps; the treated cells are then returned to the patient's systemic circulat ion. Another use of P D T is the extracorporeal inactivation of enveloped viruses in b lood products; B P D has shown promise in the inactivation of F e L V and HIV1 .287.288 Another emerging use of the technology is in the immunomodulat ion of organ grafts prior to transplantation. Oboch i and colleagues have observed that sublethal doses of P D T using B P D was sufficient to prolong the survival o f al logeneic sk in grafts in mice , 41 which was attributed to the reduction of the expression of immunost imulatory ce l l surface molecules in the Langerhan cel ls, professional antigen presenting cells wh i ch reside in the s k i n . 2 8 9 f. Bone marrow purging PDT- mediated bone marrow purging ( B M P ) , wh ich requires the extracorporeal photoirradiation of bone marrow cel ls preincubated with photosensitiser, naturally progressed f rom the other ex vivo P D T appl icat ions discussed a b o v e . 2 9 0 - 2 9 1 Preferential uptake of the photosensitiser by leukaemic cells in comparison to normal nonproliferating cel ls provides a w indow of opportunity for this modal i ty in B M P . The feasibi l i ty of using B P D - mediated P D T in purging has been investigated exhaustively in both cytotoxici ty assays and ex vivo purging s y s t e m s . 2 9 2 F luorescent microscopy and f l ow cytometry have demonstrated that leukaemic cel l l ines and primary leukaemic cells preferentially take up more B P D than normal b lood cel ls. Spec i f i ca l ly , Jamieson et a l . reported that the human leukaemic cell l ine K 5 6 2 took up 10- fo ld and primary C M L c l in ica l samples took up 7-fo ld more of B P D than normal human bone marrow c e l l s . 2 9 3 Furthermore, short- term co lony assays and long- term bone marrow cultures as we l l as murine bone marrow purging experiments al l demonstrated the uti l i ty o f B P D in B M P . 2 9 4 - 2 9 5 L e m o l i and colleagues found that mult idrug resistant ( M D R ) leukaemic cel l l ines that express the P I 7 0 membrane glycoprotein are refractory to B P D - mediated but not to dihaematoporphyrin ether (DHE)- mediated P D T ; in addit ion, other cel lular protection mechanisms may also affect the effectiveness of p u r g i n g . 2 6 1 ' 2 9 6 Therefore, the heterogeneity of the cytotoxic response to .PDT depends on. the photosensitiser, its formulat ion (eg,-liposome-encapsulated), and the type of cells targeted. Ac t i ve drug eff lux as we l l as defence mechanisms against oxidative stress can be attenuated pharmacological ly in order to enhance P D T cytotoxicity. Recently, G l u c k and colleagues demonstrated the effectiveness of B P D - mediated P D T purging o f mul t ip le mye loma cells ex vivo.291 Photosensitisers other than B P D are also being considered for purging, some notable examples include the phthalocyanines and merocyanine 540 ( M C 5 4 0 ) . 2 9 8 ' 3 0 0 P D T purging has also been shown to be effective in the eradication of cancers other than leukaemia; Sieber and colleagues demonstrated the effectiveness of M C 5 4 0 in purging contaminating 42 neuroblastoma c e l l s . 3 0 1 Therefore, P D T purging can be considered for a u B M T s in neoplasms other than the leukaemias. g. Cl inical trials Cl in i ca l bone marrow purging using P D T is sti l l relatively uncommon compared to the other methodologies such as monoclonal antibody- mediated negative selection. Sieber and associates have conducted phase I c l in ica l testing of M C 5 4 0 - mediated PDT- purging o f autologous bone marrow cel ls f rom l ymphoma and leukaemia pa t i en ts . 3 0 2 A n ongoing c l in ica l trial of B M P in acute leukaemia undergoing a u B M T using the photosensitiser B P D is being conducted in Montrea l . Initial f indings f rom the smal l group o f patients are extremely encouraging. Purg ing at 10 and 20 ng/ml of B P D with 15J/cm 2 photoactivation resulted in the prolonged survival in f ive of seven patients wi th the longest disease- free-survival interval at 349 days posttransplantation. One patient relapsed on day 84 and another patient required infusion of the unmanipulated backup marrow 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 eff icacy of the purging. A phase I trial studying the feasibi l i ty of using B P D to purge C D 3 4 + cel ls isolated f rom the autologous P B S C harvests o f patients wi th Non-Hodgk in ' s L y m p h o m a is in its f inal p lanning stage. OBJECTIVES AND RATIONALE ' In this thesis, we attempted to improve the eff icacy of B P D - mediated P D T in an in vitro setting. Th is work is based on previous f indings by Jamieson that B P D - mediated P D T was selective in the destruction of leukaemic c e l l s . 2 9 1 - 2 9 2 W e decided to approach the problem v ia two independent angles. There exists a considerable amount of literature on the various forms of P D T combinat ion t h e r a p y . 3 0 3 - 3 0 6 Several groups have already performed studies on the combinat ion of P D T and D o x , albeit wi th different r e s u l t s . 3 0 7 - 3 0 8 W e therefore decided to 43 study the combined effects of B P D - mediated P D T and D o x for the purpose of in vitro P D T , i.e. for stem cel l purging. 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 wh ich could potentially affect the efficacy of the t h e r a p y . 3 0 3 - 3 0 9 . B P D , a recently developed second generation porphyr in- based photosensitiser, is much more potent than Photofrin® and is considered for various c l in ica l a p p l i c a t i o n s . 2 6 9 - 2 8 4 B P D , unl ike its predecessor Photofrin®, is a relat ively pure photosensitiser with characterised active componen t s . 2 7 8 Therefore, P D T combinat ion therapy using B P D is a novel area of research with relevant c l in ica l potentials especially for stem cel l purging because of its in vitro nature. C incot ta et al. recently demonstrated the advantage of a P D T regimen combin ing B P D wi th another photosens i t i ser . 3 0 5 W e were also interested in the possible interactions, physica l and b iochemica l , between B P D and D o x . Mur i ne haematopoietic cel ls, normal and leukaemic, were used throughout the experiment. The standard agar co lony assay a l lows the convenient and reproducible measurement of c lonogenicity of cells as a result of the various permutations of treatment parameters. W e therefore employed the agar co lony assay as a primary assessment tool o f the effectiveness of the different combinations. W e found that the murine leukaemic cel l l ine L 1 2 1 0 was much more susceptible to the combinat ion o f Dox-> P D T than normal D B A / 2 haematopoietic progenitors (Chapter 3). Therefore, we decided to further explore some of the possible mechanisms behind the f indings (Chapter 4). The second approach involves the incorporation of the haematopoietic inhibitory peptide N - A c S D K P into B P D - mediated P D T o f normal and leukaemic cel ls. N - A c S D K P has been demonstrated to effect the selective protection of normal haematopoietic cells f rom various forms of cytotoxic treatments and therefore was considered to be an ideal protector of normal bone marrow cel ls during r ad i o chemothe r apy . 2 0 5 - 2 1 0 Coutton and col leagues showed that N - A c S D K P also selectively protected normal human haematopoietic cells f rom Photofrin®- mediated P D T in a purging se t t i ng . 2 0 9 W e therefore wanted to extend their f indings to the second generation compound B P D . W e also wished to extend the experimental system to the murine setting such that, i f needed, bone marrow purging and transplantation experiments cou ld be performed to assess the protective effect o f N-A c S D K P . A g a i n , we were able to determine the effectiveness of the scheme by using the standard agar co lony assay. W e found that a preincubation per iod of 1.5 h with 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 leukaemic cel l l ine L 1 2 1 0 f rom B P D - mediated P D T (chapter 5). Furthermore, we attempted to study some of the potential mechanisms responsible for N - A c S D K P -photoprotection (chapter 6). Our f indings suggested that cel l cyc le inhibi t ion mediated by the peptide was partly responsible for the observed protective effect. Therefore, the overal l theme of this thesis is to improve the eff icacy of B P D -mediated P D T . In addit ion, we hope that in the process we w i l l reveal f indings beneficial to the c l in ica l applications of P D T as we l l as conducive to further understanding of the molecular mechanisms of P D T . 45 E X P E R I M E N T A L P R O C E D U R E S 2.1 EXPERIMENTAL REAGENTS a. B e n z o p o r p h y r i n derivat ive monoac id r i n g A ( B P D , Verteporfin®) The monoac id ring- A analogue of benzoporphyr in derivative in a l iposomal formulat ion ( B P D , Verteporfin®) was obtained f rom Q L T PhotoTherapeutics Inc. ( Q L T , Vancouver , B C , Canada). L yoph i l i s ed B P D was reconstituted to a concentration o f 1.5 mg/ml with sterile double- dist i l led water (ddH 2 0) every two weeks and stored at 4 ° C unt i l use. Further di lutions were carried out dur ing the experiment with tissue culture med ium in a reduced light environment. b. C h e m i c a l reagents Doxorub i c in hydrochloride (Dox) and cytosine arabinoside (Ara- C ) were purchased f rom the S i gma Chemica l C o . (St. L o u i s , M O ) and were suppl ied in powder fo rm. D o x was reconstituted in sterile d d H 2 0 to a concentration of 10 m M and stored as 50 pi aliquots at -20°C. Ara- C was reconstituted in sterile d d H 2 0 to a concentration o f 500 m M and stored as 50 u M aliquots at - 20°C. A l l other chemical reagents, except where speci f ica l ly noted, were purchased f rom S i g m a Chemica l C o . (St. L o u i s , M O ) . c. Tissue culture reagents Tissue culture media Dulbecco 's M o d i f i e d Eagles M e d i u m ( D M E M ) and Iscove's M o d i f i e d Du lbecco 's M e d i u m ( I M D M ) were purchased f rom G i b c o / B R L L i f e Tech . Inc. (Grand Island, N . Y . ) and were prepared according to the manufacturer's instructions. Fetal ca l f serum (FCS ) was purchased f rom the S i g m a Chemica l C o . (St. L o u i s , M O ) and was heat- inactivated at 57°C for 15 min . D M E M was further supplemented wi th streptomycin 46 (f inal 100 pg/ml) and penic i l l in (f inal 100 U/ml ) (G ibco/BRL ) , 1 m M sodium pyruvate (G ibco/BRL ) , and 25 m M of (N- [2- Hydroxyethy l ] piperazine- N '- [2- ethanesulfonic acid]) ( H E P E S , Sigma). Supplemental glutamine in the fo rm of G l u t a M A X I (G ibco/BRL ) , a L- A l any l - L- Glutamine dipeptide, was added to a f inal concentration of 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 for performing primary haematopoietic co lony assays and long- term bone marrow med ium were purchased f r om S temCe l l Technologies ( SCT , Vancouver , B.C. , Canada). They were pokeweed mitogen- spleen cel l condit ioned med ium ( P W M - S C C M , HemoS t im M2100 ) which contained interleukin- 3 (fL- 3) and granulocyte- macrophage colony stimulating factor ( G M - C S F ) , and murine mye lo id long- term culture med ium (Mye loCu l t M5300 ) . Tissue culture grade bovine serum albumin ( B S A ) in I M D M was purchased f rom Boehringer M a n n h e i m Canada. (Lava l , Quebec, Canada). e. Fluorescence activated cell sorting (FACS) reagents Ant ibodies and f luorochromes used for F A C S analysis were purchased f rom Pharmingen, Inc. (San D i ego , C A ) and are l isted be low (Table 2.1). F A C S washing buffer consisted o f 1 % heat- inactivated F C S (HI- F C S , S i g m a Chemica l C o . , St. L u o i s , M O ) and 0 . 1 % sod ium azide (Sigma) in phosphate buffered saline (PBS) . A 4 % paraformaldehyde (Sigma) double- strength F A C S f ixat ion buffer was prepared in P B S . 47 Table 2.1 Reagents used for F A C S analysis Specif icity Ant ibody C lone Isotvpe F o r m Reference Number mouse haematopoietic progenitor cells C D 3 4 R A M 3 4 (49E8) Rat I g G 2 a , K B io t in 09432D isotypic control R35- 95 Rat I g G 2 a , K B iot in 11022C B io t in - - - Steptavidin-F I T C 13024D F I T C , f luorescein isothiocyanate f. Synthetic peptides The tetrapeptides acetylated- serine- aspartate- lysine- prol ine ( N - A c S D K P ) , acetylated- serine- aspartate- lysine- glutamate ( N - A c S D K E ) , and serine- aspartate- lysine-proline ( S D K P ) were synthesised at the Mic rosequenc ing Centre of the Univers i ty of V i c to r i a (V ic tor ia , B.C., Canada). The peptides were further pur i f ied to homogeneity by high performance l iqu id chromatography ( H P L C ) and characterised by amino acid analysis, capi l lary electrophoresis, and fast atom bombardment mass spectroscopy. Peptides were suppl ied as lyophi l ised powders and were stored at -80°C in a desiccated environment. Peptides were reconstituted to 5 m M with 1 0 % HI- F C S / I M D M and stored as single-use aliquots, a t-80°C 2.2 LIGHT SOURCE A specif ical ly constructed l ight box was used for photoactivation of cells. The unit consisted o f upper and lower banks each of eight G E F15T8- R red fluorescent tubes (Sylvannia, D rummondv i l l e , Quebec, Canada) wh i ch emit between 600- 900 nm. In addition, two electric fans were incorporated into the unit to ensure adequate coo l ing of the samples during exposure. C e l l samples were placed on a clear plexiglass platform at 48 equidistant between the two banks of l ight. The unit was warmed up for thirty minutes before irradiation and photometric measurements in three axes (x, y, z) were made with an EL 1350 Radiometer/Photometer power meter (International L ight , Inc., M A ) immediately before exposure. Exposure time (Y ) was calibrated to provide 15 J /cm 2 o f light energy using the fo l low ing formula : Y (min)= 15 J/cm 2 X m W / c m 2 x 0.06 (W s/mW min) The average of the six photometric measurements of the three axes constituted the variable X . 2.3 EXPERIMENTAL ANIMALS a. Mice PJ3A/2 (H- 2 d) mice (6- 8 weeks o f age) were purchased f rom Charles R i ve r Breeding Laboratories Canada (Montreal , Quebec, Canada) and maintained under pathogen- free conditions in the animal faci l i ty .of the Department o f M i c r o b i o l o g y & Immunology at the Univers i ty o f B r i t i sh C o l u m b i a (Vancouver , B .C. , Canada). A l l animals were housed in microisolator units and were given standard laboratory rodent diet (Ralston Purina) and HC1- acidi f ied water ( pH 2.5) ad libitum. N e w l y arrived animals were kept in quarantine for two weeks prior to introduction into the core fac i l i ty . Experiments were conducted on animals that had been in the faci l i ty for two weeks or more to ensure acclimatisation. The protocol for animal experimentation was approved by the animal care committee of U B C . 49 2.4 CELL PREPARATION a. Mouse bone marrow cells Bone marrow mononuclear cel ls ( B M M N C s ) were obtained f rom the femurs of CO2- euthanised 8-12 week o ld D B A / 2 mice . B r i e f l y , the mouse was rinsed wi th 7 0 % ethanol and a straight l ine vertical inc is ion was made on the ventral surface fo l lowed by manual separation of the skin exposing the abdomen and the legs of the mouse. The muscles surrounding the femur were removed and the femur was cut superior to the patella and inferior to the acetabulum. The marrow cavity of the femur was then f lushed with I M D M supplemented with 1 0 % HI- F C S del ivered by a 22 gauge needle connected to a 5 cc syringe (Becton- D i c k i n s o n , Rutherford, NJ ) . The marrow was then dispersed into a single cel l suspension v ia gentle manipulat ion with the syringe without the needle. The process was repeated for the other femur. One mouse normal ly y ie lded 1 x 10 7 B M M N C s f rom its two rear femurs. The harvested cel ls were then washed twice and resuspended in I M D M . Ce l l s intended for F A C S analysis underwent an additional step of erythrocyte lysis in wh i ch 5 x 10 7 bone marrow cel ls were m i x e d wi th 5 m l of Tr i s/NH 4 C1 buffer ( pH 7.3) and incubated at 37°C for 10 m in fo l lowed by two washes with I M D M . b. Mouse leukaemic cell lines The L 1 2 1 0 murine lymphocyt ic leukaemia cel l l ine ( A T C C C C L 219) was purchased f rom the Amer i can Type Culture Co l lec t ion ( A T C C , Rockyv i l l e , Mary land) and maintained in D M E M supplemented wi th 1 0 % heat- inactivated F C S (HI- F C S ) . L 1 2 1 0 was or iginal ly derived f rom the D B A / 2 mouse strain fo l l ow ing sk in paintings with 0 . 2 % methylcholanthrene in ethyl ether and has been used extensively in the screening of new chemotherapeutic agen ts . 3 1 0 The cel ls were grown at 37°C and maintained in a 5 % carbon d iox ide ( C 0 2 , Praxair, M iss i ssauga , O N , Canada) - r oom air aerated, fu l l y humid i f i ed incubator (Forma Scient i f ic , Mar iet ta , Ohio) . Ongo ing cultures of L 1 2 1 0 cel ls were routinely replaced at two month intervals with frozen stocks f rom l iqu id nitrogen storage to ensure that the cells d id not acquire any mutations in vitro wh ich cou ld affect their behaviour. 50 c. Primary human haematopoietic cells N o r m a l human bone marrow cel ls were obtained dur ing sternotomy and were k ind ly suppl ied by Dr . Lawrence Bur r at the Vancouver Genera l Hospi ta l ( V G H , Vancouver , B.C., Canada). C e l l samples were maintained in I M D M supplemented wi th 200 units of sterile sodium heparin (Fisher Sc ient i f ic , Fa i r L a w n , NJ ) at room temperature and processed on the same day of extraction. Chron ic myelogenous leukaemic ( C M L ) cells were obtained f rom venupuntures of newly diagnosed, untreated C M L patients and were k ind ly suppl ied by D r . Noe l Buskard at the Mon roe outpatient c l in ic of V G H . The leukaemic b lood samples were stored in 10 m l Vacutainers containing 143 U S P of l i th ium heparin (Becton Dick inson) and were processed on the day of extraction. T o isolate the fraction containing mononuclear cel ls, the b lood samples were m ixed with I M D M in a 1: 1 ratio and 7 m l of the resultant mixture was then layered on top of 3 m l of room temperature F i co l l - Paque Plus (Pharmacia B io tech A B , Uppsa la , Sweden) and centrifuged at 300 R C F for 15 min . The interface was harvested wi th a sterile Pasteur pipette and the cel ls were washed twice with I M D M . d. Human leukaemic cell lines The human chronic myelogenous leukaemic ce l l l ine K 5 6 2 ( A T C C C C L 243), or ig inal ly derived f rom the pleural effusion of a C M L patient in blast cr isis, was obtained f rom A T C C and was maintained in 1 0 % HI- F C S / R P M I 1640. The cells were grown at 37°C and maintained in a 5 % carbon d iox ide ( C 0 2 , Praxair , Miss issauga, O N , Canada) -room air aerated, fu l l y humidi f ied incubator (Forma Scient i f ic , Marietta, Ohio) . 2.5 CYTOTOXIC TREATMENT OF CELLS a. PDT treatment of murine bone marrow cells and L1210 cells for short term evaluations (combination experiments) Each mouse provided approximately 1 x 1 0 7 B M M N C s . Freshly extracted B M M N C s were washed twice with warm I M D M and resuspended in the same med ium. A 51 smal l aliquot of the cel ls was then stained with the vital stain Eos in Y ( 0 . 3 % in P B S ) and the cel l concentration and viabi l i ty were determined fo l lowed by di lut ion with I M D M into 2.2 x 1 0 6 cel ls/ml. A t the same time, B P D and D o x were di luted into the appropriate work ing concentrations in I M D M and 100 ul of the 10 x drug was dispensed into the sterile 5 m l polystyrene test tubes (Falcon brand, Becton D ick inson) along wi th 900 ul of the B M M N C s . The tubes were then incubated at 37°C for 60 min after gentle m ix ing . The cel ls were washed once with I M D M and resuspended in 1.0 m l of I M D M supplemented with . 1 0 % HI- F C S , dispensed into a 24 we l l tissue culture plate ( L inbro brand, F l o w Laboratories, Inc., Hamdesn, Connecticut) and were photoirradiat ion wi th red l ight at 15J/cm 2 . C lonogenic i ty o f the treated cel ls was then assessed (section 2.6a). The L 1 2 1 0 cel ls were processed in the same manner (section 2.6b). Experiments of combined simultaneous B P D and D o x treatments were conducted with both normal and leukaemic cells. Samples were incubated concomitant ly with both B P D and D o x in different combinations for one hour at 37°C. Subsequent manipulations fo l lowed the same protocol as for the single agent- treated cel ls. Exper iments wh ich involved sequential B P D / D o x or Dox/ B P D treatments involved pretreatment of cells with P D T fo l lowed by a one hour incubation period wi th D o x , or preincubation of cells with D o x fo l lowed by B P D incubation and photoirradiation. Ce l l s were washed after the first treatment and handling protocols were identical to those for single agent treatment. b. PDT treatment of normal and leukaemic murine haematopoietic cells preincubated with peptides (short- term colony assay) Mur ine B M M N C s were obtained f rom D B A / 2 mice as described previously (section 2.4a). B M M N C s or L 1 2 1 0 cells at 2.2 x 10 6 cel ls/ml in 900 pi of I M D M were mixed with 10 pi of 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 inal concentration of 100 n M . The cells were incubated at 37°C for 1.5 h fo l l owed by the addition of 100 pi of B P D at 10 x the f inal concentrations. The cel ls were then incubated for an additional 1 h at 37°C and were washed 1 x with I M D M . C e l l pellets were resuspended gently wi th 1 m l o f 10 % HI- F C S / I M D M and dispensed into wel ls of a 24 52 wel l tissue culture plate and photoirradiated at 15 J/cm 2 . C lonogenic i ty of the treated cells was then assessed (section 2.6a and 2.6b). c. PDT treatment of murine bone marrow cells for long- term bone marrow cultures Mur ine B M M N C s were obtained f rom D B A / 2 mice as described previously (section 2.4a). B M M N C s at concentration of 2.2 x 10 6 cel ls/ml in 900 pi o f I M D M were mixed with 10 pi of N - A c S D K P to a f inal concentration o f 100 n M . The cells were incubated at 37°C for 1.5 h fo l lowed by the addition o f 100 pi of B P D at 10 x the f inal concentrations. The cells were then incubated for an additional 1 h at 37°C and were washed 1 x with I M D M . C e l l pellets were resuspended gently with 1 m l of 10 % HI-F C S / T M D M and dispensed into wel ls of a 24 wel l tissue culture plate and photoirradiated at 15 J/cm 2 . The treated cel ls were then assessed us ing the L T B M C assay (section 2.6d). d. PDT treatment of human normal haematopoietic and leukaemic cells preincubated with peptides (short- term colony assay) Norma l or leukaemic cells at 2.2 x 10 6 cells/ml in 900 pi of I M D M were mixed with 10 pi of 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 inal concentration of 100 n M . The cel ls were incubated at 37°C for 1.5 h fo l l owed by the addit ion of 100 pi of B P D at 10 x the f inal concentrations. The cells were then incubated for an additional 1 h at 37°C and were washed 1 x with I M D M . Ce l l pellets were resuspended gently with 1 m l of 10 % HI- F C S / I M D M and dispensed into wel ls of a 24 we l l tissue culture plate and photoirradiated at 15 J/cm 2 . C lonogenic i ty of the treated cel ls was then assessed (section 2.6e and 2.6f). e. ara- C treatment of DBA/2 bone marrow cells preincubated with peptides Mur ine B M M N C s were obtained f rom D B A / 2 mice as described previously (section 2.4a). B M M N C s at concentration of 2.2 x 10 6 cel ls/ml in 900 pi o f I M D M were 53 mixed with 10 ul of N - A c S D K P to a f inal peptide concentration of 100 n M . The cel ls were incubated at 37°C for 1.5 h fo l lowed by the addition of 100 pi of ara- C at 10 x the f inal concentrations. The cells were then incubated for an additional 1 h at 37°C and were washed 1 x with I M D M . Ce l l pellets were resuspended gently with 1 ml of 10 % HI-F C S / I M D M and 140 pi (2.8 x 10 5 cel ls) was m i x e d with the other ingredients of the standard agar co lony assay for c lonogenic i ty determination (section 2.6a). f. PDT of DBA/2 bone marrow cells preincubated with 50 uM ara- C Mur ine B M M N C s were obtained f rom D B A / 2 mice as described previously (section 2.4a). B M M N C s at concentration of 2.2 x 10 6 cel ls/ml in 900 pi of I M D M were m i x e d wi th 100 pi of ara- C to a f inal concentration of 50 u M . The cel ls were incubated at 37°C for 1 h, washed once with I M D M , and resuspended in 900 pi of I M D M . Next , 10 x solutions of B P D (100 pi) were then added to the appropriate sample tubes to a f inal vo lume of 1.0 m l . The cells were then incubated for an additional 1 h at 37°C and were washed 1 x with I M D M . Ce l l pellets were resuspended gently with 1 m l of 10 % HI-F C S / I M D M and dispensed into wel ls of a 24 wel l tissue culture plate and photoirradiated at 15 J/cm 2 . C lonogenic i ty of the treated cel ls was then assessed (section 2.6a). 2.6 CYTOTOXICITY ASSAYS a. Agar colony assay of treated murine bone marrow cells Standard agar- based co lony assays were used to assess cytotoxic i ty on normal murine bone marrow mononuclear cel ls ( B M M N C s ) . The protocol for the murine haematopoietic granulocyte- macrophage progenitor assay has been described elsewhere 294,311 u j 0 f treated D B A / 2 bone marrow cel ls was added to a plat ing mixture wh ich consisted o f 2235 p i I M D M , 750 pi of pretested HI- F C S , 375 pi of 100 mg/ml (w/v) bovine serum albumin ( B S A ) in I M D M (Boehringer Mannhe im , Lava l , Quebec), and 100 pi o f Hemos t im M 2 1 0 0 (see section 2. Id) as a source of cytokines. F ina l l y , 400 pi o f prewarmed 3 % agar Nob le solution (w/v) in sterile d d H 2 0 (D i fco Laboratories, Detroit , 54 Mich igan ) was dispensed into the ce l l mixture to achieve a f inal agar concentration of 0 . 3 % and the resulting mixture was dispensed immediately in 1.0 m l volumes to triplicate 35 m m tissue culture dishes (Sarstedt, Newton , N C ) wi th approximately 70000 cel ls dispensed per tissue culture dish. T w o of the agar containing plates were then placed inside a 100 m m petri dish (Fisher Sc ient i f ic , Edmonton , A lber ta , Canada) a long wi th a 35 m m dish containing 5 m l d d H 2 0 . The plates were then placed inside a 37°C incubator for seven days prior to co lony enumeration. A Zeiss Ax iove r t 35 inverted microscope (Carl Zeiss Canada, , D o n M i l l s , Ontar io, Canada) was used for co lony count ing and a cluster consist ing of forty or more cel ls was counted as a granulocyte- macrophage co lony fo rming unit ( C F U - G M ) . C l o n i n g eff ic iency for D B A / 2 C F U - G M was approximately 0.126 % . b. Colony assay of L1210 cells Aga r colony assay of the leukaemic ce l l l ine L 1 2 1 0 was slightly different f rom the previously described assay. A 334 pi al iquot of di luted ce l l suspension was mixed with 2247 pi of I M D M , 800 pi of pretested HI- F C S , 400 pi of B S A , and f ina l l y 400 pi o f 3.0 % Agar- Nob le . T o fu l l y appreciate to range of L 1 2 1 0 log reduction, seeding numbers of cel ls was customised for ind iv idua l drug concentrations. A g a i n , 1.0 m l was dispensed into triplicate 35 m m tissue culture dishes. Co lon ies consist ing of forty or more cells were enumerated on day 6 us ing the inverted microscope. C l on ing eff ic iency of L 1 2 1 0 C F U - L in this system was approximately 36 % . c. Short-term cytotoxicity assay (MTT assay) Cel l s in log phase growth, were washed twice with D M E M to remove residual serum and 900 p i of cel ls at 1.4 x 1 0 6 cel ls/ml were dispensed to 5 m l polystyrene test tubes (Falcon). B P D and other chemotherapeutic agents at 10 x f inal concentrations were then added to each tube in volumes o f 100 p i . The tubes were then gently agitated and a l lowed to incubate for 1 hour. The treated cel ls were then washed once and resuspended in 900 p i of D M E M . The content of a single test tube was then distributed into eight wel ls of a 96 wel l tissue culture plate (Falcon) in 100 p i aliquots fo l lowed by 15 J /cm 2 o f red l ight photoirradiation as described in section 2.2. The plate was then incubated overnight at 37°C in a 5 % CO2 incubator. The next day, 10 p i o f 5 mg/ml 3-[4,5-Dimethylthiazol-2-yl]-2,5-55 diphenyltetrazol ium bromide ( M T T , S igma) in P B S was then dispensed into the wel ls with an Eppendor f Repeator Pipette dispenser (B r inkmann Instruments, Miss issauga , Ontar io, Canada) and a l lowed to incubate for 60 m in at 37°C before f ixat ion with 150 pi of 0.05 N HC1/ i. Mi tochondr ia l dehydrogenase in viable cells convert the ye l low M T T tetrazolium salt to purple formazon crystals, wh ich cou ld be quantif ied spectrophotometrically at 595 n m ( O D 5 9 5 ) . 3 1 2 ' 3 1 3 The plate was read on a Spect raMax 250 scanning mul t iwe l l spectrophotometer (Molecular Dev ices , Sunnyvale, C A ) . O D 5 9 5 measurements of control cells were averaged and arbitrarily set as 1 0 0 % survival and viabi l i ty of the treated cells were calculated based on their relative values. d. One step long- term bone marrow culture (LTBMC) of murine haematopoietic cells Long- term bone marrow culture ( L T B M C ) was initiated to determine the fitness of earlier haematopoietic progenitors and stem cells. Mur ine L T B M C culture med ium (Mye loCu l t M 5 3 0 0 , S temCel l Technologies Inc.) was used to initiate and maintain the growth of PDT- treated D B A / 2 B M M N C s for six weeks. A one step L T B M C protocol was adapted in wh ich the inocu lum was used to start the stromal layer as we l l as to initiate haematopo ies i s . 3 1 4 Hydrocort isone (S igma Chemc ia l Co. ) d issolved in 100 % ethanol was added to the L T B M C med ium to a f inal concentration of 1 0 " 6 M before use. The PDT-treated cel ls were d iv ided into two equal portions of 1 x 10 6 cel ls and dispensed into wel ls of a 24 we l l tissue culture plate (Corn ing Costar Corp . , Co rn i ng , N Y ) in vo lume of 1 m l . .The plates were incubated at 33°C during the first week and moved to a 37°C 5 % C 0 2 incubator for the subsequent duration of the experiment. Ce l l s were harvested at weekly intervals during which the entire content of the appropriate we l l was used for ce l l number determination fo l lowed by incorporation of the cells into the short- term agar co lony assay (section 2.6a). The rest of the cel ls were demidepopulated, i.e. gently agitated fo l l owed by aspiration of 500 pi of the cells (50 % ) , and replenished with 500 pi o f murine L T B M C culture med ium containing 1 0 " 6 M . The cells were then returned to the incubator (37°C, 5 % C 0 2 , fu l l y humidi f ied) unti l the next harvest seven days later. 56 e. C o l o n y assay of treated n o r m a l a n d leukaemic h u m a n haematopoietic c e l l s Short- term colony assays of human haematopoietic cells were ini t ia l ly described by Messner et a / . 3 1 5 PDT- treated or untreated cells at 400 pi vo lume were m i x e d wi th 400 pi phytohaemagglutinin- stimulated leucocyte condit ioned med ium ( PHA- L C M ) , 1200 pi pretested HI- F C S , 1600 pi I M D M , and 400 pi 10 % methyl cel lulose d issolved in I M D M (Methocel M C , F l u k a B iochemika , Switzerland) to a total assay vo lume o f 4.0 m l . P H A -L C M was harvested f rom the peripheral b lood of a healthy volunteer. B r i e f l y , buffy coat (mononuclear cells and plasma), wh ich settled to the upper layer of " res ted" whole b lood containing 50 U sod ium heparin (Fisher) per m l , was removed by aspiration. The mononuclear cells were then separated f rom the plasma by centrifugation for 10 m in at 400 x R C F . The cel ls were washed 2 x wi th I M D M and the resuspended at 1 x 10 6 cel ls/ml in 1 % P H A (v/v)/ I M D M and incubated at 37°C for 7 days. The condit ioned med ium (PHA-L C M ) , separated f rom the ce l ls , was dispensed and stored at -80°C. P H A - L C M contains a variety of haematopoietic growth factors and supports the growth o f different colonies. The mixture was then combined and 1 m l each was dispensed into triplicate 35 m m dishes (Sarstedt) using a 16 gauge needle connected to a 3 cc syringe (Becton- Dickenson) . Norma l human B M M N C s and primary leukaemic cells f rom C M L patients were plated at 1 x 10 5 cel ls/ d ish. Co lon ies consist ing o f forty or more cel ls were counted after 14 days of culture at 37°C using an inverted microscope (Carl Zeiss Canada). f. C o l o n y assay of the h u m a n leukaemic cel l l ine K 5 6 2 * PDT- treated or untreated K 5 6 2 cells were assayed s imi lar ly as the pr imary human haematopoietic cells described above (section 2.6e) with the f o l l ow ing modif icat ions. K 5 6 2 cells were plated at a f inal concentration of 1 x 10 4 cells/ dish and P H A - L C M was replaced with I M D M in 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 analysis Stock B P D and D o x were d issolved in phosphate buffered saline (PBS ) to concentrations of 1 pg/ml and 10 u M , respectively. Equa l amounts o f B P D and D o x were dispensed into a spectrophotometric cuvette and then analysed with the H P 8 4 5 2 A spectrophotometer (Palo A l t o , C A ) . b . Spectro f luor imetr ic analysis of the interact ion between 1BPD a n d D o x The protocol for B P D uptake analysis has been described elsewhere 2 8 5 . B r i e f l y , L 1 2 1 0 cells were incubated with B P D alone, B P D in the presence of D o x , and B P D after one hour preincubation wi th D o x . Ce l l s were washed twice wi th P B S and then lysed in the presence of 500 pi o f 2 % Tr i ton X- 100 (S igma) /PBS. Samples were snap frozen in dry ice/ methanol bath and stored at -80°C i f readings were not planned on the same day of the experiment. Pr ior to spectrofluorimetric readings, the lysates were put through three rounds of freeze/ thawing. A n additional vo lume of 500 pi P B S was added to the rest of the ce l l lysate and the resultant mixture was used for analysis with the A m i n c o S L M 500 C sprectrofluorimeter ( S L M instruments Inc., Urbana, IL) . A l l measurements were performed using the continuous wave ( C W ) setting and at excitation and emission bandpass of 0.5 nm. Determination o f B P D concentration was achieved using excitation reference at 440 n m and emission reference at 700 nm. M e a n fluorescence intensity (MF I ) was measured at 694 nm for each sample. Em i s s i on reference was adjusted to 600 n m for D o x concentration determination and M F I was measured at 590 nm. A l l experiments .were done in duplicates. 2.8 CELL ANALYSIS BY FLUORESCENCE ACTIVATED CELLS SORTING (FACS) The protocol for the determination of cel lular content of B P D in murine splenocytes has been described e l sewhere . 2 8 5 Essential ly the same protocol has been adopted for analysis of D B A / 2 B M M N C s . One mi l l i on of freshly isolated B M M N C s in 1.0 m l I M D M 58 were preincubated with N - A c S D K P or S D K P (negative control) to a f inal concentration of 100 n M . Tissue culture med ium was used in place of peptide in a second negative control . The cells were incubated at 37°C for 1.5 h fo l lowed by 1 x wash with I M D M . C e l l pellets were resuspended in 900 pi I M D M and m ixed with 100 pi of 10 x B P D (100 ng/ml). The cel ls were incubated wi th an addit ional 0.5 h fo l l owed by 2 x washes w i th F A C S buffer and transferred to a round bottom 96 we l l plate (Costar, Cambr idge, M A ) . The cells were label led with 10 pi of biotin- C D 3 4 antibody (clone R A M 3 4 ) or 2 pi of biot in- isotypic control for 0.5 h at 4 °C . The secondary F ITC- strepavidin reagent was added after 2 washes w i th F A C S buffer. A n addit ional incubat ion per iod of 0.5 h was fo l l owed by 2 f inal washes wi th F A C S buffer. The ce l l pellets were then transferred to 5 m l polystyrene tubes (Falcon) in 500 pi of F A C S buffer and 500 pi o f 4 % paraformaldehyde/PBS and stored at 4 °C unti l analysis. A Coul ter E P I C S X L ® f l ow cytometry system (Coulter C o r p . , M i a m i , F L ) was used for dual colour F A C S analysis o f B P D uptake and surface antigen expression. The excitation wavelength employed for F A C S analysis invo lv ing B P D and F I T C was 488 n m whi le a 690 n m emission (longpass) filter was uti l ised to detect B P D (red) fluorescence and a 525 n m emission (longpass) filter was used to detect F I T C (green) fluorescence. One hundred thousand cells were analysed for each sample. Data f rom three independent experiments are presented and source of error is der ived f rom the standard error o f the mean. 2.9 FLUORESCENT MICROSCOPY K 5 6 2 Ce l ls were resuspended at a concentration of 1 x 10 6 cel ls/ml in D M E M and incubated at 37°C with 10 pg/ml of B P D for 60 min . Ce l l s were then washed once and resuspended in 1 m l o f D M E M . A 50 pi drop (5 x 10 4 cells) was placed on a glass sl ide (Fisher Sc ient i f ic , Edmonton, A lber ta , Canada) and subsequently covered wi th a cover sl ip (Fisher). The cells were observed under an O l ympus N e w V a n o x microscope wi th the A H 2 - F L transmitted light fluorescence attachment on " B l u e " setting (excitation @ 380-490 nm, observation @ 515 n m +). Pictures were taken wi th the attached camera using Fuj ichrome 400 I SO f i l m at automatic settings. 59 3.0 ANALYSIS OF INTRACELLULAR GLUTATHIONE (GSH) CONTENT a. Tietze enzymatic assay Cel lu lar glutathione (GSH ) was determined using enzymatic cyc l ing and E l lman ' s reagent (5,5'- dithiobis- (2- nitrobenzoic acid), D T N B ) as described by Eyer and P o d h r a d s k y . 3 1 6 B r i e f l y , cel ls were lysed in 0.6 % su lphosa l icyc l i c acid/ 0.5 m M E D T A (Sigma) at a f ina l concentration of 100 ul lysis buffer per 1 x 10 6 cells for 1 h at 4 °C . The supernatant was then separated after centrifugation at 16000 x R C F for 10 min (max imum speed setting on the Beckmann microcentrifuge) at 4 °C . The c lar i f ied lysates were dispensed into a 96 we l l microtitre plate (Falcon) and the vo lume made up to 180 ul with P B S . A mixture containing 4 ug/ul N A D P H (Ca lb iochem, L a Jo l la , C A ) and 1.2 ug/ul D T N B (Sigma) was dispensed into each wel l using a Eppendorf repeating pipetter dispensing at 10 ul (Br inkmann) . Last ly , 10 ul of yeast- der ived glutathione reductase at 0.012 U/ul (Ca lb iochem) in P B S was dispensed into each we l l . The plate was then read at O D 4 1 2 n m f o r 6 min in 1 min intervals using a SpectraMax 250 scanning mul t iwe l l spectrophotometer (Molecu lar Dev ices , Sunnyvale, C A ) . The difference between the 1 min and 6 min readings was calculated and the concentration of G S H was determined in compar ison to known G S H standards (S igma). 3.1 STATISTICAL ANALYSIS a. Analysis of colony assay data from PDT/Dox combination experiments Data were inputted into the N C S S 6.0.21 statistical analysis package ( N C S S Statistical software, Kaysv i l l e , Utah). The various treatment and control group means were compared using the Student's t- test wi th paired sample for means. The data were further analysed using 2- way analysis of variance (2- way A N O V A ) wi th the two B P D concentrations (2.5 and 5.0 ng/ml) and the three different combinat ions (Dox/PDT, PDT-> D o x , Dox-> P D T ) set as the two parameters. Add i t iona l statistical analyses were carried out with the term signif icant at alpha (p value) = 0.05 and intragroup differences were examined using the Bonferron i (all- pairwise) mult iple compar ison test. 60 b. Analysis of colony assay data from the inhibitory peptide pretreatment experiments Data were inputted into the N C S S 6.0.21 statistical analysis package ( N C S S Statistical software, Kaysv i l l e , Utah). The various treatment and control group means were compared using the Student's t- test wi th paired sample for means. The data were further analysed using two- way analysis o f variance (two- way A N O V A ) with 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 we l l as the no peptide control groups. Add i t iona l statistical analyses were carried out with the term significant at alpha (p value) = 0.05 and intragroup differences were examined using the Bonferron i (all-pairwise) mult iple comparison 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 STUDY 3.1 ABSTRACT Benzoporphyr in derivative monoac id r ing A ( BPD) , a porphyr in based photosensitiser, shows ideal properties for the purpose of photodynamic therapy (PDT)-based bone marrow purging and is currently undergoing c l in ica l testing for stem cel l purging in Canada. T o enhance the eff icacy of B P D - mediated P D T , a combinat ion approach wi th Doxorub i c in (Dox) was pursued. Short term co lony assays were used to measure the frequency of co lony forming unit- granulocyte/macrophage progenitors ( C F U -G M assay) of normal D B A / 2 bone marrow cells and colony forming unit- leukaemia ( C F U - L ) of the murine leukaemic cel l l ine L I 2 1 0 after drug treatment. B P D alone in the absence of l ight resulted in no reduction of C F U - G M ; however, 10 ng/ml o f B P D fo l lowed by 15 J/cm 2 of red l ight activation resulted in the e l iminat ion o f 0.5 logs o f the C F U - G M and 4 logs of C F U - L. D o x at 5.0 u M resulted in one log reduction o f both normal as we l l as leukaemic L 1 2 1 0 colony fo rming units; in addit ion, the cytotoxic i ty o f D o x was neither enhanced nor d imin ished in the presence of 15 J /cm 2 o f red l ight exposure. Simultaneous treatment of normal D B A / 2 bone marrow cel ls or the L 1 2 1 0 cel l l ine with P D T and D o x (PDT/Dox).resulted in the predicted log reductions. However , when cel ls were preincubated with D o x for one hour prior to P D T (Dox-> P D T ) , the resultant cytotoxici ty was 1.5- 2.5 fo ld more than that observed in the simultaneous treatment (PDT/Dox) and in the reverse sequence (PDT-> Dox ) . S igni f icant ly , L I 210 cel ls were more susceptible to the Dox-> P D T treatment than normal C F U - G M resulting in enlargement of the therapeutic w indow. This augmentation is treatment sequence dependent as we l l as drug dose dependent. The above f indings suggest that care must be taken when P D T is used in conjunction with other chemotherapeutic agents in the c l in ica l setting and that judic ious use 62 of P D T combinat ion therapy can lead to the augmentation of the therapeutic w indow between normal and neoplastic cells. 3.2 INTRODUCTION H i g h dose radiochemotherapy coupled wi th haematopoietic stem cel l rescue is an effective fo rm of treatment for various kinds of human malignancies. Bone marrow f rom allogeneic human leukocyte antigen ( H L A ) - matched donors is an ideal source of haematopoietic stem cells for leukaemic patients because of the associated yet beneficial graft- versus- leukaemia ( G v L ) effect. Unfortunately, the dearth of H L A - matched donors restricts the use of allogeneic bone marrow transplantation ( a l B M T ) . In addit ion, many older patients exhibit a high incidence of age- related intolerance to graft- versus- host-disease ( G v H D ) . Therefore, autologous bone marrows are used in older cancer patients or those without H L A - matched donors. Cancer patients undergoing autologous bone marrow transplantations ( a u B M T ) have higher relapse rates than those receiving allogeneic bone m a r r o w . 1 5 2 Through retroviral mark ing studies, Brenner and colleagues have unequivocal ly demonstrated the presence of contaminating neoplastic cells in autologous bone marrow harvests and the contr ibution of these cel ls to relapses post t ransplantat ion. 1 5 4 Bone marrow purging therefore attempts to address this problem by the selective removal or destruction of the contaminating cancer cells whi le preserving enough of the normal haematopoietic progenitor and stem cells to effect haematopoietic reconstitution. Bone marrow purging using photodynamic therapy ( PDT ) represents an ideal application o f this technology. The ex vivo nature o f purging eliminates problems associated with l ight del ivery and in vivo pharmacokinet ics. Var ious photosensitisers have been proposed for purging inc lud ing the benzoporphyr in derivatives, phthalocyanines and Merocyan ine 540 ( M C 5 4 0 ) . 2 9 0 ' 2 9 1 In this laboratory, the mono- acid r ing A analogue o f benzoporphyr in derivative ( BPD ) was shown to be a promis ing purging agent because o f preferential accumulation by leukaemic cells in addition to their higher susceptibility to P D T mediated by B P D . 2 9 2 Jamieson has also demonstrated the effectiveness of B P D purging in an ex vivo mur ine purging m o d e l . 2 9 4 In addit ion, B P D has an unique absorption peak at 63 690 nm wh ich is outside the absorption range of haemoglobin ; therefore, photoactivation of B P D is not affected by the presence of erythrocytes, a common occurrence in haematological s amp l e s . 3 1 7 Cancer therapies wh ich combine different drugs have been in use for many years. Combinat ion therapy is especially effective in the management of leukaemia and lymphoma. P D T has been used in combinations wi th traditional chemotherapeutic agents or other photosensitisers to achieve improved k i l l i n g of cancerous c e l l s . 3 0 5 - 3 1 8 Ideally, combinat ion therapy aims to util ise drugs wi th different mechanisms o f action such that the probabil i ty of the cancer developing drug resistance to all the drugs in the regimen is min imised. In addit ion, drugs used in combinat ion therapy should have additive anticancer cytotoxici ty but not adverse effects against normal tissues. In this chapter, B P D - mediated P D T was combined wi th D o x in various concentration as we l l as sequence combinations. The relative sensitivities of the murine leukaemic cel l l ine L I 2 1 0 and its normal counterpart, haematopoietic progenitor cells f rom D B A / 2 mice, were determined using standard agar based co lony assays. Th is chapter addresses the question as to whether a combinat ion approach using D o x and B P D can be applied to P D T purging and whether the differential susceptibil ity of normal and leukaemic cells can be altered with such a combinat ion approach. 64 3.3 RESULTS Single agent cytotoxicity In order to establish baseline susceptibil ity profi les to single- agent treatments. L 1 2 1 0 cells and haematopoietic progenitor cells derived f rom D B A / 2 mice were treated s ingly wi th either B P D - mediated P D T or D o x (with red l ight irradiation). In figure 3.1, normal and leukaemic cells were incubated with various concentrations of B P D fo l lowed by red l ight irradiation at 15 J/cm 2 . C lonogenic i ty of the cells was then examined using standard agar based co lony assays, wh ich measured co lony fo rming units- leukaemic ce l l ( C F U - L ) and co lony fo rming units- granulocyte/ macrophage ( C F U - G M ) of L I 210 and the D B A / 2 haematopoietic progenitors, respectively. The number of colonies which consisted of 50 or more cel ls were enumerated at day 6 (leukaemic cells) or day 7 (normal progenitor cells) w i th an inverted microscope. Results are expressed as survival fraction relative to control cel ls that were exposed to red l ight in the absence of B P D or D o x . Under identical condit ions, both L I 2 1 0 cells and D B A / 2 haematopoietic cel ls responded to treatment in a dose- dependent manner. However , the L 1 2 1 0 cel ls were much 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 of 10 ng/ml B P D and red light (15 J/cm 2) resulted in 4- log reduction of L 1 2 1 0 clonogenic cel ls whi le the same dose of B P D only reduced D B A / 2 progenitors by 0.5 log. S imi lar differential susceptibil ity between L 1 2 1 0 and D B A / 2 progenitor cytotoxicity was observed, albeit to a lower degree, at 5 ng/ml of B P D . Nevertheless, the data (figure 3.1) clearly showed that the leukaemic ce l l l ine L 1 2 1 0 was much more susceptible to B P D -mediated P D T than normal murine haematopoietic cells. B P D alone in the absence of direct l ight exposure was not cytotoxic as determined by the M T T assay, wh i ch measures mitochondrial dehydrogenase activity in viable cells (figure 3.2). Doxo rub i c i n hydrochlor ide (Dox) , un l ike B P D and l ight, showed essentially no selective k i l l i ng of L 1 2 1 0 cells over normal progenitor cells (figure 3.3). In addit ion, D o x cytotoxic i ty on L 1 2 1 0 cells was unaltered w i th or without direct l ight exposure (figure 3.4). 65 Figure 3.1 The effect of BPD- mediated PDT on the clonogenicity of normal DBA/2 haematopoietic progenitors and the leukaemic cell line L1210 Differential susceptibil ity o f L I 210 leukaemic cells and D B A / 2 haematopoietic progenitors to P D T mediated by B P D . Ce l l s received different doses of B P D and were exposed to 15 J /cm 2 o f red light (600- 900 nm). C o l o n y fo rming units o f normal and leukaemic cells were determined with standard assays and colonies were scored on day 6 ( C F U - L ) and day 7 ( C F U - G M ) o f culture us ing an inverted microscope. On ly colonies with 50 or more cells were counted. Untreated L 1 2 1 0 cells and D B A / 2 progenitors y ielded 137.85 ± 9.57 and 97.35 ± 7.78 co lonies , wh i ch translated to clonogenicity o f 41 % and 0.126 % respectively. Surv iva l fractions of treated cells were calculated based on the number o f colonies generated d iv ided by the number of colonies f rom the untreated contro l . Furthermore, di lut ion or correction factors were incorporated into the calculations for L I 2 1 0 cells in order to cover the 4- log surv ival fraction range. Data obtained f r om 12 (L1210) and 11 (DBA/2 progenitors) independent experiments is presented. Error bars are derived f rom standard errors of the mean. 66 survival fraction 0 . 0 H 0.00 H 0.0001 2.5 j — i — i — i — • • • • T 7.5 i 10 DBA/2 B M O L1210 [BPD] ng/ml 67 100 • 75 A %OD595 of respective no BPD controls 50 4 25 J 10 %OD595 of control-no light •O- %OD595 of control • 15J/cm 2 red light 100 [BPD] ng/ml Figure 3.2 Cytotoxicity of B P D 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 with different doses o f B P D for 1 hr fo l lowed by red light exposure at 15 J / cm 2 or no i l luminat ion. V iabi l i ty o f the cells was determined after overnight incubation at 37° C us ing the M T T viabil ity assay. O D 5 9 5 o f cel ls that received no B P D was arbitrarily designated to be 100 % . B P D - mediated P D T is only cytotoxic when activated by light and inhibited 50 % of the L 1 2 1 0 cells ( I C 5 0 L 1 2 1 0 ) at 6 ng/ml. Ce l ls wh i ch had received B P D but no light remained viable. Data f rom a single representative experiment is presented. 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 susceptibil ity o f L I 210 leukaemic cells and DBA/2 haematopoietic progenitors to D o x and light is m in ima l . Ce l ls received different doses of D o x and exposed to 15 J /cm 2 o f red light (600- 900 nm) . Co lony fo rming units o f normal and leukaemic cells were determined with standard assays and colonies were scored on day 6 ( C F U - L ) and day 7 ( C F U - G M ) of culture using an inverted microscope. Untreated L I 2 1 0 cells and D B A / 2 progenitors yielded 119.33 ± 9.67 and 97.35 ± 7.78 colonies, wh ich translated to clonogenicity o f 36 % and 0.126 % respectively Surv iva l fractions o f treated cells were calculated based on the number of colonies generated div ided by the number o f colonies f rom the untreated control . Data obtained f rom 5 (L1210) and 6 (DBA/2 progenitors) independent experiments is presented. Er ror bars are derived f rom standard errors o f the mean. / 69 70 100 - i 75 J %OD595 of control-no light %OD595 of respective no Dox controls %OD595 of control-15J/cm2 red light 50^ 25 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 wi th different doses of D o x fo l lowed by red light exposure at 15 J / cm 2 o r , no i l luminat ion. V iabi l i ty o f the cells was determined after overnight incubation at 37°C using the M T T viabi l i ty assay. O D 5 9 5 o f cells that received nd D o x was arbitrarily designated to be 100 % . L ight activation subsequent to D o x incubation d id not alter the viabil i ty o f L 1 2 1 0 cel ls. The concentrations of D o x wh i ch inhibited 50 % of L 1 2 1 0 cells therefore were s imi lar at 20 u M , in the presence or absence of 15 J /cm 2 red light exposure. Data f rom a single representative experiment is presented. 71 Combination experiments with BPD- mediated PDT and Dox: simultaneous and sequenced treatments of DBA /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 marrow cells were treated with various combinat ions o f the above two agents, simultaneously and sequentially. Coincubat ion of D B A / 2 haematopoietic progenitor cel ls wi th 5.0 ng/ml B P D and 2.5 p M D o x fo l lowed by 15 J /cm 2 red l ight exposure (Dox/ P D T ) resulted in a modest increase in cytotoxicity compared to P D T or D o x single agent treatments (Figure 3.5). Cytotoxic i ty d id not change appreciably f rom the simultaneous regimen when the cells were treated first with P D T fo l l owed by one hour incubation with 2.5 p M D o x (PDT-> Dox ) . 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 of the surv iva l fraction of normal C F U - G M f rom 0.47 ± 0.08 to 0.15 + 0.04 or a 3.2- fo ld increase in k i l l i ng compared to reverse sequence was observed (PDT-> D o x ) . Therefore, preincubation with D o x appeared to presensitise cells to the subsequent P D T treatment. A s shown in f igure 3.6, s imi lar f indings were made when 5.0 ng/ml B P D was combined with 1.25 p M D o x in the different combinat ions. F igures 3.7 and 3.8 show that the sequence of Dox-> P D T using 2.5 ng/ml of B P D in combinat ions wi th 2.5 or 1.25 p M D o x effected superior k i l l i ng compared to PDT-> D o x and the simultaneous use of D o x / P D T . It should be noted that a less than one log reduction in ce l l numbers was mediated by the treatments shown in f igures 3.6 and 3.7 since a lower dose o f B P D was used. Combination experiments with BPD- mediated PDT and Dox: simultaneous and sequenced treatments of L 1 2 1 0 leukaemic cells The clonogenicity of the leukaemic cel l l ine L 1 2 1 0 was s imi lar ly affected by the combinat ion treatments as normal haematopoietic progenitors. A s demonstrated in figure 3.5- 3.8, preincubation of L 1 2 1 0 cel ls w i th either 1.25 or 2.5 u M of D o x for one hour y 72 increased the efficacy of subsequent PDT- mediated cel l k i l l i ng . The resultant cytotoxicity was signif icantly greater than the reverse sequence PDT-> D o x and simultaneous treatment. In addit ion, L I 2 1 0 cells were more susceptible to the Dox-> P D T combinat ion regimen than D B A / 2 haematopoietic progenitors. A s shown in f igure 3.5, the surv ival fract ion of L 1 2 1 0 cel ls was signif icantly reduced f rom 0.10 + 0.07 to 0.0035 ± 0.005, or a 28.8- fo ld increase in k i l l i ng , when the cells were exposed to 2.5 p M D o x prior to P D T mediated by 5.0 ng/ml P D T (Dox-> P D T ) compared to the reverse sequence PDT-> D o x . Combination Dox->PDT PDT->Dox PDT/Dox Dox only • BPD only I T T 0.001 0.01 0.1 survival fraction £3 DBA/2 BMMNC H L 1 2 1 0 Figure 3.5 Survival fraction of DBA/2 haematopoietic progenitors and L1210 cells to combinations of 5 ng/ml BPD and 2.5 p M Dox in conjunction with 15 J/cm2 of red light exposure-Simultaneous Dox/ P D T and PDT-> D o x treatments us ing 5 ng/ml of B P D and 2.5 p M of D o x yielded s imi lar reductions in the c lonogenici ty of D B A / 2 C F U - G M and L 1 2 1 0 C F U - L. Howeve r , L I 2 1 0 cells were more susceptible to the Dox-> P D T treatment than its nonmalignant counterpart, resulting in the enlargement of the therapeutic w i n d o w . Data obtained f rom 6 (Dox/PDT of L 1 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T and PDT-> D o x of L I 2 1 0 and D B A / 2 cells) independent experiments is presented. Er ror bars are derived f rom standard errors of the mean. 73 Combination Dox->PDT PDT-->Dox PDT/Dox Dox only BPD only 0.001 T 0.01 0.1 Survival fraction ED DBA/2 BMMNC HI L12I0 Figure 3.6 Survival fraction of DBA/2 haematopoietic progenitors and L1210 cells to combinations of 5 ng/ml BPD and 1.25 p M Dox in conjunction with 15 J/cm2 of red light exposure Simultaneous D o x / P D T and PDT-> D o x treatments us ing 5 ng/ml o f B P D and 1.25 p M o f D o x yielded similar reductions' in the clonogenicity o f D B A / 2 C F U - G M and L1210 . .CFU- L. H o w e v e r , : I J 2 1 0 cells were more susceptible to the Dox-> P D T treatment than its nonrhalignant counterpart, resulting in the enlargement o f the therapeutic w i n d o w . Data obtained f rom 6 (Dox/PDT of L l 210 and D B A / 2 cells) and 3 (Dox-> P D T and PDT-> D o x of L 1 2 1 0 and D B A / 2 cells) independent experiments is presented. Er ror bars are der ived f rom standard errors o f the mean. 74 Combination Dox->PDT PDT->Dox PDT/Dox Dox only BPD only i—i • DBA/2 BMMNC H 1.1210 0.1 Survival fraction Figure 3.7 Survival fraction of DBA/2 haematopoietic progenitors and L1210 cells to combinations of 2.5 ng/ml BPD and 2.5 p M Dox in conjunction with 15 J/cm2 of red light exposure Preferential k i l l ing . o f - L l 2 1 0 cells .of the Dox-> P D T treatment protocol disappeared when a' lower dose of B P D was used. .Clonogenicity of normal C F U - G M and leukaemic C F U - L were reduced by the same amount when 2.5 ng/ml of B P D was used wi th 2.5 u M of D o x . Data obtained f rom 6 (Dox/PDT of L 1 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T and PDT-> D o x o f L 1 2 1 0 and D B A / 2 cells) independent experiments is presented. E r ror bars are derived f rom standard errors o f the mean. 75 Combination Dox->PDT PDT->Dox PDT/Dox Dox only BPD only • i i m [3 DBA/2 BMMNC H L1210 0.1 Survival fraction Figure 3.8 Survival fraction of DBA/2 haematopoietic progenitors and L1210 cells to combinations of 2.5 ng/ml BPD and 1.25 p M Dox in conjunction with 15 J/cm2 of red light exposure Preferential k i l l ing o f L 1 2 1 0 cells o f the Dox-> P D T treatment protocol disappeared when a lower dose o f B P D was used. Clonogenic i ty Of normal C F U - G M and leukaemic C F U - L were reduced similar ly when 2.5 ng/ml of B P D was used wi th 1.25 u M of D o x . Data obtained f rom 6 (Dox/PDT o f L 1 2 1 0 and D B A / 2 cells) and 3 (Dox-> P D T and PDT-> D o x o f L 1 2 1 0 and D B A / 2 cells) independent experiments is presented. Er ror bars are derived f rom standard errors of the mean. 76 Differential susceptibility of L1210 cells and DBA/2 haematopoietic progenitors to Dox-> PDT sequenced combination treatment To further investigate the effects that treatments wh ich combine D o x and B P D -mediated P D T had on the cytotoxicity therapeutic w indow between D B A / 2 haematopoietic progenitors and L 1 2 1 0 cel ls , data f rom the above combinat ions were represented as ratios of L 1 2 1 0 C F U - L survival fractions over D B A / 2 C F U - G M survival fractions (Figure 3.9). A therapeutic ratio o f 1 impl ies equivalent k i l l i ng of normal and leukaemic cells. On the other hand, a ratio higher than 1 denotes preferential k i l l i ng of the leukaemic cells in l ieu of normal haematopoietic cel ls. S ingle agent P D T treatment using B P D at 5.0 ng/ml was moderately effective in the selective eradication of L I 210 clonogenic cells with a therapeutic ratio o f 3.30. D o x , however, was not selective in its k i l l i ng of leukaemic cel ls with therapeutic ratios o f 0.94 and 0.98 at 2.5 u M and 1.25 m M , respectively. The advantage o f treating cells with D o x prior to P D T (Dox-> P D T ) was clearly demonstrated in combinat ions wh i ch ut i l ised 5.0 ng/ml B P D with 2.5 p M or 1.25 p M D o x ; however, preferential k i l l i ng o f L 1 2 1 0 cel ls was not realised when the lower dose of B P D at 2.5 ng/ml was used. Combinat ion treatments which invo lved the simultaneous administration of P D T and D o x (Dox/PDT) as wel l as P D T before D o x (PDT- D o x ) y ie lded similar therapeutic ratios. A g a i n , advantageous therapeutic ratios were achieved only at the higher B P D dose of 5.0 ng/ml but not at 2.5 ng/ml. Statistical analysis of combination treatments The signif icance of results f rom the cytotoxic i ty assays was determined using the two- way analysis of variance ( A N O V A ) test with term signif icant at alpha (p- value) = 0.05. Data f rom combinat ion treatments of 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 ignif icance of the resultant surv ival fractions o f the two different doses of B P D (2.5 and 5.0 ng/ml) and the three 77 combinat ion treatment regimens were used (Dox/PDT, PDT-> D o x , Dox-> P D T ) . A s shown in table 3.1a, there were signif icant differences between 2.5 and 5.0 ng/ml B P D as we l l as amongst the three combinat ion treatment regimens when 2.5 u M D o x was used in cytotoxici ty assays invo lv ing D B A / 2 haematopoietic progenitor cel ls ; however, at the lower dose of D o x (1.25 u M ) , only the combinat ion regimens were signif icantly different f rom each other. Next , intergroup differences were compared with the Bonferroni (all- pairwise) mult iple comparison test, a more powerful analysis tool wh ich provided a more detailed picture of the signif icance o f differences between the groups. A s shown in table 3.1b, cytotoxici ty results f rom combinat ion treatments which incorporated the higher dose of D o x of 2.5 u M were signif icantly different f r om each other in terms of the dose of B P D used (2.5 and 5.0 ng/ml). M o r e importantly, Bonferron i analysis revealed that the regimen Dox-> P D T was signif icantly different f rom the other combinat ion regimens, PDT-> D o x and Dox/PDT . A t the lower D o x concentration of 1.25 u M , significant intergroup difference was only demonstrated between Dox-> P D T and PDT-> D o x . The same analyses were performed on clonogenicity data of L 1 2 1 0 cells treated with the different combinations. In table 3.2a, two- way A N O V A demonstrated signif icance of the results of combinat ion experiments which uti l ised the higher D o x dose of 2.5 u M . Exper iments wh i ch used the lower D o x dose of 1.25 u M were only signif icant in terms of treatment sequence but not in the dose of B P D used. A g a i n , Bonfer ron i (all-pairwise) mult ip le comparison test was used to provide more statistical details. Table 3.2b shows that the Dox-> P D T combinat ion was signif icantly different f rom PDT-> D o x and D o x / P D T when 2.5 u M of D o x was used. However , the three combinat ions were not s ignif icant ly different when a lower dose of D o x was used. 78 so Therapeutic ratio 40 J 30-^ 20 J 1 0 J f— Q DM A X z O 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 Treatment regimen F i g u r e 3.9 D i f f e r e n t i a l k i l l i n g o f L1210 ce l l s o v e r D B A / 2 p r o g e n i t o r s i n 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 Preferential k i l l i ng of L 1 2 1 0 cells in the various PDT/Dox combinations are expressed as therapeutic ratios and are derived f rom the ratios o f survival fractions o f D B A / 2 CFU- G M over the survival fractions of L 1 2 1 0 C F U -L. Therefore, a therapeutic ratio of 1 denotes equal k i l l ing of normal and leukaemic cells. Ratios over 1 suggest preferential k i l l i ng of the L 1 2 1 0 cells over normal D B A / 2 bone marrow cells. Preferential k i l l ing o f L 1 2 1 0 cel ls in the Dox-> P D T regimen was dependent on the B P D concentration since the lower B P D dose o f 2.5 ng/ml effected no enhanced cytotoxici ty. 79 Table 3 .1a Two- way analysis of variance (ANOVA) of cytotoxicity data from DBA/2 haematopoietic progenitor cells treated with the three different drug combinations involving Dox and B P D -mediated PDT: significance of treatment sequence and B P D dose B P D dose (2.5 vs 5.0 ng/ml) Treatment sequence (Dox/PDT , PDT-> D o x , Dox-> P D T ) 2.5 u M D o x + 1.25 p M D o x -Two- way analysis o f variance (ANOVA) 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 examining the signif icance o f B P D doses (2.5 vs 5.0 ng/ml) as wel l as the three treatment regimens (Dox/ P D T , PDT-> D o x , Dox-> P D T ) . The plus sign (+) wi th in shaded cell indicates significant difference with probabil i ty level less than a predetermined a value of 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 (all- pairwise) multiple- comparison testings of B P D concentrations and treatment sequences B P D dose (2.5 vs 5.0 ng/ml) Treatment sequence 2.5 u M D o x i i i i i i i f i i l i i i i i f s i H l l j i l i Dox/FDT PDT- > Dox Dox->PDT Dox/FDT PDT-> Dox Dox->PDT ++ -H-1.25 u M D o x Dox/FDT PDT- > Dox Dox->PDT Dox/FDT PDT- > Dox Dox->PDT ++ Bonferroni (al l-pairwise) mult ip le compar ison analysis was performed on cytotoxicity data f rom D B A / 2 haematopoietic progenitor cells treated with 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 was arbitrarily designated the constant in the analysis. The sign (<|>) within the shaded cell in the middle co lumn indicates an significant intergroup difference between the two B P D concentrations of 2.5 and 5.0 ng/ml w i th in a constant D o x concentration. The plus sign (++) wi th in the cells in the rightmost co lumn indicate intergroup significant difference between the represented treatment regimen wi th in a constant D o x concentration of either 1.25 or 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 BPD- mediated P D T : significance of treatment sequence and BPD dose B P D dose (2.5 vs 5.0 ng/ml) Treatment sequence (Dox/PDT, PDT-> D o x , Dox-> P D T ) 2.5 u M D o x +" + 1.25 p M D o x + -Two- way analysis of 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 uM ) as the constants and examining the significance of B P D doses (2.5 vs 5.0 ng/ml) as wel l as the three treatment regimens (Dox/ P D T , PDT-> D o x , Dox-> P D T ) . The plus sign (+) within shaded cell indicates significant difference with probabil i ty level less than a predetermined a value of 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 BPD concentrations and treatment sequences B P D dose (2.5 vs 5.0 ng/ml) Treatment sequence 2.5 p M D o x Dox/FDT PDT- > Dox Dox->PDT Dox/FDT PDT- > Dox Dox->PDT ++ ++ 1.25 p M D o x Dox/FDT PDT- > Dox Dox->PDT Dox/ FDT PDT- > Dox Dox-> PDT Bonferron i (all- pairwise) mult iple compar ison analysis was performed on cytotoxicity data f rom,L1210 leukaemic.xells treated with the different combinations of B P D - mediated P D T and D o x . A g a i n , the concentration of. D o x was arbitrarily-designated as the constant in the analysis. The <j> sign (<j>) w i th in the shaded cel ls in the middle Column indicate ah intergroup significant difference between the two B P D concentrations of 2.5 and 5.0 ng/ml wi th in a constant D o x concentration. The plus s ign (++) within the cells in the rightmost co lumn indicate intergroup significant difference between the represented treatment regimen with in a constant D o x concentration of either 1.25 or 2.5 u M . 83 DISCUSSION The util ity of benzoporphyrin derivative- mediated photodynamic therapy ( PDT) in autologous bone marrow purging was extensively explored by Jamieson and c o l l e a g u e s . 2 9 2 - 2 9 4 These investigators found that the monoac id ring- A analogue o f benzoporphyrin derivative ( BPD ) was selectively taken up by leukaemic cells of human and mouse or ig in . O n average, a six- fo ld increase in photosensitiser uptake over normal haematopoietic cells was observed. Further studies examin ing the cytotoxic i ty of B P D -mediated P D T on the clonogenicity of normal haematopoietic cells and leukaemic cells also demonstrated the selectivity of this modal i ty of treatment. In addit ion, using the long- term bone marrow assay ( L T B M C ) , wh i ch measures the growth o f less developed cel ls of the haematopoietic system, these authors demonstrated that B P D - mediated P D T had min imal toxic i ty on human early haematopoietic progenitor cel ls. Photosensitisers other than B P D , such as merocyanine 540 ( M C 5 4 0 ) and the phthalocyanines are also being considered for PDT- mediated purging of contaminated bone marrow 2 7 5 > 2 9 1 . P D T has also been used in conjunction with different treatment modalit ies such as heat and ion is ing radiat ion for in vivo a p p l i c a t i o n s . 3 0 3 - 3 1 9 ' 3 2 0 In addi t ion, various groups have combined P D T with chemotherapeutic agents, cytokines, and even mult iple photosensitisers in an effort to improve the eff icacy and selectivity o f t reatment . 3 0 5 ' 3 1 8> 321-323 Doxorub i c in or D o x is an anthracycline- based antibiotic derived f rom the Streptomyces species and has been used in the management o f various forms of c a n c e r . 3 2 4 Daunorubic in and idarubicin are also members of the anthracycline fami ly with extensive uses in ahti leukaemia :therapy. D o x was chosen because its photophysical ,prof i le and cytotoxic mechanisms were unique f rom those of B P D . The ex vivo nature of pharmacological purging bypasses many of the constraints imposed by systemic pharmacokinetics in vivo and therefore permits greater latitude in dose and sequence experimentation in combinat ion therapy. The pr incipal goal o f the present project was to increase the eff icacy o f B P D -mediated photodynamic purging. In this system, when either normal D B A / 2 haematopoietic progenitors or L 1 2 1 0 leukaemic cells were treated simultaneously with P D T and D o x (Dox/ P D T ) , the resultant cytotoxici ty was roughly additive compared to P D T or D o x single agent 84 treatment o f the same dose. The same observations were made when cel ls were treated first with P D T fo l l owed by one hour incubation wi th D o x (PDT-> D o x ) . Interestingly, preincubation of the cells with D o x prior to P D T (Dox-> P D T ) signif icantly increased the cytotoxicity of the treatment in both normal and leukaemic cel ls. M o r e signif icantly, the L 1 2 1 0 cel l l ine was much more susceptible to the Dox-> P D T regimen than normal D B A / 2 haematopoietic progenitor cells wi th a resultant enlargement of the therapeutic w indow. A s demonstrated in f igure 3.9, the enhancement of differential k i l l i ng of L 1 2 1 0 cel ls was only evident when the higher B P D dose of 5.0 ng/ml was used and the cel ls were exposed to D o x before P D T treatment. The results for D B A / 2 haematopoietic progenitors and L 1 2 1 0 leukaemic cel ls treated with different combinat ion regimens incorporating the higher D o x concentration of 2.5 u M were signif icantly different f rom each other as demonstrated by two- way A N O V A with p< 0.05. Importantly, there was signif icant intergroup difference between the Dox-> P D T sequence and the other two combinat ion regimens of PDT-> D o x and D o x / P D T in both normal and leukaemic cel ls exposed to 2.5 u M of D o x . Th is phenomenon could be attributed to several events wh ich w i l l be the subject o f chapter 4 of this thesis. It is possible that pretreatment of cel ls wi th D o x cou ld increase the subsequent uptake of the photosensitiser or reduce the amounts of cel lular antioxidants such as glutathione. Daunorub ic in , a congenor of D o x , increases ceramide production through its modulat ion of the enzyme ceramide syn thase . 3 2 5 Ceramide is a sphingol ip id wh i ch mediates several signal transduction pathways such as those downstreams of the tumour necrosis factor type I receptor (TNFr ) and F A S ; furthermore, addit ion of exogenous short chain ceramide analogue induced apoptotic ce l l d e a t h . 3 2 6 Separovic and colleagues recently demonstrated ceramide production in response to P D T mediated by the phthalocyanine photosensitiser Pc 4 . 2 6 8 Pretreatment of cel ls wi th D o x cou ld therefore raise intracellular ceramide to a crit ical level with exacerbation of subsequent P D T cytotoxicity. Recently, several groups have implicated the transcriptional factor N F - K(3 in anthracycline- mediated cytotoxici ty. Inhibit ion o f N F - K p nuclear translocation enhanced the cytotoxic i ty of daunorubic in, T N F a , and y- radiation in H T 1 0 8 0 human f ibrosarcoma cel ls wh i ch impl icated NF- K(3 activation as part of the normal response to environmental stresses wh i ch threaten cel lular s u r v i v a l . 3 2 7 However , contradictory studies have shown that N F -K(3 activation precedes cel l death therefore the exact role of this ubiquitous transcriptional 85 factor in cel lular demise cou ld be quite i d iosync ra t i c . 3 2 8 Das and Whi te demonstrated that both doxorubic in and daunorubic in effected N F - K(3 activation v i a protein kinase C . 3 2 9 Since P D T has been shown to activate N F - Kp\ pretreatment of cel ls wi th D o x cou ld conceivably 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, combinat ion treatment o f D o x and B P D - mediated P D T preferentially affected the leukaemic cel l l ine L 1 2 1 0 over normal D B A / 2 haematopoietic cells. Selective k i l l i ng was only realised when D o x was used prior to P D T (Dox-> P D T ) and at a dose of 5.0 ng/ml B P D and 15 J /cm 2 red l ight. 86 C H A P T E R 4: C O M B I N E D T R E A T M E N T O F M U R I N E N O R M A L H A E M A T O P O I E T I C A N D L E U K A E M I C C E L L S W I T H D O X O R U B I C I N A N D P D T : M E C H A N I S T I C S T U D Y 4.1 ABSTRACT Benzoporphyr in derivative monoac id ring- A ( B PD ) is a porphyrin- based photosensitiser with potential in the c l in ica l application of photodynamic therapy (PDT) . Prev ious ly , we have demonstrated the effectiveness o f B P D in P D T purging of contaminated leukaemic cells f rom autologous haematopoietic stem cel l harvests. In order to improve its eff icacy, we combined B P D - mediated P D T with doxorubic in (Dox) and found that the sequence of Dox-> P D T was superior to simultaneous D o x / P D T and the reverse sequence of PDT-> D o x . In addit ion, the murine leukaemic cel l l ine L 1 2 1 0 was much more susceptible to the Dox-> P D T regimen than normal D B A / 2 haematopoietic progenitor cel ls. In this chapter, we studied some of the interactions between D o x and B P D and specif ical ly the possible mechanisms responsible for the augmentation of P D T cytotoxicity effected by D o x preincubation. The unique photophysical and b io logica l properties of B P D and D o x permit their cytotoxic cooperativity in the simultaneous D o x / P D T regimen. Fluorescent microscopy showed that B P D was restricted to the cytoplasm and the plasma membrane whereas D o x local ised to both cytoplasmic and nuclear compartments. Dox- mediated inhibit ion of B P D photobleaching was observed at the excitation wavelength of 440 nni but not at 630.nm. Since a red light source emitting-between 600- 900 n m was used in the cytotoxic i ty assays described in chapter 3, the presence of D o x should not affect B P D photoactivation. Spectrofluorimetric measurements of L 1 2 1 0 cel l lysates revealed that uptake of B P D was reduced in the presence of D o x ; however, preincubation of L 1 2 1 0 with D o x restored B P D accumulat ion to levels of control cells incubated with only B P D . Next , we proceeded to examine the cel lular level of glutathione (GSH ) , an ubiquitous protective th io l . L 1 2 1 0 cel ls incubated with 5 u M of D o x for 1 h resulted in slight but reproducible reduction o f cel lular G S H . The above factors, in 87 conjunct ion wi th other mechanisms, cou ld be responsible for the presensitisation o f L 1 2 1 0 cel ls by D o x . 4.2 INTRODUCTION Numerous factors can affect the eff ic iency of photodynamic therapy (PDT) . Considerably more variables are invo lved when P D T is used in vivo; for example, the extent of first pass hepatic metabol ism and the degree of b ind ing to various serum proteins affect the delivery of the photosensitiser to the target site. Other factors of particular importance in oncology include tissue oncotic pressure, regional b lood f l ow , and the hypox ic state of the tumour, wh ich are governed by tumour size. The above concerns are not unique to P D T and are l ikewise of importance in the del ivery of other forms of therapies such as chemotherapeutic agents and an t ibod ies . 3 3 0 A t the cel lular leve l , photosensitiser uptake is also governed by s imi lar factors wh i ch affect the uptake o f other chemotherapeutic compounds. For example, molecular size and partition coeff ic ient of the photosensitiser influence the cel lular accumulat ion of the compound. Environmental factors such as the rate of cel lular proliferation, intracellular and extracellular p H , cel l size, and sen im concentration al l influence the uptake of the pho tosens i t i s e r . 2 5 9 - 3 3 1 The photosensitiser init ia l ly associates with the cel l membrane then gradually relocalises to the different intracellular compartments such as the mitochondr ia and l y s o s o m e s . 3 3 2 In addit ion, private attributes such as charge characteristics and.presence or absence o f sidechain moieties further contribute to the intracellular local isat ion of the compound. 3 - 3 3 Cat ion ic dyes such as merocyanine 540 ( M C 540) and rhodamine 123 (Rh 123) preferentially accumulate in the mitochondria as a result of the electrochemical gradient whereas the porphyrin- based photosensitiser benzoporphyr in derivative monoac id ring- A ( BPD ) , wh ich is l ipophi l i c , init ial ly localises to the p lasma membrane and then rapidly to various cel lular organelles with longer incubation. In addit ion to photosensitiser accumulat ion, other factors also determine the effectiveness o f P D T cytotoxici ty. O f particular importance is the ratio of cel lular pro- and antioxidants since P D T cytotoxici ty is 88 pr imar i ly mediated by reactive oxygen species (ROIs) and radical f o rma t i ons . 2 5 5 Therefore, the amount of cel lular antioxidants such as glutathione ( G S H ) and a- tocopherol can influence the extent of P D T damage and their modulations can alter P D T suscep t ib i l i t y . 2 6 1 - 3 3 4 > 3 3 5 P D T combinat ion therapy, through the incorporation o f P D T wi th other treatment modalit ies or therapeutic agents, attempts to effect modulat ion on the ce l l or the organism in order to maximise cytotoxic effects on the target cel l populat ion. Manipulat ions of the t iming, chemical and physical characteristics of the photosensitiser and drug dose, l ight fluence rate, as wel l as the, activation wavelength can substantially affect the eff ic iency of PDT- mediated cytotoxici ty. Therefore, introduction of another fo rm of treatment into a P D T regimen can signif icantly increase its complexi ty and also the interpretation of data. Hyperthermia and ionis ing radiation have similar cytotoxic mechanisms as P D T and were naturally used in different variations of combinat ions with P D T . Several groups have demonstrated the beneficial effects of P D T prior to hyperthermia treatment and sequence speci f ic i ty of this regimen was a result o f tumour and vascular effects o f P D T . 3 0 3 ' 3 0 9 - 3 2 0 Chemotherapeutic agents such as etoposide and doxorubic in are also used in conjunction with P D T . 3 0 6 ' 3 0 8 M a et al. observed that presence o f m i tomyc in C ( M M C ) during Photofrin®- mediated P D T signif icantly increased its antitumour effects in vitro and in vivo.304'318 Some of the most innovative regimens invo lved combinat ions of different photosensitisers and activation wavelengths; for example, C incot ta and colleagues reported synergistic antitumour effects of P D T in vivo mediated by B P D and 5- ethylamino- 9-diethylaminobenzo[a] phenothiazinium chloride ( E t N B S ) . 3 0 5 Coadministrat ion of cytokines such as G M - C S F and T N F a potentiated the antitumour eff icacy o f P D T in vivo.322'336 Myers-er al. observed that the addit ion of the. immunost imulant , C.'parvum, signif icantly enhanced P D T eff icacy in an animal bladder tumour m o d e l . 3 3 7 These experiments highl ighted the participatory role of the immune system in in vivo antitumour action mediated by P D T . Var ious mechanisms have been proposed for the different forms of P D T combinat ion therapy. In regimens invo lv ing combinat ions wi th other photosensitisers, two groups have noted that synergistic k i l l i ng of tumour cells in vitro and in vivo was achieved with differential local isat ions of the a g e n t s . 3 0 5 - 3 3 8 C o m b i n i n g photosensitisers or chemotherapeutic agents with unique photophysical and biochemical characteristics can 89 invariably ensure an increase in cel lular targets. M a et al. reported that cel l cyc le arrest fo l lowed by S- phase accumulat ion was responsible for the increased uptake of Photofrin® in the presence M M C . 3 0 4 Therefore, one can understand why simultaneous incubation of cells wi th M M C and Photofrin® was superior to administration of M M C post- P D T (P->light-> M M C ) . In this chapter, we attempted to study the interactions between B P D and D o x and specif ical ly , to investigate the mechanisms responsible for the enhanced k i l l i ng of L 1 2 1 0 cel ls when D o x was administered pr ior 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 propert ies of B P D a n d D o x The molecular structure of benzoporphyr in derivative monoac id r ing A (one regioisomer, B P D ) is g iven in f igure 4.1. B P D , a member of the porphyr in f ami l y , is a second generation photosensitiser wh ich is more potent and purer than its predecessor Photofrin®. The structure of the anthracycline antibiotic doxorubic in (Dox, adriamycin) is shown in f igure 4.2. D o x is a 14- hydroxy analogue of daunorubic in isolated f rom Streptomyces peucetius (var. caesius) and is used extensively in anticancer chemotherapy. The absorption spectra o f B P D and D o x in 10 % HI- F C S / P B S are g iven in f igure 4.3 and T p g / m l (1-.376 p M ) of B P D and 10 p M of D o x were used'during the'analysis: The presence o f the unique B P D absorption peak at 690 nm, in addition to several other peaks above 600 nm, permits B P D photoactivation at higher wavelength and is an unique feature of this photosensitiser. The spike observed at 656 nm, wh ich is present in both D o x and B P D spectrum, is an artefact of the deuterium lamp used for the spectrophotometric scan. D o x has a prominent broad absorption peak at 500 n m and a smaller peak at 579 nm. Fluorescence emission spectra (A,ex = 440 nm) o f 10 ng/ml (13.76 n M ) B P D and 5 p M D o x are presented in figure 4.4a. Note that this is the same B P D : D o x molar ratio that was used in the cytotoxic i ty assays described in chapter 3. A t this ratio, D o x fluorescence emiss ion 90 virtually concealed almost all o f the B P D profi le save the unique B P D emission peak at 693 nm, wh i ch remained detectable even in a solution containing 363.5 fo ld more o f D o x . Th is is of particular signif icance for the interpretation of the photobleaching data in a subsequent section as we l l in the appreciation of the general distinctiveness between B P D and D o x . F igure 4.4b, wh ich restricts the emiss ion spectra to 650- 700 nm, highl ights this unique property of B P D . 91 C O O H COOCH3 Figure 4.1 Chemical structure of the benzoporphyrin derivative monoacid ring- A ( B P D , Verteporfin) The molecular structure of one regioisomer of benzoporphyr in derivative monoac id r ing A ( B P D , Verteporf in) is shown. 92 Figure 4.2 Chemical structure of Doxorubicin (Dox) The molecular structure o f Doxorub i c in (Dox) is shown. 93 Absorbance 0.1250 0.1000 0.07504 0.0500-T 0.0250 0.0000 400 500 600 wavelength (nm) 700 1 pg/ml B P D 10 u M Dox Figure 4.3 Absorption spectra of 1 pg/ml (1.376 pM) B P D and 10 p M Dox in 10 % H I - FCS/ PBS The absorption spectra of 1 pg/ml (1.376 p M ) B P D and 10 p M D o x in P B S conta in ing 1 0 % HI- F C S are presented. The 656 n m spike observed in both samples was an artefact of the deuterium lamp f rom the. spectrophotometer. 94 Fluorescence intensity 5 uM Dox only 5 uM Dox & 10 ng/ml BPD 550 600 650 wavelength (nm) 700 Figure 4.4a Fluorescence emission spectra of B P D in the presence of Dox (550 - 700 nm) Super imposit ion of the fluorescence emission profi le of 5 u M D o x and a mixture consist ing of 5 u M D o x with 10 ng/ml B P D in P B S containing 1 0 % HI- F C S . B P D has an emiss ion peak at 693 n m which permits its activation even in the presence of D o x . Note that the molar ratio o f B P D to D o x (1: 363.5) is the same that was used in the cytotoxic i ty assays. Settings for the spectrofluorimeter: continuous wavelength ( C W ) excitation at 440 n m wi th bandpass of 4 n m , emiss ion at 700 nm with bandpass o f 4 n m , 1 nm/s scan rate, photomult ip l ier voltage ( P M T ) was set at 650 V . 95 1.5-, Fluorescence intensity 5 uM Dox 5 uM Dox & 10 ng/ml BPD 0 . 5 H 650 1 I i I 1 660 670 680 690 700 wavelength (nm) Figure 4.4b Fluorescence emission profile of B P D in the presence of Dox (650 - 700 nm) Superposit ion o f the fluorescence emission profi le o f 5 u M D o x and a mixture consist ing o f 5 u M D o x with 10 ng/ml B P D in P B S containing 1 0 % HI- F C S . B P D has a emiss ion peak at 693 n m , accentuated in this emission scan between 650- 700 n m , wh ich permits its activation even in the presence of D o x . Note that the molar ratio o f B P D to D o x (1: 363.5) is the same that was used in the cytotoxicity assays. Settings for the spectrofluorimeter: continuous wavelength ( C W ) excitation at 440 nm with bandpass o f 4 nm, emiss ion at 700 n m with bandpass of 4 n m , 1 nm/s scan rate, photomult ipl ier voltage ( P M T ) was set at 650 V . 96 Intracellular localisations of BPD 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 compounds were established by fluorescent microscopy. The ce l l l ine K 5 6 2 , or ig ina l ly derived f rom the pleural effusions of a chronic myelogenous leukaemia ( C M L ) patient in blast cr is is, was used for this study. F luorescence was visual ised with an O lympus N e w V a n o x microscope attached to the A H 2 - F L transmitted fluorescence unit, a blue range barrier f i l ter was used for both B P D and D o x observations. Pictures were taken with the attached camera using Fu j ichrome 400 I SO f i l m . A s presented in f igure 4.5, K 5 6 2 cel ls that were incubated wi th 10 pg/ml B P D for 60 m in showed intense yet dif fused cytoplasmic fluorescence around but not wi th in the nuclear poles. In addit ion, discrete and punctate fluorescence patterns, suggesting B P D accumulat ion in cytoplasmic organelles, were also observed. Th is is consistent with the f indings o f C incot ta et al. us ing the murine mammary sarcoma cel l l ine E M T - 6 . 3 0 5 D o x , at 10 u M , demonstrated both nuclear and cytoplasmic fluorescence in a diffused pattern (figure 4.6). Interestingly, nuclear fluorescence of D o x was much stronger than cytoplasmic fluorescence wh ich impl ies significant nuclear accumulation of D o x . The fluorescence pattern o f D o x is in agreement wi th known properties o f this compound wh ich targets the l ip id membrane bi layers, various cel lular enzymes, and the genetic materials of t h e c e l l . 3 2 4 In f igure 4.7, K 5 6 2 cel ls were incubated simultaneously with 10 pg/ml B P D and 10 p M D o x for 60 min and observed under the fluorescent microscope at the same settings as before. A l l the cells demonstrated the red cytoplasmic fluorescence pattern of B P D whi le D o x nuclear fluorescence was observed in some of the cel ls. Di f ferent ia l localisations o f B P D and D o x to the different compartments of the K 5 6 2 cel l l ine, in addition to their unique but parallel cytotoxic mechanisms of action, support their use in combinat ion P D T therapy. 97 F i g u r e 4.5 F l u o r e s c e n c e m i c r o g r a p h o f K 5 6 2 l e u k a e m i c ce l l s i n c u b a t e d w i t h B P D K 5 6 2 Ce l l s were resuspended at a concentration of 1 x 10 6 cells/ml in serum- free D M E M and incubated with 10 pg/ml of B P D for 60 m i n . Cel ls were then washed once and resuspended in 1 ml o f medium. A drop containing 50 pi or 5 x 10 4 o f cells was placed on a glass slide and subsequently covered with a cover s l ip. The cells were observed under an O lympus N e w Vanox microscope with the A H 2 - F L transmitted light fluorescence attachment on " B l u e " setting (excitation @ 380- 490 n m , observation @ 515 n m +). 98 F i g u r e 4.6 F l u o r e s c e n c e m i c r o g r a p h o f K 5 6 2 l e u k a e m i c ce l l s i n c u b a t e d w i t h D o x K 5 6 2 Ce l ls were resuspended at a concentration o f 1 x 10 6 cells/ml in serum- free D M E M and incubated with 10 u M of D o x for 60 m i n . Ce l ls were then washed once and resuspended in ! ml o f med ium. A drop containing 50 pi or 5 x 10 4 o f cells was placed on a glass slide and then covered with a cover s l ip. The cel ls were observed under an Olympus N e w Vanox microscope with the A H 2 - F L transmitted light fluorescence attachment on " B l u e " setting (excitation @ 380- 490 n m , observation @ 515 nm +). 99 F i g u r e 4.7 F l u o r e s c e n c e m i c r o g r a p h o f K 5 6 2 l e u k a e m i c ce l l s i n c u b a t e d w i t h B P D a n d D o x K 5 6 2 Ce l ls were resuspended at a concentration of 1 x 10 6 cells/ml in serum-free D M E M and incubated wi th 10 pg/ml (13.76 p M ) B P D and 10 u M D o x for 60 min . Ce l l s were then washed once and resuspended in 1 ml of medium. A drop containing 50 pi of cells was placed on a glass slide and thereafter covered wi th a cover s l ip . The cells were observed under an Olympus N e w Vanox microscope with the A H 2 - F L transmitted light fluorescence attachment on " B l u e " setting (excitation @ 380- 490 n m , observation @ 515 n m +). 100 The role of excitation wavelength (kex) has on the photobleaching of BPD in the presence of Dox To further investigate the interactions between B P D and D o x , the two compounds were m ixed together in a solvent consist ing o f 10 % fresh human p lasma in phosphate buffered saline (10 % H P / P B S ) and photobleaching of B P D in the presence of different concentrations of D o x was measured. Photobleaching is the process of irreversible autodegradation of the excited photosensitiser and originates f rom the triplet excited state of the compound as a result o f intersystem crossing f rom the activated singlet state. However , it is inherently a complex phenomenon and can also occur f rom the singlet state. B P D does not photobleach readily in organic solvents such as methanol or dichloromethane; however, photobleaching is signif icantly enhanced in the presence of human plasma or foetal calf serum (data not shown). Th is is consistent wi th f indings by Ave l i ne et al.219 In the present study, B P D photobleaching was used as a surrogate marker of photosensitiser activation. A s demonstrated in f igure 4.8, the mean fluorescence intensity o f 10 ng/ml of B P D at 693 nm ( M F I 6 9 3 n m) decreased steadily when the compound was subjected to continuous l ight exposure at the excitat ion wavelength (X e x) of 440 n m (0 p M D o x , bottommost curve). In fact, the M F I 6 9 3 n m a t the end o f a 15 m in exposure per iod was 43.2 % o f the starting level . Interestingly, B P D photobleaching was ameliorated when 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 of B P D photobleaching, different concentrations of D o x were subjected to the same photoactivation protocol and D o x fluorescence emissions at 593 n m (fluorescence emission peak unique to Dox ) and 693 n m (fluorescence emission peak unique to B P D ) were monitored for 15 m in . In both cases, M F I was not s ignif icant ly altered at the end of the observation period suggesting D o x itself was not affected by the light exposure and the reduction of B P D photobleaching in the presence of D o x was caused by some form o f molecular interaction between D o x and B P D . F igure 4.9 summarises the results of three independent B P D photobleaching experiments in terms of percent reductions of M F I 6 9 3 n m a t the end of the 15 m in exposure per iod compared to starting M F I 6 9 3 n m . In the experiments described above, photobleaching or photoactivation of B P D was found to be inversely correlated with the concentration o f D o x present when A,ex o f 440 101 nm was used. Since a red l ight source emitt ing between the range of 600- 900 n m was used for B P D photoactivation in the cytotoxicity assays described in chapter 3, the photobleaching experiments were repeated wi th ?i e xset at 630 nm. F igure 4.10 compares the effects of D o x had on B P D photobleaching between ^ e x o f 440 n m and 630 nm. The data show that at A,ex of 630 nm, reduction of B P D photobleaching by D o x was much less obvious than at Xex o f 440 nm. In fact, photobleaching of B P D was dramatical ly lower at the former wavelength. F igure 4.11 illustrates that 1 m M of D o x in 10 % HI- F C S / P B S does not appreciably interfere wi th l ight transmission above 600 nm. A t 630 nm, l ight transmittance was 89.39 % and reached a plateau at 720 n m (97.83 % ) . S ince a much lower concentration of D o x (5- 10 p M ) was used in the cytotoxic i ty assays (chapter 3) and in the spectrofluorimetric analyses described in this section, the presence of D o x should have minima] negative effect on light transmission to the B P D molecules in the solution when A,ex o f above 600 n m was used. The results, in sum, suggest that the presence of D o x d id not affect the eff ic iency of red light activation of B P D during the cytotoxici ty assay and the observed attenuation of B P D activation by D o x at 440 nm was most probably secondary to shielding of l ight by the abundance of D o x in solution. 102 1.25-, Mean fluorescence intensity @ 693 n m 0.5n. 0.25-0.75-f* ( 5 ==«ta *t ; 10 uM Dox 5.0 uM Dox 2.5 uM Dox 1.25 uM Dox 0 uM Dox 900 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 693 nm was monitored for 15 min whi le the sample was photoactivated at 440 nm in a solvent consist ing of 10 % human plasma/ P B S . The presence of increasing concentrations of D o x in the reaction signif icantly reduced the degree of B P D photobleaching. Settings for the spectrofluorimeter: continuous wavelength ( C W ) excitation at 440 nm with bandpass of 16 n m , emiss ion at 700 n m with bandpass of 4 n m , M F I at 693 nm continuously monitored for 900 s, photomult ipl ier voltage ( P M T ) was set at 650 V 103 100 n 75 J % BPD fluoresecence intensity of original [ T 50 J 25 J _1 1—•—•—|—•—•—p 0 1.25 2.5 5 —r-10 % BPD fluorescence @ 693 nm [Dox] uM Figure 4.9 Summary of BPD photobleaching in the presence of different concentrations of Dox (440 nm excitation) Photobleaching of B P D as measured by its f luorescence intensity at 693 nm was proportionally decreased in the presence o f increasing concentrations of D o x . Mixtures consist ing of 10 ng/ml of B P D and various concentrations o f D o x were mixed in 10 % human plasma/ P B S and were subjected to continuous wavelength ( C W ) activation and fluorescence emission at 693 n m was monitored for 900 s. Percent photobleaching of B P D was obtained by d iv id ing B P D fluorescence at the end o f the 900 s exposure period by B P D fluorescence at the beginning o f the monitor ing per iod. Data f rom 3 independent experiments is presented. E r ror bars are derived f rom standard errors of the mean. 104 100-, 440 630 Excitation wavelength (nm) Figure 4.10 The effect of A,ex on BPD photobleaching in the presence and absence of 5 p M Dox The inhibitory effect o f D o x had on B P D photobleaching was observed at the lower excitation wavelength (kex) o f 440 n m but was not evident at the higher /Vex o f 630 n m . B P D photobleaching was assessed by comparing M F I 6 9 3 n m at the end of 900 s o f exposure to that at the beginning of the monitor ing per iod. Results f rom a single representative experiment are s h o w n . 105 100 n 95 90 Percent light transmittance 85 80 75 70 I 1 1 1 1 i 1 1 1 1 i 1 1 1 1 i 600 650 700 750 wavelength (nm) Figure 4.11 Light transmittance of 1 m M Dox in 10 % HI- FCS/ PBS 1 m M of D o x does not appear to significantly, interfere wi th .light transmittance in a solution of 10 % HI- F C S / P B S . Therefore, the presence of 5- 10 u M of D o x in a solution containing B P D should have no adverse effects on the photoactivation of the photosensitiser wi th A.ex above 600 nm. 106 Uptake of BPD by L1210 cells as affected by the presence of Dox in different combination regimens Spectrofluorimetric measurements of lysates f rom L I 210 cells that were incubated wi th either B P D and D o x are presented in figures 4.12a and 4.12b. Sensit ive detection o f the two compounds was accomplished by measuring B P D and D o x mean fluorescence intensity (MF I ) at their unique fluorescence emiss ion peaks (Xem) at 593 n m (Dox) and 693 nm (BPD ) . A l l experiments were performed us ing 1 x 10 6 cel ls as described in the experimental procedure section. A linear correlation was observed between M F I at Xem o f 693 n m and the concentration o f B P D (figure 4.12a). Interestingly, f luorescence intensities of L I 210 cel l lysates were similar between samples that were incubated with the photosensitiser for 10 min and 60 m in . F igure 4.12b shows that a l inear relat ionship also existed between M F I at ^ e m o f 593 n m and the concentration of D o x ; in addit ion, uptake of the drug was also quite rapid because of the similarit ies between the M F I measurements f rom the 10 min and 60 min cel l lysate samples. F igure 4.13 illustrates the effects that 10 u M D o x coincubation (Dox/ B P D ) or 10 u M D o x preincubation (Dox-> B P D ) had on the uptake of B P D . L 1 2 1 0 cells that were coincubated with D o x (Dox/ B P D ) appeared to take up less B P D than control cells incubated wi th only B P D ; this phenomenon became more prominent with increasing concentrations of the photosensitiser. In summary, B P D uptake by L I 210 cells was not affected by prior incubation with D o x ; however, presence o f D o x (10 p M ) during B P D incubation appeared to reduce the uptake of the photosensitiser. 107 lOrnin incb 60min incb [ l BPD ]ng/ml Figure 4.12a Uptake of BPD by L1210 cells as determined by spectrofluorimetric measurements of cell lysates M F I 6 9 8 n m o f lysates f rom 1 x 10 6 L 1 2 1 0 cells was directly correlated with the concentration o f B P D . Uptake o f B P D was rapid since M F I 6 9 8 n m o f lysates f rom cells incubated with B P D for 10 min were similar to those of the 60 min group. 108 lOmin incb 60min incb [DOX] u M Figure 4.12b Uptake of Dox by L1210 cells as determined by spectrofluorimetric measurement of cell lysates M F I 5 9 3 n m o f lysates f rom 1 x 10 6 L 1 2 1 0 cells was directly correlated wi th the concentration of D o x used dur ing the incubation. Uptake of D o x was rapid since M F I 5 9 3 n m o f lysates f r om cel ls incubated wi th D o x for 10 min were s imi lar to those of the 60 m i n test group. 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 cel ls appeared to take up less B P D in the presence o f 10 u M of D o x compared to the respective B P D only controls : Reduct ion of B P D mean fluorescence intensity at 698 n m ( M F I 6 9 8 n m) was more prominent at higher B P D doses. Preincubat ion of cel ls wi th JO u M o f D o x appeared to have no inhibitory effects on the subsequent uptake of the photosensitiser. Data obtained f rom 3 independent experiments is presented. E r ror bars are derived f rom standard errors of the mean. 110 Depletion of cellular glutathione (GSH) in L1210 cells by preincubation with Dox To further investigate the effects of D o x preincubation had on subsequent P D T cytotoxicity on L 1 2 1 0 cel ls, cel lular glutathione (GSH ) levels were measured after 1 h incubation with D o x . A g a i n , the same experimental conditions as the cytotoxic assays described in chapter 3 were used for this set of experiments in order to ensure relevancy of the data. Results are summarised in f igure 4.14. Preincubation o f L I 2 1 0 cel ls wi th D o x resulted in a dose- related, albeit insignif icant (student's t- test, p > 0.05), reduction of ce l lu lar G S H . Fo r example , 11.93 ± 0.76 p M G S H / 1 x 10 6 cel ls was obtained after 1 h incubation with 5 p M D o x compared to 13.54 + 1.51 nmole G S H / 1 x 10 6 cel ls f rom the untreated control group or 88.16 % of the untreated control . U s i n g the Tietze enzymatic method, that preincubation of L I 2 1 0 cells with 5 p M of D o x d id not s ignif icantly reduced the amount of cel lular G S H . Therefore, this ruled out the role of G S H in the enhancement of P D T cytotoxicity v i a D o x preincubation. I l l 2 0 - , [GSH ] u M per 1 X 1 0 A 6 L 1 2 1 0 cells 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 with D o x for 1 h resulted in a dose- related reduction o f cellular glutathione ( G S H ) in L 1 2 1 0 cel ls . G S H was determined us ing the Tietze enzymatic assay. Data f rom a three independent experiments is presented. Error bars are derived f rom standard errors of the mean. 112 4.4 DISCUSSION The eff ic iency of P D T is inf luenced by mult iple intrinsic and extrinsic factors. Obv ious ly , variables such as first- pass hepatic metabol ism and relative tissue distribution are o f concern for in vivo applications of P D T ; however, the picture is no less compl icated for in vitro P D T . B iophys i ca l and b iochemica l properties of the photosensitiser such as hydrophobic i ty , extinct ion coefficient, singlet oxygen y ie ld , as we l l as its absorption prof i le are some of the examples of the determinants of P D T behaviour in vitro and in vivo. 3,33 Susceptibi l i ty to P D T cytotoxic i ty is addit ional ly established by cel lular growth rate, expression of surface receptors such as the receptor for l ow density l ipoprotein ( L D L r ) , and relative concentrations of prooxidants and antioxidants in the c e l l . 2 5 5 Combinat ion therapy has been in use for the management of various forms of medical condit ions; in addit ion, it is indispensable in the treatment many oncologic diseases. The pr incipal goals of combinat ion therapy are to use agents with different mechanisms of action so as to reduce the probabil i ty o f drug resistance development; furthermore, the cytotoxic potentials of the drugs should complement and not nu l l i f y each other. Ideally, the side effects of the drugs should not be additive such that the patient but not the tumour remains tolerant to the drug combinat ion. A successful example o f such therapy is the use of corticosteroid with vincristine for the treatment of paediatric acute lymphoblast ic leukaemia ( A L L ) . 1 1 S ince the advent of the c l in ica l use of P D T , numerous groups have attempted to combine P D T with other treatment modalit ies such as hyperthermia, ionis ing radiation, and surgery, with different chemotherapeutic agents or with different combinations of photosensitisers. Henderson and colleagues in i t ia l ly documented that the combinat ion of Photofrin®- mediated P D T and hyperthermia was synergistic in tumour k i l l i ng only when P D T was used before hyperthermic treatment (PDT-> heat); the authors further demonstrated that pretreatment with hyperthermia resulted in extensive haemorrhage of the tumour site wh ich interfered with the subsequent del ivery of the photosensitiser as wel l as reduced the amount of l ight reaching the target s i t e . 3 0 3 Add i t iona l studies supported the early f indings of the influence that treatment sequence had on the eff icacy of P D T / hyperthermia combinat ion therapy . 3 0 9 S igni f icant ly , Chen etal. noticed that P D T resulted in tumour acidity and hypox ia wh ich sensitised the tumour to subsequent hyper the rmia . 3 3 9 113 The experiments described above were performed on implanted tumours in murine hosts, wh ich introduced in vivo factors affecting pharmacokinetics in addition to unique determinants such as tumour vasculature and oxygenat ion status. Paradoxica l ly , the reverse sequence of heat-> P D T was found to be more cytotoxic than PDT-> heat on L 1 2 1 0 cel ls in vitro. 340 P D T was also used in combinations wi th chemotherapeutic agents inc luding etoposide V P 16, taxol , v incr ist ine, and ET-18- O C H 3 , an a lky l- l ysophospho l ip id to effect additive antitumour e f f e c t s . 3 0 6 ' 3 4 1 - 3 4 2 The bioreduct ive a lky lat ing agent m i tomyc in C ( M M C ) has also been extensively studied in P D T combinat ion therapy. M a et al. reported that simultaneous administration of M M C and Photofrin® fo l lowed by l ight exposure 16 h later (MMC/P-> light) resulted in synergistic k i l l i ng of the human co lon adenocarcinoma cel l l ine W i D r in vitro and in vivo in nude mice ; the authors also concluded that M M C effected ce l l cyc le inhibi t ion wh ich increased the tumour S- phase fraction and consequently the uptake of Photo f r in® . 3 0 4 ' 3 1 8 Th is is consistent w i th earlier f ind ings by Christensen et al. that susceptibi l i ty to P D T in several ce l l l ines ( N H I K 3025, H e L a S 3 , N H I K 1922, and V 7 9 ) is correlated wi th S- p h a s e . 3 4 3 Baas et al. repeated the experiment on the E 0 9 mouse tumour model and conf i rmed the sequence- specif ic synergy of the M M C / P - > l ight regimen since M M C given after P D T d id not enhance tumour k i l l i n g . 3 4 4 The sequence combinat ion of M M C / P - > l ight was later successfully used in a l imi ted c l in ica l trial on patients with skin metastases o f pr imary mammary c a r c i nomas . 3 2 3 C lear l y , numerous parameters can affect the interpretation of the different variations of P D T combinat ion therapy, chief among them include the photosensitiser and drug used, the sequence of treatment, and the experimental system (tumour type, in vitro or in vivo). In this study, D o x was observed to effect'sequence- specif ic potentiated k i l l i ng of the murine leukaemic cel l l ine L 1 2 1 0 but not normal D B A / 2 haematopoietic progenitors when used in combinat ion wi th B P D - mediated P D T . The sequence of Dox-> P D T was found to be superior to simultaneous D o x / P D T , the reverse combinat ion sequence of PDT-> D o x , as we l l as single agent P D T or D o x treatments. In addit ion, effectiveness of the Dox-> P D T sequence was found 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 of this thesis). Several other groups have also studied the use o f D o x in P D T combinat ion therapy. Niahabedian and colleagues successfully used a combinat ion of haematoporphyrin derivative- mediated P D T with D o x 114 to treat B A L B / c mice carrying the E M T - 6 t u m o u r . 3 0 8 They attributed the potentiation of cytotoxicity to photochemical activation of D o x by the red laser l ight (630 nm) used for P D T or alternatively to hyperthermic enhancement of D o x as a result of laser irradiation. The first explanation was dubious because D o x wi th an absorption peak of 579 n m absorbs l ight min ima l l y beyond 600 n m . 3 4 5 Interestingly, Lanks et al. was able to demonstrate photodynamic enhancement of D o x cytotoxicity in the L 9 2 9 cel l l ine in vitro at the A e x o f 514.1 n m and 488 n m . 3 4 6 One year earlier, C o w l e d et al. publ ished a report c i t ing the discrepancy between in vitro and in vivo results of haematoporphyrin derivative (HPD)-mediated P D T combinat ion therapy invo lv ing D o x . 3 0 7 The authors found that the simultaneous P D T / D o x regimen was superior to PDT-> D o x sequence treatment on subcutaneously implanted Lew i s lung carc inoma in vivo; however, they found that D o x inhibited P D T cytotoxicity when the same regimen (PDT/Dox) was tested in vitro on the same cel l l ine. In addit ion, they showed that D o x inhibited the uptake of H P D in vitro. In this study, fluorescence microscopy was used to establish the intracellular localisations of B P D and D o x . Us ing the human leukaemic cel l l ine K 5 6 2 as a mode l , B P D was found to localise to the cytosol and the cel l membrane whereas D o x was found in both cytoplasmic and nuclear compartments. W e believe that differential localisations of the two compounds coupled with their sl ightly different cytotoxic mechanisms contributed to the observed additivity in cel l k i l l i ng of the P D T / D o x regimen. W e also performed a series of spectrofluorimetric experiments to further study the molecular interactions between these two compounds. B P D photobleaching, monitored as mean fluorescence intensity at 693 n m ( M F I 6 9 3 n m ) , was used as a surrogate marker of the degree of its photoact ivat ion. U s i n g physio logica l ly relevant concentrations of B P D and D o x , we determined that D o x inhibited B P D activation when X e x o f 440 nm was used (figure 4.9- 4.11). Th is was probably caused by interference of B P D l ight absorption by D o x wh ich was present at 363.5 fo ld molar excess of B P D . W h e n an A. e x of 630 n m was used, the presence of D o x had a negl igible effect on B P D photobleaching. Th is is expected because D o x absorbs weakly beyond 600 n m and B P D , even at a concentration 363.5 fo ld less than that of D o x , has three distinct absorption peaks above 600 nm. The f indings f rom the photobleaching experiments suggest that in P D T combinat ion therapy, the photophysical characteristics of the compound and their possible interactions must be investigated thoroughly. S ince a red light source emitt ing between 600- 900 n m was used for the cytotoxici ty experiments in 115 chapter 3, the presence of D o x should not affect the photoactivation of B P D in the D o x / P D T regimen. C o w l e d et al. found that D o x at 200 ug/ml (345 u M ) completely inhibited the uptake of H P D in the L ew i s lung carc inoma and Raj i ce l l l ines in vitro. 301 W e observed that 10 u M of D o x decreased the accumulation of B P D in the D o x / B P D regimen. However , L 1 2 1 0 cel ls that were preincubated with 10 u M D o x fo l lowed by 1 h incubation with B P D (Dox-> B P D ) displayed normal levels of the associated photosensitiser. M i l d inhibit ion of B P D uptake by the presence of D o x cou ld explain the additive but not synergistic k i l l i ng of L 1 2 1 0 cel ls. On the other hand, "no rma l i sed " B P D accumulat ion in Dox-> P D T probably accounted for the significant improvement in cytotoxici ty of this regimen. Nevertheless, one must note that the difference in B P D accumulat ion between the two regimens (Dox/BPD vs Dox-> B P D ) was only evident at the higher B P D doses wh i ch therefore questions the relevancy of uptake modulat ion in the combinat ion experiments. A l s o , we believe that the moderate effect on B P D uptake by D o x preincubation cou ld not, by itself, account for the improvement in cel l k i l l i ng seen at 5 ng/ml B P D in the Dox-> P D T sequence. T o explain the superiority of the Dox-> P D T sequence compared to D o x / P D T and PDT-> D o x regimen, we postulated that pretreatment of L 1 2 1 0 cel ls might result in the depletion of cel lular glutathione (GSH ) wh ich wou ld then predispose the cel ls to subsequent oxidative damage mediated by P D T . GSH, .an abundant tripeptide su lphydry l , is invo lved in a variety of cel lular p rocesses . 3 4 7 A l o n e or in conjunct ion wi th the enzymes glutathione S- transferase and selenium- glutathione peroxidase, G S H scavenges free radicals, reduces H 2 0 2 and organic peroxides. Xenobiot i cs and other chemotherapeutic agents such as cisplatin can cause oxidative stress to tumour cel ls wh i ch is buffered by cel lular G S H and numerous studies have shown an inverse correlation between the concentration of G S H and susceptibil ity to chemotherapy. Cancer cells wh ich exhibit the mult idrug resistance ( M D R ) phenotype often have higher amounts of G S H or demonstrate heightened G S H m e t a b o l i s m . 3 4 8 Converse ly , art i f ic ia l depletion of cel lular G S H is achievable v ia the use of buthionine sulphoximine (BSO) which specif ical ly inhibits y-glutamylcysteine synthase, a rate- l imi t ing enzyme in the G S H synthesis cascade. B S O has been successfully used, in conjunct ion with verapamil , to overcome the M D R phenotype in the M C F - 7 breast carc inoma ce l l l i n e . 2 4 5 Furthermore, doxorubic in and cisplat in cytotoxic i ty are enhanced by B S O . 3 4 9 ' 3 5 0 S imi l a r l y , G S H wou ld be expected to play a 116 significant role in determining cel lular susceptibil ity to P D T because of the central role of oxidat ive stress in 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 by the Dox-> P D T sequence cou ld not be related to modulat ion of cel lular G S H by D o x . Us ing the Tietze enzymatic assay, pre- incubation of L 1 2 1 0 cells with 2.5 p M D o x d id not effect a s ignif icant reduction of ce l lu lar G S H . Nevertheless, G S H reduction was observed in a dose- dependent manner wi th respect to the concentrations of D o x used. In summary, the current assay method (Tietze assay) d id not show a significant alteration of cel lular G S H wh i ch cou ld potentially be secondary to the l ow resolution of the assay itself. A more sensitive method of measurement, such as one that is based on the fluorescence detection of G S H - bound monochlorobimane or monobromobimane, cou ld possibly reveal any alterations o f cel lular G S H as a result of D o x pre incubat ion. 4 1 1 The unique photophysical characteristics o f B P D and D o x , in addition to their distinct intracellular localisations, are responsible for the additivity in cytotoxici ty when L 1 2 1 0 cells were treated simultaneously with B P D - mediated P D T and D o x (PDT/Dox) . The apparent synergistic k i l l i ng of L I 2 1 0 cells in the D o x pretreatment regimen (Dox-> P D T ) d id not effect apparent changes in the uptake of B P D nor reduction in G S H ; therefore, other factors such as the generation of ceramide and alteration of activation threshold o f the nuclear transcription factor N F - K(3 cou ld expla in the b io log ica l f indings. Al ternat ive ly , smal l changes in the above measured parameters, in sum, could potentially affect P D T cytotoxici ty in the Dox-> P D T regimen. Nevertheless, more sensitive detection assays are needed in order to ascertain the role of G S H in D o x pretreatment and other possible mechanisms as list above must be considered. 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 H A E M A T O P O I E T I C C O M M I T T E D P R O G E N I T I O R C E L L S F R O M P D T W I T H N - A C S D K P : C Y T O T O X I C I T Y S T U D Y 5.7 ABSTRACT Several studies have demonstrated that the tetrapeptide N - A c S D K P (Seraspenide) selectively inhibits cel l cycle progression of normal haematopoietic cells thereby protecting them f rom the cycle- specif ic cytotoxic i ty of chemoradiation. It was found that N-A 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 marrow cells f rom B P D - mediated P D T cytotoxicity. W h e n D B A / 2 bone marrow cells were incubated wi th 100 n M of N - A c S D K P for 1.5 hours pr ior to P D T , subsequent C F U - G M survivals were improved by 1.5-2 folds over cells that were preincubated wi th the control peptides or with medium. The leukaemic cel l l ine L 1 2 1 0 was not protected f rom P D T with N - A c S D K P preincubation. S imi la r results were observed in normal and leukaemic human haematopoietic cells and in the leukaemic cel l l ine K 5 6 2 . That is, N - A c S D K P effected selective photoprotection of normal human C F U - G M formation but not leukaemic C F U - L formation f rom patient isolates or f rom the K 5 6 2 ce l l l ine. Therefore, results f rom the in vitro short- term co lony assays suggest that N - A c S D K P cou ld be used for the selective protection of the normal committed progenitor cel ls during P D T purging. W e then used the one step long- term bone marrow culture ( L T B M C ) assay to assess whether the photoprotective effect of N - A c S D K P also extends to earlier haematopoietic progenitor and stem cells. Direct quantif ication of ce l l numbers and C F U - G M product ion over a five-week period showed no difference between D B A / 2 bone marrow cells treated with P D T after preincubation with 100 n M o f N - A c S D K P or wi th medium. W e therefore concluded that the photoprotective effect of N - A c S D K P as used here appl ied only to committed progenitor cells wh i ch give rise to C F U - G M colonies. However , this level o f protection, albeit l imited, cou ld sti l l be of c l in ica l advantage in the improvement of the early phase of engraftment mediated by the committed progenitor populations. 118 5.2 INTRODUCTION Haematopoiesis is a highly dynamic yet coordinated process control led by a plethora of posit ive and negative regulators; the bone marrow, site of pr inc ipal haematopoiesis in humans, must be able to function in a steady state equ i l ib r ium in order to replace mature b lood cells that are lost to turnovers for the l ifespan of the organism. In addit ion, the bone marrow must be able to support and maintain heightened haematopoiesis in the course of increased demands such as dur ing infect ions or b lood l o s s . 2 0 0 - 3 5 2 The soluble factors wh ich posit ively regulate haematopoiesis are also termed growth factors, cytokines, or co lony st imulat ing factors (CSFs ) . The first C S F ident i f ied was erythropoietin ( EPO) by Reissmann and colleagues; however, murine granulocyte-macrophage colony stimulating factor ( G M - C S F ) was the first haematopoietic cytokine to be c l o n e d . 3 5 3 ' 3 5 4 Identif ication of C S F s was also aided by the maturation of the hierarchical paradigm of haematopoiesis and technical developments in the isolation of stem cel ls as we l l as their in vitro p ropaga t i on . 3 5 2 ' 3 5 5 > 3 5 6 Industrial- scale product ions of various recombinant human C S F s have revolutionised c l in ica l medic ine, especial ly in the management of cancer pa t i en ts . 3 5 7 E P O , G- C S F and G M - C S F are such examples and are indicated for the amelioration of anaemia and leukopaenia, respec t i ve l y . 3 5 8 In addit ion, thrombopoietin (TPO) , a recently isolated cytokine, shows great promise in managing the thrombocytopaenic condit ion in cancer patients undergoing myeloablat ive the rapy . 3 5 9 Rat ional use of C S F s in the c l in ic is sti l l far f rom realisation, however. Recent ly , several multicentre trials have demonstrated the abilities of G- and G M - C S F s to improve objective criteria without signif icant improvements in the c l in ica l endpoints of the p a t i e n t s . 1 1 3 ' 1 1 4 ' 3 6 0 Hoe lzer suggested that C S F s can play important and relevant roles in the c l in i c , given the appropriate i nd i c a t i ons . 3 6 1 Maintenance of a dynamic equi l ibr ium in haematopoiesis also requires the activities of mult iple feedback mechanisms wh ich negatively modulate the system, some of which include soluble regulatory or inhibitory factors, adhesion molecules, and intrinsic programming wi th in the c e l l s . 2 0 0 Inhibitory factors such as tumour necrosis factor- a (TNF- a ) , t ransforming growth factor- (3 (TGF- (3), and interleukin- 1 (IL- 1) have been wel l charac ter i sed . 3 6 2 Macrophage inhibitory factor- l a (MIP- l a ) is a member of the C-C chemokine fami ly and was or iginal ly identif ied as a stem cel l inhibitor by Graham and 119 co l l eagues . 2 0 1 M IP- l a also mediates the inhibi tory effects of interferon- a ( IFN- a ) on C M L progenitors, through the restoration of [31 integrin mediated adhesion of the leukaemic progenitor cells to the bone marrow stroma, hence effecting restitution of the growth inhibi tory signals f rom the s t r o m a . 2 1 5 A new class of peptide- based haematopoietic inhibitors which include N - A c S D K P , p E E D C K , and S K & F 108636 were ident i f ied w i th various m e t h o d s . 3 6 3 - 3 6 4 The biochemical as we l l as the bio logica l properties of N - A c S D K P has since then been characterised in d e t a i l . 2 0 5 Numerous studies have shown that the peptide as we l l as cytokine inhibitors selectively affect only normal haematopoietic cells, a fact of c l in ica l s i gn i f i c ance . 2 0 4 ' 3 6 5 - 3 6 6 Haematopoiet ic inhibitors act pr inc ipa l ly through the prevention of stem cel l cyc le entry f rom Gj/G, to S phase; therefore, these factors can protect haematopoietic cel ls f rom cycle- specif ic c h e m o t h e r a p y . 2 1 0 - 3 6 7 - 3 6 8 Furthermore, the inhibitors also effect other cel lular changes that result in the protection of normal haematopoietic cells f rom y- radiation, and hyperthermia, in addition to chemotherapy. Fo r example, IL- 1 and T N F - a induce the expression of protective enzymes . 3 6 9 - 3 7 0 N-A c S D K P has been demonstrated to selectively protect normal human haematopoietic progenitors f rom PDT- mediated phototoxicity by the first generation photosensitiser Photofr in®. 2 0 9 In this chapter, the photoprotective effects of N - A c S D K P or Seraspenide was further investigated on P D T mediated by the second generation photosensitiser B P D . Relative susceptibility of D B A / 2 haematopoietic progenitors and L 1 2 1 0 leukaemic cells to P D T was studied after preincubation wi th N - A c S D K P , or the control peptides S D K P and N - A c S D K E . In addit ion, the long- term bone marrow culture ( L T B M C ) assay was used to study whether the photoprotective effect also extended to earlier normal haematopoietic progenitors. T o complement the murine studies, N - A c S D K P was used in P D T experiments invo lv ing normal and leukaemic human haematopoietic cells. 120 5.3 RESULTS Photoprotective effect of N-AcSDKP on DBA/2 haematopoietic progenitors Figure 5.1 demonstrates that preincubation of D B A / 2 bone marrow cells wi th 100 n M of N - A c S D K P for 1.5 hr protected the haematopoietic progenitors f rom subsequent B P D - mediated P D T , as measured by the short- term co lony assay. In addit ion, 100 n M of the control peptides N - A c S D K E and S D K P , as we l l as tissue culture med ium, d id not offer protection. The photoprotective effect mediated by 100 n M N - A c S D K P was especially prominent at B P D concentrations of 2.5, 5, and 10 ng/ml wi th approximately 1.5-2 fo ld higher survival of D B A / 2 C F U - G M compared to cells preincubated with 100 n M of control peptides or medium. Contro l peptides were not tested at the highest B P D concentrations of 15 and 20 ng/ml; however, N - A c S D K P was sti l l able to offer approximately 2- fo ld protection of the progenitors in comparison to medium preincubation. Un l i ke its effects on human haematopoietic cel ls , N - A c S D K P d id not suppress murine C F U - G M f o r m a t i o n s . 2 0 4 W e showed that preincubation for 1.5 hr wi th 100 n M of N - A c S D K P , S D K P , and N - A c S D K E d id not decrease the numbers of day 7 colonies in standard agar based co lony assays (figure 5.2). Absence of N-AcSDKP mediated photoprotection of L1210 cells The murine leukaemic cel l l ine L 1 2 1 0 was not protected f rom subsequent P D T by 1.5 hr of preincubation wi th 100 n M N - A c S D K P . A s shown in f igure 5.3, cel ls that were preincubated with N - A c S D K P d id not show any survival advantage post- P D T in comparison with cells that were preincubated with the control peptides N - A c S D K E and S D K P or in the absence of any peptides. F igure 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 of day 6 L 1 2 1 0 colonies. The above results suggest that N - A c S D K P has no photoprotective effects on L 1 2 1 0 ce l l l ine. 121 F i g u r e 5.1 Selective cytoprotect ive effects of N - A c S D K P on D B A / 2 haematopoietic progenitor cells subjected to B P D - mediated P D T D B A / 2 haematopoietic progenitors were preincubated wi th 100 n M of 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 med ium for 1.5 hr fo l lowed by P D T treatment. Clonogenic i ty was determined with the standard agar- based co lony assay and colonies ( C F U - G M ) were scored on day 7 o f culture us ing an inverted microscope. On ly colonies w i th 40 or more cells were counted. Percent surv ival o f treated cells was calculated based on the number o f colonies generated d iv ided by the number of colonies f r om the no B P D control wi th in the same peptide group Average number f rom the 0 ng/ml B P D control samples of the four pretreatment groups is 100.815 day 7 colonies f rom 70000 cells plated. Data obtained f rom f ive independent experiments (n= 5) is presented. Er ror bars are derived f rom standard errors o f experimental means. Results are significant wi th p< 0.05 as determined by two way-analysis of variance test ( A N O V A ) . 122 i 5 0 - r i 03 lOOnM N - AcSDKP • lOOnM SDKP • lOOnM N - AcSDKE Q no peptide [BPD] ng/ml 123 150 Yl # D a y 7 C F U - G M per 70000 cells CU Q Q Q on < < 1 1 2; Peptide treatment Figure 5.2 Effects of N-AcSDKP, control peptides N-AcSDKE 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 wi th 100 n M of N-A c S D K P , 100 n M of the control peptides N - A c S D K E and S D K P , or med ium prior to mock P D T treatment, i.e. exposure to 15J/cm 2 red light in the absence of the photosensitiser B P D . Day 7 C F U - G M formations in each group were assessed us ing the standard agar- based colony assay. A n inverted microscope was used to enumerate colonies consist ing of 50 or more cel ls. The number o f day 7 C F U - G M was not signif icantly altered in each of the peptide- treated group. Data f rom five independent experiments is presented (n= 5). Source o f error is derived f rom the standard errors o f experimental means. 124 F i g u r e 5.3 Absence of cytoprotect ive effects of N - A c S D K P on L 1 2 1 0 leukaemic cell l ine subjected to B P D - mediated P D T L 1 2 1 0 leukaemic cells were preincubated with 100 n M o f N - A c S D K P (Seraspenide), 100 n M of the control peptides S D K P or N - A c S D K E , or medium for 1.5 hr fo l lowed by P D T treatment. Clonogenic i ty was determined with the standard agar- based colony assay and colonies ( C F U -L ) were scored on day 6 o f culture us ing an inverted microscope. Only colonies with 50 or more cel ls were counted. L o g reductions of treated cells were calculated with respect to the no B P D control wi th in the same peptide group. Data obtained f r om three independent experiments (n= 3) is presented. Error bars are derived f rom standard errors o f experimental means. Results are significant wi th p< 0.05 as determined by two way-analysis of 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 leukaemic cells were incubated with 100 n M of N - A c S D K P , 100 n M of the control peptides N - A c S D K E and S D K P , or medium only pr ior to mock P D T treatment, i.e. exposure to 15J/cm 2 red light in the absence of B P D . Day 6 C F U - L formation in each group were assessed us ing the standard agar- based co lony assay. A n inverted microscope was used to enumerate colonies consist ing o f 50 or more cel ls . The number o f day 6 C F U - L was not signif icantly altered in each o f the peptide- treated group. Data f rom three independent experiments is presented (n= 3). Source o f error is derived f rom the standard errors of experimental means. 127 Statist ical analyses of progeni tor assay data Two- way analysis of variance ( A N O V A ) and Bonferroni (all- pairwise) mult iple comparison tests were employed to test for the signif icance of data f rom the progenitor assays. Two- way A N O V A demonstrated signif icant differences in co lony survivals (p <0.05) between the three B P D doses o f 2.5, 5, and 10 ng/ml in 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 of D B A / 2 cells preincubated with the different peptides were signif icantly different f rom each other yet there was no significant difference amongst PDT- treated L 1 2 1 0 cells preincubated with the different peptides. This suggested that selective photoprotective action was being exerted on the normal D B A / 2 haematopoietic cel ls, but not on the leukaemic cel l l ine L1210 . T o further delineate the difference between the various groups, a more powerfu l statistical analytical tool was used. The Bonfer ron i (all-pairwise) mult iple comparison 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 , and medium) and B P D (2.5, 5, 10 ng/ml) groups in both D B A / 2 haematopoietic cel ls and L 1 2 1 0 leukaemic cel ls. Results f rom normal cel ls are presented in tables 5.1a and 5.1b. Table 5.1a shows that a signif icant intergroup difference exists between 10 ng/ml B P D and the two lower doses of 5 and 2.5 ng/ml. There was no signif icant difference between 5 and 2.5 ng/ml. Table 5.1b shows that the haematopoietic inhibitory peptide N - A c S D K P acted selectively on the normal haematopoietic cells since progenitor assay data of N - A c S D K P preincubated cells treated with P D T was signif icantly different f rom PDT- treated cells that were preincubated with the control peptides N - A c S D K E , S D K P , .as we l l as with med ium control . Data f rom the L 1 2 1 0 experiments are presented in tables 5.2a and 5.2b. A l l three B P D doses are signif icantly different f rom each other (table 5.2a). M o r e importantly, there was no significant intergroup difference in P D T survival data amongst L 1 2 1 0 cells preincubated wi th 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 leukaemic cel ls f rom 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 BPD doses [ BPD] ng/ml 10 5 2.5 10 — ++ ++ 5 ++ — — 2.5 ++ — — Table 5.1a Bonferroni (all- pairwise) mult iple compar ison test of absolute numbers of day 7 C F U - G M o f D B A / 2 haematopoietic progenitor cel ls treated wi th 10, 5, and 2.5 ng/ml of B P D and 15J/cm 2 o f red- l ight irradiation. The plus signs (++) wi th in the shaded cells indicate intergroup significant differences between the represented B P D concentration regardless o f the type of peptide used dur ing the preincubation. 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 - A c S D K P S D K P N - A c S D K E no peptide N - A c S D K P — ++ ++ ++ S D K P ++ — — — N - A c S D K E ++ — — — no peptide ++ — — — F igure 5.1b Bonferroni (all- pairwise) mult iple comparison test of absolute numbers of day 7 C F U - G M of D B A / 2 haematopoietic progenitor cells preincubated wi th 100 n M of N - A c S D K P , 100 n M of control peptides S D K P or N - A c S D K E , or med ium only for 1.5 hr fo l lowed by B P D - mediated P D T . The plus sign (++) wi th in the shaded cel ls indicate intergroup significant differences in the survival of D B A / 2 haematopoietic progenitor cells between the represented peptide used regardless of the concentration of B P D used. 130 Table 5.2a- Bonferroni (all- pairwise) multiple comparison test of clonogenicity data of L1210 leukaemic cells: significance of BPD doses [ BPD] ng/ml 10 5 2.5 10 ++ ++ ': ++ 5 ++ ++ ++ 2.5 ++ ++ '++.-Table 5.2a Bonferron i (all- pairwise) mult iple comparison test of absolute numbers of day 6 C F U - L o f L 1 2 1 0 leukaemic cells treated wi th 10, 5, and 2.5 ng/ml of B P D and 15J/cm 2 of red- l ight irradiation. The plus sign (++) wi th in the shaded cel ls indicate intergroup significant differences between the represented B P D concentration regardless of the type of peptide used dur ing the preincubation. 131 Table 5.2b- Bonferroni (all- pairwise) multiple comparison test of clonogenicity data of L1210 leukaemic cells: significance of peptides Peptide N - A c S D K P S D K P N - A c S D K E no peptide N - A c S D K P — — — — S D K P — — — — N - A c S D K E — — — — no peptide — — — — Table 5.2b Bonferroni (all- pairwise) mult ip le comparison test of absolute numbers of day 6 C F U - L of L 1 2 1 0 leukaemic cel ls preincubated with 100 n M of N - A c S D K P , 100 n M of control peptides S D K P or N - A c S D K E , or med ium only for 1.5 hr f o l l owed by B P D -mediated P D T . There was no significant difference in cytotoxic response of L 1 2 1 0 cel ls preincubated wi th N - A c S D K P , the control peptides, or med ium. 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 marrow 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 cel ls. In this L T B M C assay, freshly harvested bone marrow cells were again preincubated with 100 n M N - A c S D K P or tissue culture med ium for 1.5 hr prior to B P D - mediated P D T . The cells were then m ixed wi th L T B M C culture med ium and plated into duplicate wel ls of 24 we l l tissue culture plates and assessed as described in the experimental procedures section. Dur ing the course of in vitro culture, stromal precursors adhered to the bottom surface of the wel ls and established a stromal microenvironment s imilar to that found in the bone marrow; haematopoietic progenitor cells and stem cel ls undergo maturation in close contact with the stromal layer (figure 5.5). The one- step L T B M C assay uses cells f rom a single harvest for both the establishment of the stroma as we l l as for subsequent haematopoiesis. Co lony formations f rom long- term progenitors are apparent at week four and f ive of the assay whereas colonies f rom week one to three harvests are derived f rom the more mature progenitor ce l l popu la t i ons . 3 7 1 In table 5.3, results in the fo rm of cel l concentrations f rom weekly harvestings of a f ive week L T B M C assay are presented. C e l l numbers of L T B M C suspension harvests f rom both peptide- treated and control groups inversely correlated with B P D concentrations and was most obvious in cultures f rom week one to three (figure 5.6). Later t ime points (week 4 and 5, data.not shown).showed less definit ive difference; the.lack of correlation between harvested cel l number and B P D dose later in culture was caused by dehiscence of cells f rom the stromal layer. In addit ion, contributions of cel ls f rom the dehisced stromal layer greatly inflated the ce l l counts in the control as wel l as the peptide- treated group in week four and f ive. N- A c S D K P does not signif icantly affect suspension ce l l numbers in the PDT- treated cel ls, especial ly in samples harvested at week one to three, suggesting that 100 n M N - A c S D K P d id not appear to offer photoprotection to the population(s) of cells measured by the L T B M C assay. 133 Suspension cel ls f rom the weekly harvests were subsequently put into short- term colony assay which measures the late haematopoietic progenitors such as those that f o rm C F U - G M s . A s shown in table 5.4, there was again an inverse correlation between the numbers of colonies f rom the harvested cel ls and the dose of B P D . However , the numbers were not signif icantly different between the peptide- treated and the control group; therefore, results f rom 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 cel ls . 134 Table 5.3- Suspension cell numbers of weekly harvests of long- term bone marrow culture of DBA /2 haematopoietic cells preincubated in 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 Denticle control 100 nM N-AcSDKP [BPD] 20 10 5 0 20 10 5 0 ng/ml week 1 15000 ± 17300± 23000 ± 27400 ± 5 6 0 0 ± 15600± 21600 ± 28300 ± 300 6300 800 3400 500 2700 2500 6900 week 2 3900 ± 5400 ± 8000 ± 9800 ± 1600 ± 7500 ± 6600 ± 9500 ± 300 800 1100 300 200 2000 400 1200 week 3 5600 ± 6700 ± 12000 ± 14000 ± 3900 ± 2700 ± 6100 ± 23600 ± 1600 3000 400 5900 800 1400 600 900 Table 5.3 Freshly isolated bone marrow cells f rom D B A / 2 mice were preincubated with 100 n M N - A c S D K P or med ium for 1.5 hr pr ior to B P D - mediated P D T . Treated cel ls were resuspended in L T B M C med ium and plated into 24 wel l tissue culture plates in duplicates. A t weekly intervals, nonadherent cel ls f rom each wel l were harvested by gentle agitation and aspiration using an Eppendorf pipetter. The number of cel ls f rom each sample was counted under a haemocytometer by Eos in Y staining of an aliquot of cel ls f rom each we l l . The concentrations of cells f rom the samples were then calculated. Each number, rounded of f to two signif icant f igures, represent an average of two independent samples (n= 2) and source of error is standard error o f the mean. 135 F i g u r e 5.5 O n e s tep l o n g - t e r m b o n e m a r r o w c u l t u r e ( L T B M C ) o f D B A / 2 b o n e m a r r o w ce l l s Freshly harvested bone marrow cells f rom D B A / 2 mice were resuspended in long- term bone marrow culture ( L T B M C ) medium (Mye loCu l t 5300) containing 10"6 M hydrocortisone and dispensed into 24 wel l tissue culture plates in 1.0 m l volumes containing 1 x 10 6 cel ls. Establishment of the stromal layer by stromal precursor cells was fo l lowed by initiation and maintenance o f haematopoiesis. The nonadherent cells wh i ch contain haematopoietic cells at various phases o f maturation were harvested weekly v ia gentle agitation and aspiration o f the media , leaving the stroma behind and intact. Th is picture was taken at week 2 o f a f ive week L T B M C culture us ing a N i k o n F3 camera connected to the Zeiss Axiovert 35 inverted microscope. The lOx objective with phase contrasting setting 1 (P I ) was selected. Note the confluent stromal layer as we l l as the clusters of cells that appeared to rest on top o f the it; active haematopoiesis occurs at these clusters. 136 Figure 5.6 Comparison of week 1 long- term bone marrow culture ( L T B M C ) harvests of nonadherent cells from samples preincubated with 100 n M N - A c S D K P or medium followed by P D T Graphic representation o f the first row of table 5.3' compares the cell numbers of the harvested nonadherent populations of week 1 L T B M C . Ce 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 /cm 2 red l ight. The difference observed at 20 ng/ml d id not extend to later harvests (week 2- 5). 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 in 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 peptide control 100 nM N-AcSDKP [BPD] ng/ml 20 10 5 0 20 10 5 0 week 1 6.80 ± 1.20 12.30 ± 2.30 16.00 ± 1.70 19.80 ± 2.20 2.20 + 0.50 11.20 ± 0.20 14.50 ± 0.50 13.00 ± 1.00 week 2 1.80 ± 0.80 2.80 ± 0.20 5.70 ± 3.00 7.20 ± 3.50 0.70 ± 0.70 5.80 ± 3.80 7.50 + 3.80 11.50 ± 0.20 week 3 7.70 ± 0 7.30 ± 1.00 23.50 ± 15.20 15.70 + 3.00 2.30 ± • 0.30 '5.80 ± 4.20 19.70 ± 13.70 32.50 ± 2.80 Table 5.4 Cel ls f r om the weekly harvestings of nonadherent populations of L T B M C were resuspended at a standard vo lume and grown in standard short- term co lony assays. The numbers of day 7 C F U - G M consist ing of 40 or more cel ls were enumerated using an inverted microscope. Each number, rounded o f f to one signif icant f igure, represent an average o f two independent samples (n= 2) and source o f error is standard error of 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 Human bone marrow cells or primary leukaemic cells f rom a newly diagnosed C M L patient were preincubated with 100 n M N - A c S D K P or tissue culture med ium for 1.5 138 hr prior to P D T mediated by 10 ng/ml B P D and 15 J /cm 2 red l ight. The treated cel ls were then put into short- term colony assays wh ich measured late human haematopoietic progenitors wh ich give rise to C F U - G M or C M L leukaemic precursor cel ls wh i ch give rise to C F U - L. In addit ion, the human C M L ce l l l ine K 5 6 2 was also examined. Numbers of colonies f rom the peptide group were d iv ided by the number of colonies in the tissue culture med ium group to obtain a photoprotection ratio. A ratio of more than 1 signif ies photoprotection and the results is shown in f igure 5.7. Ce l l s f rom two independent human bone marrow samples benefited f rom preincubation wi th 100 n M N - A c S D K P wi th photoprotection ratios of 4.5 and 8. However , the leukaemic cel l l ine K 5 6 2 and cel ls f rom the pr imary leukaemic cel l sample d id not benefit f rom N - A c S D K P because both samples demonstrated photoprotection ratios of 1. Results f rom the human experiments mirrored those of the earlier murine assays; that is, N - A c S D K P selectively protected late haematopoietic progenitors but not leukaemic cells f rom B P D - mediated P D T . 139 10-, Ratio of photo- protection conferred by lOOnM N-AcSDKP * 6H 2H 1 H u B M M N C K562 C M L • A 4 BM1 BM2 K562-1 K562-2 C M L Figure 5.7- 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 of normal human bone marrow cells wi th l O O . n M of N-A c S D K P for 1.5 hr selectively protected them f rom subsequent P D T treatment mediated by 10 ng/ml of B P D and 15 J / cm 2 o f red l ight. The human myelogenous leukaemic cel l l ine K 5 6 2 and pr imary untreated human C M L cells d id not respond to the photoprotective effects o f N - A c S D K P . The ratios of photoprotection o f the respective cell type were derived f rom the ratio of the percentage of colonies f rom PDT- treated cells that were preincubated with 100 n M N - A c S D K P over the percentage of colonies of PDT- treated cells that were preincubated with med ium. 140 DISCUSSION The process of haematopoiesis is a h ighly regulated process invo lv ing both posit ive and negative s i g n a l s . 2 0 0 Var ious co lony st imulating factors or cytokines have been identif ied and cloned and some of these factors show promise in the c l in ica l management of the cancer pa t ient . 3 5 7 Funct ional haematopoiesis also depends on its eff icient control v ia negative regulators such as IL- 1, T G F - p\ and chemokines such as M IP- l a . 3 6 2 A new class of oligopeptide haematopoietic inhibitors wh ich include the tetrapeptide N-Acetyl-Ser- Asp- Lys- Pro ( A c S D K P ) and pyroG lu- G lu- Asp- Cys- L y s ( p E E D C K ) was recently charac ter i sed . 2 0 5 N - A c S D K P is der ived f rom proteolyt ic processing of the N- terminus o f thymosin (34, a protein wh i ch binds F- a c t i n . 3 7 2 > 3 7 3 Se rum level o f N - A c S D K P changes during chemotherapy in leukaemic patients and in mice after treatment with cytosine arabinoside (ara- C ) . 3 7 4 - 3 7 5 Numerous studies have demonstrated that N - A c S D K P selectively inhibits cel l cycle progression of normal haematopoietic cel ls, preventing the entry f rom GQ/G , to S- p h a s e . 2 0 4 - 3 6 5 Select ive cyc le inh ib i t ion of normal cel ls by N-A c S D K P protects them f rom chemotherapy, hyperthermia, radiation, as we l l as P D T t r e a t m e n t s . 2 0 6 - 2 0 9 However , other mechanisms mediated by N - A c S D K P cou ld be invo lved as we l l . Large scale production of the peptide for proposed trials for human applications prompted subsequent human pharmacokinet ic s t ud i e s . 3 7 6 N - A c S D K P therefore jo ins the other haematopoietic inhibitory cytokines and peptides and becomes part of the armamentorium in the new f ie ld of stem cel l protection, in wh ich b io logica l factors are used to effect selective protection of normal haematopoietic cells f rom chemotherapy and radiation therapy. 2 1 0 - 3 6 7 < 3 6 8 - 3 7 7 Ih addit ion, other candidates such as the thiol- based compound amifostine showed promise in ameliorating the harmful side effects of chemotherapy on normal haematopoietic c e l l s . 3 7 8 Fo r example, incorporation of amifostine in the ex vivo purging of autologous bone marrow f r o m breast cancer patients with 4 -hydroperoxycyclophosphamide signif icantly improved subsequent haematological r e c o v e r y . 3 7 9 In this study, N - A c S D K P was used to effect selective photoprotection on normal haematopoietic cells. A previous study has shown that N - A c S D K P cou ld be used to protect normal human bone marrow cells f rom P D T mediated by the first generation 141 photosensitiser Photofrin®. 2 0 9 S ince B P D is a much more 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 . Freshly isolated bone marrow cells f rom D B A / 2 mice or L 1 2 1 0 leukaemic cel ls were incubated with 100 n M of N - A c S D K P for 1.5 h prior to exposure to P D T mediated by B P D . Co lony fo rming units of granulocyte/ macrophage ( C F U - G M ) and colony fo rming units of leukaemic cel ls ( C F U - L ) were determined using standard colony assays. Neither the tetrapeptide N - A c S D K P nor the control peptides S D K P and N-A c S D K E inhibited the growth of C F U - G M and C F U - L. The absence of inhibi tory effect on late murine progenitors supported earlier experimental f indings using 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 rom P D T , especial ly at the lower doses o f B P D o f 2.5 and 5.0 ng/ml. The control peptides N-A c S D K E and S D K P as wel l as the med ium control had no protective effects on the cel ls. The same protective effect was not observed in L 1 2 1 0 cells preincubated wi th N - A c S D K P or the control peptides. In addit ion, Bonferron i (all- pairwise) mult ip le compar ison test of the data f rom the progenitor assays demonstrated significant intergroup differences between PDT- treated D B A / 2 haematopoietic cells that were preincubated with N - A c S D K P and the PDT- treated cells that were preincubated with the control peptides or medium. N o significant intergroup difference was observed in PDT- treated L 1 2 1 0 cells preincubated with 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 rom B P D -mediated P D T . Furthermore, one step long- term bone marrow culture ( L T B M C ) assay revealed that the photoprotective effect on the late progenitors measured by the colony assays d id not extend to earlier progenitors. C e l l numbers of week ly harvests as we l l as the ^numbers of C F U - G M produced f rom the harvests were not s ignif icantly different ;between the peptide and control group treated wi th P D T at different doses. Bo th harvested ce l l number and colony number correlated inversely with the dose of B P D and this correlation was more obvious in the earlier harvests of week 1 to 3. The loss of adherence of the stromal layer during harvesting of the suspension cells contributed to the inflated ce l l numbers and therefore colony numbers in the harvests of weeks 4 and 5 (data not shown), wh ich also contributed to the loss of correlation between the B P D doses and the results of the L T B M C assay. A n apparent contradict ion exists between the results in F igure 5.1 and table 5.4. Spec i f ica l ly , short- term agar co lony assay of D B A / 2 bone marrow cel ls 142 demonstrated the photoprotective effect o f 100 n M N- A c S D K P (figure 5.1); however, the numbers of C F U - G M derived f rom week 1 L T B M C harvests of control and peptide treated cells showed no sigif icant difference. In fact, the same trend was noted in the direct ce l l counts of the week 1 harvests (table 5.3). N - A c S D K P at 100 n M was able to selectively protect normal human bone marrow cells f rom B P D - mediated P D T . This conf i rms earlier results by Coutton and colleagues using Photofrin®- mediated P D T . 2 0 9 The procedures described here dif fered somewhat f rom the ones used in the above mentioned paper. Spec i f ica l ly , we were able to achieve photoprotection of the normal human haematopoietic progenitors by 1.5 h incubation with 100 n M of N - A c S D K P whereas a 20 h incubation per iod was used in C o u t t o n ' s paper. W e also observed inhibitory activity against human in vitro C F U - G M in wh ich human bone marrow cel ls incubated with 100 n M N - A c S D K P for 1.5 h resulted in the production of 54- 67 % o f day 14 colonies compared to the no peptide controls. In addit ion, the inhibitory effects of N - A c S D K P were not un i form in the different human bone marrow samples which suggested idiosyncratic susceptibil ity to the peptide. Photoprotection against B P D was not observed when N - A c S D K P was used dur ing preincubat ion of K 5 6 2 cel ls or pr imary C M L cells. The uti l ity o f selective photoprotection mediated by N - A c S D K P is sti l l obvious even though it does not extend to earlier haematopoietic progenitor and stem cel ls. Norma l haematopoietic cel ls, especially the earlier progenitor and stem cel ls, 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 of the human L T B M C assays . 2 9 2 Pretreatment of murine bone marrow cells effected significant protection of the C F U - G M populat ion f rom P D T . Th is permits B P D dose escalation in purging without significant damage to the haematopoietic ce l l compartment responsible for 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 of D B A / 2 bone marrow cells with 100 n M of N - A c S D K P selectively protected of the committed granulocyte/macrophage progenitors ( C F U - G M ) but not cells of earlier lineage f rom P D T . Peptide pretreatment, however, d id not protect the murine leukaemic ce l l l ine L1210 . F A C S analysis was used to determine B P D uptake in the cells responsible for short- term haematopoietic reconstitution. Doub le label l ing of D B A / 2 bone marrow cells preincubated with control med ium or 100 n M of N - A c S D K P wi th anti- C D 3 4 antibody and 10 ng/ml B P D demonstrated no significant difference in mean fluorescent intensity (MF I ) of B P D in the C D 3 4 + populat ion. In addit ion, peptide incubation d id not alter the proportion of C D 3 4 + c e l l s . The bioact iv i ty o f N - A c S D K P was ver i f ied by standard ara- C suicide assay. Incubation of D B A / 2 bone marrow cells with 100 n M N - A c S D K P for 1.5 h or 5.0 h resulted in substantial protection of C F U - G M format ion, v i a prevention o f S- phase entry, f rom subsequent exposure to 50 or 100 p M of ara- C . S ince 1.5 h incubation wi th N - A c S D K P was able to effect cyc le inhibi t ion in C F U - G M progenitors, the observed photoprotective effect cou ld therefore be the result of N - A c S D K P - mediated cycle inhib i t ion. This possibi l i ty was proven by pretreating the bone marrow cel ls with 50 u M Ara- C wh ich resulted in the destruction of S- phase cel ls. The surv iv ing cel ls 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. Examinat ion of cel lular glutathione content revealed no significant difference between the peptide- treated cells and the control cel ls. Therefore, the specif ic mechanisms responsible for N - A c S D K P - mediated photoprotection are sti l l not clear. However , it is possible that N - A c S D K P - mediated cel l cyc le inhibi t ion could affect mult iple pathways (i.e. drug uptake, levels of protective thiols, subcellular localisation of target proteins, etc) that together result ultimately in protection f rom P D T . 144 6.2 INTRODUCTION Selective protection of normal haematopoietic stem cells and progenitor cells during in vivo and ex vivo antineoplastic radiochemotherapy is a therapeutic goal long sought after by cl inic ians (and patients al ike). Other normal tissues, such as the regenerating epithel ium of the gastrointestinal tract and hair fo l l icu lar cel ls, are also susceptible to the side effects of therapy; in addit ion, organ- specif ic toxicit ies f rom potent yet indispensable drugs such as doxorubic in and vincrist ine l imi t the uti l i ty o f these agents. Nevertheless, bone marrow toxicity encourages the development of life- threatening episodes of thrombocytopaenia, anaemia, as we l l as immunosuppress ion and therefore remains one of the major l imi t ing factors in the use of therapeutically efficient doses of radiochemotherapy. Therefore, one ideally wou ld l ike to selectively protect normal haematopoietic cells during therapy so as to enlarge the therapeutic w indow. In vivo bone marrow protection can be effected through the use of posit ive haematopoietic regulators, such as granulocyte and granulocyte- macrophage co lony st imulat ing factor (G- C S F , G M -C S F ) and erythropoietin (EPO) , wh ich stimulate the prol iferation plus differentiation of normal haematopoietic cells to counteract therapy- induced mye losuppress ion . 2 0 0 The usefulness of these factors in the c l in ica l setting, especial ly in effecting myeloprotection f rom dose and schedule intensif icat ion o f radiochemotherapy, is c o n t r o v e r s i a l . 1 1 4 - 1 1 5 > 3 6 0 Nevertheless, judic ious and rational use coupled wi th improv ing c l in ica l and scientif ic knowledge w i l l increase the appl icabi l i ty o f these factors in cancer therapy . 3 6 1 Converse ly , cytokines such as tumour necrosis factor- a ( T N F a ) and interleukin- 1 (IL- 1), the chemokine macrophage inf lammatory factor- l a (MIP- l a ) , and smal l peptides such as acetyl- N- Ser- Asp- Lys- Pro ( N - A c S D K P ) and pG lu- G lu- Asp- Cys- L y s ( p E E D C K ) can also effect stem cel l protection through the inhibit ion of cel l c yc l ing and possibly other m e a n s . 2 1 0 - 3 6 8 The phosphorylated su lphydry l compound amifost ine (WR- 2721) has been demonstrated to afford haematopoietic protection to murine hosts exposed to y- radiation or cytotoxic agents, probably v ia the modulat ion of cel lular thiol concentrat ions . 3 8 1 Amifos t ine , in combination with G- C S F , can signif icantly accelerate myelopoiet ic recovery in lethally irradiated m i c e . 3 8 2 - 3 8 3 145 Haematopoiet ic stem cel l transplantation is essentially a fo rm of ex vivo bone marrow protection and the transplantation of bone marrow- derived stem cells constituted one of the earliest application of such t h e r a p y . 3 7 - 3 8 4 Auto logous haematopoietic stem cel l transplantations (bone marrow or peripheral b lood stem cells) el iminate the need for H L A -compatible al logeneic donors and hence reduce the r isk of graft- versus- host d i sease . 1 5 2 Nevertheless, contamination of autologous stem cel l harvests by neoplastic cells leads to an increase in the incidence of disease relapse post t ransplantat ion. 1 5 4 Ex vivo manipulat ion or purging of the harvests with pharmacological agents attempts to eradicate a significant number of the contaminating cancer cells whi le preserving enough of the normal haematopoietic cells for reconstitution. The same pr inciple that is applicable for in vivo stem cel l protection is also suitable for ex vivo purging in that the procedure attempts to enlarge the therapeutic w indow v ia the selective protection of normal haematopoietic cel ls, wh i ch permits dose escalation of the purg ing r e g i m e n . 2 0 5 - 2 1 0 Mechanist ica l ly , the current paradigm of stem cel l protection mediated by inhibitory molecules revolves around the ce l l cycle. M o s e r and Paukovits , in their review article, conceded that cytostatic drug- induced haematopoietic damage is effected on the proliferating compartments of the bone marrow which contain the mitotical ly active haematopoietic progenitor and precursor cel ls, whose depletions result in the recruitment of normal ly quiescent stem cells into c y c l e . 3 6 7 Unfortunately, the cyc l i ng stem cel ls, in an effort to replenish the progenitors, in turn become vulnerable to damage and repeated rounds of chemotherapy can lead to the irreversible impairment or .quantitative impairment of the stem cel l p o o l . 3 8 5 Gardner et al. recently demonstrated that chemotherapy causes the exhaustion of the pluripotent pr imit ive haematopoietic stem cel l ( P H S C ) poo l , resulting in prolonged;marro .W 'reconstitutive defects . 1 3 8 6 Stem ce l l inhibitprs.such.as M IP- l a , N-A c S D K P and p E E D C K selectively prevent cel l cyc le entry of normal haematopoietic stem cells and precursor cel ls. N - A c S D K P was or ig inal ly characterised to prevent the recruitment o f murine P H S C s into the D N A synthetic or S- phase after ara- C chemotherapy as determined by spleen co lony fo rming units ( C F U - S) and administration of the peptide protected mice f rom lethal doses o f ara- C . 3 6 3 - 3 8 7 Furthermore, N - A c S D K P was shown to inhibit the S- phase entry of human haematopoietic progenitor and precursor cel ls in vitro in short- term progenitor assays and long- term bone marrow c u l t u r e . 3 8 8 - 3 8 9 Leukaemic cells do not appear to respond to the regulatory effects of N - A c S D K P and 146 therefore are not afforded protection by the p e p t i d e . 2 0 4 - 3 6 5 M IP- l a was found to be another mediator of stem cell protection with s imilar target populations and mechanisms as N - A c S D K P . 2 0 1 - 2 0 2 > 3 6 6 Interestingly, Cashman et al. presented evidence that the N-A c S D K P - mediated inhibitory effect cou ld be abrogated by the simultaneous addition of M IP- 1/3, an antagonist of M IP- l a therefore suggesting that M IP- l a is a.downstream mediator of N - A c S D K P b i o a c t i v i t y . 2 0 4 A different form of stem cel l protection described by Zuca l i and colleagues concerns the cytoprotective effects of T N F a and fL- 1 on human and murine haematopoietic cells f rom ionis ing radiation and the purging agent 4- hydroperoxycyc lophosphamide (4-H C ) . 3 9 0 ' 3 9 1 Furthermore, they presented evidence that cytokine induct ion o f the enzymes manganese- superoxide dismutase ( M n S O D ) and aldehyde dehydrogenase ( A L D H ) was responsible for their cytoprotective effects . 3 6 9 - 3 7 0 > 3 7 7 M n S O D detoxif ies the h igh ly reactive superoxide anion, generated dur ing cel lular exposure to ionis ing radiation or the drug doxorub ic in , v i a its transmutation into hydrogen p e r o x i d e . 3 9 2 A L D H , whose transcription in human marrow cel ls is increased three- fo ld with IL- 1 or T N F a , detoxifies 4- H C . 3 6 9 Stem cel l protection can be mediated by cel l cyc le inhibi t ion, induction o f enzymes, and augmentation of the cel lular thiol pool or, conversely through the use of posit ive regulators wh ich numerical ly expand b lood cells to counteract the deleterious effects of therapy. In this chapter, we examined some of the mechanisms responsible for the selective photoprotection of normal haematopoietic cells by N - A c S D K P ; specif ical ly , the effects of N - A c S D K P on cel l c yc l ing , photosensitiser uptake, and cel lular glutathione were investigated. 147 6.3 RESULTS B P D uptake by DBA/2 bone marrow cells preincubated with 100 n M N -A c S D K P Freshly isolated femoral bone marrow cells f rom D B A / 2 mice were incubated in the presence or absence o f 100 n M N - A c S D K P in serum- free I M D M for 1.5 h fo l l owed by incubation w i th 10 ng/ml B P D for 0.5 h. F A C S analysis was then used to assess for the percentage of C D 3 4 - posit ive cel ls. C D 3 4 is a surface s ia lomucin expressed by populations of haematopoietic progenitor and stem c e l l s . 3 9 3 Next , the level of B P D uptake or association in the peptide- treated population was determined by analysing B P D mean fluorescence intensity (MF I ) in C D 3 4 + cells (described in the Experimental procedure chapter). A s shown in table 6.1, preincubation with N - A c S D K P d id not alter the proportion of C D 3 4 - expressing cel ls in the peptide- treated group (16.13 + 0.94 %) compared to the culture medium- treated control group (16.83 + 0.43 % ) . Furthermore, there was no significant difference in B P D M F I between the peptide- treated group (4.67 + 0.25) and the control group (4.84 + 0.18). Therefore, in vitro preincubation of D B A / 2 bone marrow cells with 100 n M N - A c S D K P for 1.5 h d id not appreciably alter the proportion of C D 3 4 + c e l l s nor d id it change the degree of B P D association wi th in the CD34 + popu l a t i on . Nevertheless, as demonstrated in chapter 5, the same peptide preincubation protocol was able to effect cytoprotection o f D B A / 2 C F U - G M progenitors f r om 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 of D B A / 2 bone marrow cells with 100 n M N - A c S D K P for 1.5 h d id not signif icantly alter the relative proport ion o f C D 3 4 + cells nor B P D uptake in these cel ls. T w o colour F A C S analysis o f cells labelled with biotin- C D 3 4 antibody (clone R A M 3 4 ) / F I TC- strepavidin and 10 ng/ml B P D . R A M 3 4 , a monoclonal antibody o f rat o r ig in , is specif ic for the mouse haematopoietic stem cell antigen C D 3 4 . F A C S analysis was based on 100000 events per sample. N - A c S D K P does not signif icantly alter the percentage of C D 3 4 + c e l l s (Figure 6.1a) and the peptide does not affect B P D association and uptake in B M M N C s (Figure 6.1b). In addit ion, N - A c S D K P does not alter B P D association and uptake in C D 3 4 + c e l l s (Figure 6.1c). 149 No peptide control 100 nM N- AcSDKP Table 6.1 BPD uptake in the CD34 expressing subpopulation of DBA/2 bone marrow cells % CD34 expressing bone marrow cells BPD MFI in CD34+cells inhibitor treatment no peptide 100 n M N-A c S D K P no peptide 100 n M N-A c S D K P 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 Table 6.1 Preincubation o f D B A / 2 bone marrow cells wi th 100 n M N - A c S D K P for 1.5 h did not significantly alter the relative proport ion of C D 3 4 + cells or B P D uptake in these cel ls. T w o colour F A C S analysis o f cells labelled with biotin- C D 3 4 antibody (clone R A M 3 4 ) / F ITC- strepavidin and 10 ng/ml B P D . R A M 3 4 , a monoclonal antibody of rat or ig in , is specific for the mouse haematopoietic stem cell antigen C D 3 4 . F A C S analysis was based on 100000 events per sample. Data f rom three independent experiments are presented and source of error is derived f rom the standard error o f the mean. 151 Inhibition of the proliferative activity of murine CFU- GM progenitors by 100 nM N-AcSDKP F igure 6.2 shows that incubation of freshly harvested D B A / 2 bone marrow cel ls with 100 n M N - A c S D K P for 1.5 h resulted in the inhib i t ion of S- phase entry of the C F U -G M progenitors. Ce l ls that were exposed to N - A c S D K P became resistant to the cytotoxic activity of ara- C , an S- phase specif ic tox in . Inhibit ion of S- phase entry was also demonstrated in cel l samples that were incubated with N - A c S D K P for 5.0 h (figure 6.3). Interestingly, the absolute numbers of day 7 colonies were not appreciably reduced by N-A c S D K P preincubation. W e therefore demonstrated that the fraction of S- phase D B A / 2 C F U - G M progenitors cou ld be reduced wi th in vitro exposure to 100 n M N - A c S D K P for 1.5 h. In addit ion, our data suggests that the observed photoprotective effect mediated by N - A c S D K P could be due to its abil ity to inhibit ce l l cyc le progression into the D N A synthetic or S- phase. W e proceeded to examine the relationship between cel l cyc l ing and P D T susceptibil ity in 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 Exposure of freshly harvested D B A / 2 bone marrow cells to 100 n M N - A c S D K P for 1.5 h effected cel l cyc le inhibit ion of the C F U - G M fraction, rendering the cells resistant to the S- phase specif ic cytotoxici ty of ara- C . The observation also suggest that the photoprotective effect reported in chapter 5 was mediated v ia the inhibit ion of cycle entry. F igure 6.4 further illustrates the relationship between cel l c yc l ing and susceptibil ity to B P D - mediated P D T in the C F U - G M progenitor populat ion. Harvested marrow cel ls were incubated with 50 p M ara- C for 1.0 h wh i ch , as shown in f igure 6.2, resulted in the destruction of approximately 50 % of the C F U - G M progenitor populat ion. Ara- C effected 152 selective cytotoxicity to cells in S- phase which resulted in the enrichment of quiescent cells ( G 0 /G, ) . Subsequent exposure to B P D - mediated P D T revealed differential susceptibi l i ty to P D T cytotoxic i ty . Even though 50 u M ara- C caused a reduction of the absolute number of day 7 C F U - G M , the surv iv ing fraction was signif icantly more resistant to P D T than the control cel ls not pretreated with ara- C (figure 6.4). 153 125- , H X H 7 5 -Day 7 C F U - G M 5 0 -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 had on the cycling status of C F U - G M from DBA/2 mice C F U - G M formation is protected f rom ara- C cytotoxicity by N - A c S D K P ; however , exposure to the peptide alone had no effects on the number of C F U - G M . D B A / 2 bone marrow cells were incubated wi th 100 n M N-A c S D K P for 1.5 h in serum free I M D M at 37°C. The cel ls were washed and incubated with 0, 50 , or 100 u M of ara- C for 1 h and put into standard agar- based colony assay in triplicates. Co lon i e s , wh i ch consisted of 50 or more cel ls , were counted on day 7 of the experiment with an inverted microscope. Approximately 70000 cells were seeded per plate wh ich translated to a plating eff iciency of 0.129 % . Data obtained f rom 3 independent experiments is presented. Er ror bars are derived f rom standard error of the mean ( S EM ) . 154 100-, 75 J D 7 C F U - G M 50 4 25 0- mm. 100 100 n M N- A c S D K P - 5hr no peptide control- 5hr [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 DBA/2 mice C F U - G M formation is protected f rom ara- C cytotoxicity b y ' N - A c S D K P ; however , exposure to the peptide alone had no effects on the number o f C F U - G M . D B A / 2 bone marrow cells were incubated wi th 100 n M N-A c S D K P for 5.0 h in serum free I M D M at 37°C. The cel ls were washed and incubated wi th 0, 50 , or 100 p M o f ara- C for 1 h and put into standard agar- based colony assay in triplicates. Co lon ies , wh i ch consisted of 50 or more cel ls , were counted on day 7 o f the experiment wi th an inverted microscope. Approximate ly 70000 cells were seeded per plate wh ich translated to a plating eff iciency o f 0.129 % . Data obtained f rom 3 independent experiments is presented. Error bars are derived f rom standard error of the mean ( S EM ) . 155 150 - I Figure 6.4 Correlation between cell cycling and susceptibility to B P D -mediated PDT in DBA/2 C F U - G M progenitors C F U - G M progenitors preincubated wi th 50 u M were less susceptible to ;BPDr mediated PDT. Even though ara- C resulted in a. reduction o f the absolute number o f day 7 C F U - G M , cells that were preincubated with 50 p M ara- C showed higher relative survival f rom subsequent PDT treatment than control cells preincubated with med ium. Cont ro l DBA/2 progenitors y ie lded 185.67 ± 14.57 colonies f rom 70000 cells plated (0.264 % c loning efficiency) whereas progenitors cells treated wi th 50 u M ara- C yielded 169.33 + 9.07 colonies f rom 140000 cells plated (0.121 % c loning eff ic iency) . Results f rom 3 independent experiments are presented. Error bars are derived f rom standard errors of the mean ( S EM ) . 156 Glutathione level in DBA/2 bone marrow cells treated with 100 n M N-A c S D K P The amount of intracellular glutathione (GSH ) was established using the Tietze method as described in the Experimental procedure chapter. The G S H content f rom cel l extracts derived f rom 2 x 10 6 D B A / 2 bone marrow cel ls incubated wi th 0 (51 .67 + 11.46 p M G S H ) , 10 (47.01 ± 12.59 p M G S H ) , or 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 ignif icantly different f rom one another (figure 6.5). W e therefore cou ld not attribute the photoprotective effect of N - A c S D K P to an increase of the intracellular antioxidant G S H . However , a more sensitive method o f measurement, such as one that is based on the fluorescence detection of G S H - bound monochlorobimane or monobromobimane, cou ld possibly reveal any alterations of cel lular G S H as a result o f N-A c S D K P incubation. In addit ion, we d id not consider other cel lular antioxidants wh i ch cou ld mediate photoprotection in this experiment. 157 [N- A c S D K P ] n M Figure 6.5 Cellular glutathione content in DBA/2 bone marrow cells incubated with 0, 10, or 100 n M N-AcSDKP Incubation of D B A / 2 bone marrow cells with N - A c S D K P for 1.5 h had no effect, on cellular glutathione ( G S H ) content as determined by the Tietze method. Ce l l extracts f rom 2 x 10 6 cells incubated wi th .0, 10, 1 0 0 ' n M N-A c S D K P were processed as described in the Experimental Procedure chapter and G S H content in u M was measured. Results f rom two independent experiments are shown. Standard errors o f the means ( S E M ) are used as source of error. 158 6.4 DISCUSSION Bone marrow toxicity is one of the major l imi t ing factors in the effective use of radiochemotherapy in cancer control , and dose escalation in therapy is invar iably accompanied by life- threatening episodes of marrow aplasia and end- organ toxicit ies. Interruptions of treatment regimens are frequent occurrences in the c l in ic and ult imately contribute to inefficient control of cancer. Bone marrow toxic i ty is especially problematic in allogeneic and autologous haematopoietic stem cel l transplantations but it is, however perverse, a necessary part o f the therapy. H i g h dose radiochemotherapy is required during the condit ioning regimen to prepare the host for the acceptance of donor (allogeneic) haematopoietic cells and the destruction o f a significant number o f neoplastic cel ls in vivo. Long- term defects in marrow reconstitutive capabil ity as a result of the intensive therapy are evident in human patients and cou ld be due to irreversible damage to the marrow microenvironment wh i ch remain of 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 in the haematopoietic progenitor populations of patients 8 years after their t ransplantat ions. 3 9 4 In addit ion, the prob lem o f iatrogenic damage to normal haematopoietic cells is also encountered ex vivo in the purging o f autologous stem cel l harvests f r om cancer patients. Overzealous purging w i l l result in effective destruction of the contaminating neoplastic cel ls, at the expense of normal haematopoietic cells in the harvest. Th is can result in faulty engraftment manifested either by a temporal delay in achieving haematopoietic normalisation or an absence of permanent engraf tment . 2 2 0 Haematopoietic stem cel l protection attempts to selectively preserve normal haematopoietic cells f rom cytotoxic damage, wh ich theoretically permits dose escalation of therapy-to effect eff icient destruction o f the neoplastic cel ls in vivo or ex vivo. 210 A t the present moment, toxicities to other normal tissues such as the l in ing o f the gastrointestinal tract and hair fo l l icular cells are not obviated by stem cel l protection. However , improvements in chemotherapeutic agents and treatment protocols in addition to developments of novel protective compounds w i l l help to ameliorate the iatrogenic damage to normal tissues and alleviate organ- specif ic toxicity. Fo r example, sod ium 2- mercaptoethanesulfonate ( M E S N A ) , in conjunct ion with prophylact ic hydration, is used to reduce the incidence of acute haemorrhagic cystitis in patients receiving cyc lophosphamide treatment. 3 9 5 159 Stem cel l protection effected by negative regulators of haematopoiesis is a promis ing area of research. O f special s ignif icance are two low molecular- weight ol igopeptide inhibitors, the pentapeptide pG lu- G lu- Asp- Cys- L y s ( p E E D C K ) and the tetrapeptide acetyl- N- Ser- Asp- Lys- P ro ( N - A c S D K P ) . 2 0 5 The acknowledged pr inc ipa l mechanism of protection is the inhibi t ion of cel l cycle entry into S- phase, where the cel l becomes vulnerable to a plethora of cytotoxic agents . 3 6 7 Another apparently unique feature o f these inhibitors is in the ability to preferentially affect normal haematopoietic cells hence specif ic i ty in their protective a c t i o n . 2 0 4 - 3 6 5 A l ready , several groups have demonstrated that N - A c S D K P mediated stem cel l protection in vivo in mice as we l l as monkeys and dogs . 2 0 5 -387, 396 In this study, we showed that exposure of freshly harvested D B A / 2 bone marrow cel ls to 100 n M (10" 7 M) N - A c S D K P for 1.5 h in serum free I M D M resulted in the inhib i t ion of S- phase entry of C F U - G M progenitors, manifested by resistance to cycle-specif ic toxicity of ara- C . N - A c S D K P , however, d id not alter the total number of C F U -G M . Together, this demonstrated that inhibi t ion of S- phase entry was achieved without toxic i ty to the C F U - G M progenitors. S igni f icant ly , the incubation protocol was identical to the one used in the cytotoxicity experiments described in chapter 5, suggesting a possible explanation for the observed photoprotection of D B A / 2 C F U - G M . Our results are in agreement with those publ ished by W ie renga and K o n i n g s . 3 9 7 U s i n g a sl ightly different protocol , they showed that incubation wi th 10~7to 10~12 M of N - A c S D K P for 8 h in the presence of 30 % foetal calf serum and 1 u M captopril resulted in protection o f C F U - G M f rom the S- phase- specif ic toxin hydroxyurea. The authors rationalised, that due to random distribution of cells over the cel l cyc le , an incubation period of 8 h was necessary to realise optima] inhibitory effect on the C F U - G M progenitors; this in turn necessitated inc lus ion of serum in the medium and the addition of 1 u M captopri l . G r i l l on et al. showed that the bioact iv ity of N - A c S D K P is s ignif icantly reduced by serum proteases, whose proteolytic activities could be abrogated by the addit ion of captopri l , a metalloproteinase i n h i b i t o r . 3 9 8 Since we were able to achieve cycle inhibi t ion with an incubation per iod of 1.5 h, we d id not include serum or captopril in the tissue culture med ium. In addit ion, we observed no adverse effects on C F U - G M numbers result ing f rom our incubatory condit ion up to 5 h. Other groups could not demonstrate N - A c S D K P - mediated inhibit ion o f the proliferative activity of murine C F U - G M in vitro.3S0>399 W e , however, bel ieve that the discrepancies 160 cou ld be due to the different experimental condit ions as we l l as the source of bone marrow cel ls . The observed photoprotection of D B A / 2 C F U - G M progenitors cou ld therefore be a result of N - A c S D K P - mediated ce l l cyc le inhibi t ion. W e showed data supporting this hypothesis by depleting the S- phase fraction f rom harvested bone marrow cel ls wi th 50 u M o f ara- C fo l lowed by P D T . Overa l l , the absolute numbers o f day 7 C F U - G M in the group that was pretreated with ara- C were approximately 50 % of the control group; nevertheless, the relative percent survival in response to different doses o f B P D were signif icantly higher in the experimental group pretreated with ara- C. Ce l l s in S- phase were susceptible to ara- C cytotoxici ty wh ich resulted in the enrichment of non S- phase cel ls in the survival fraction and the C F U - G M progenitors in ara- C- enriched fraction were found to have higher tolerance to B P D - mediated P D T cytotoxici ty (figure 6.4). Studies have shown that cel ls in S and G 2 / M phases are more susceptible to P D T than cells that are in the quiescent G</ G , phase . 3 4 3 - 4 0 ° - 4 0 1 Furthermore, Gantchev and col leagues have documented that P D T cytotoxic i ty f rom low dose Photofrin® and l ight exposure resulted in preferential damage to cells in S- phase; whereas high photosensitiser load resulted in general cytotoxicity without cel l cyc le se lec t i v i t y . 4 0 2 Oboch i et al. recently demonstrated that activated murine splenic lymphocytes take up more B P D than resting lymphocytes, wh ich subsequently translated to increased susceptibi l i ty to P D T . 2 8 5 In addit ion, previous studies in this laboratory have shown that pr imary leukaemic cells and leukaemic ce l l l ines take up more B P D than normal haematopoietic c e l l s . 2 9 3 Increased uptake of photosensitiser by proliferating cel ls cou ld be related to ce l l volume and the surface expression of l ow density l ipoprote in 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 of B P D by .DBA/2 bone marrow-.cells preincubatediwith 100 n M N - A c S D K P using two colour fluorescent activated ce l l sorting ( F A C S ) analysis. A rat monoclonal antibody, R A M 3 4 , was used to label cel ls wh ich express the murine equivalence of the human haematopoietic stem and progenitor cel l antigen C D 3 4 . 4 0 4 M o r e l et al. isolated C D 3 4 - expressing murine haematopoietic cells f rom femoral bone marrow label led with R A M 3 4 wh ich resulted in signif icant enrichments of various types of pr imit ive haematopoietic c e l l s . 3 9 3 In vitro incubation of D B A / 2 bone marrow cells with 100 n M N - A c S D K P d id not alter the proport ion of 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 med ium control) . Th is was expected since the known 161 characteristics of N - A c S D K P do not include the alterations of the percentage of C D 3 4 + cells nor the surface expression of the C D 3 4 glycoprotein. In addit ion, it is doubtful whether the short incubatory per iod of 1.5 h has any effects on these parameters. Unfortunately , we also showed no significant difference in the level of B P D mean fluorescence intensity (MF I ) in 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 control cel ls (4.84 + 0.18). The absence of demonstrable difference in B P D uptake between the two groups, however, does not d imin i sh the s ignif icance o f our earlier f indings. W e have shown in chapter 4 that uptake of B P D is rapid and reaches an equ i l ibr ium qu ick l y ; therefore, it is not surprising that B P D uptake or association was s imi lar between the two groups. In the experiments described by Oboch i and colleagues, murine splenocytes were incubated with 2.5 pg/ml of concanaval in A (Con A ) for 72 h wh ich resulted in blast cel l formation and increased expressions of the activation markers IL- 2 R (5- fo ld x control) and C D 7 1 (2.67- fo ld x con t ro l ) . 2 8 5 Bohmer and colleagues demonstrated a direct correlation between ce l l size and photosensitiser u p t a k e . 2 5 9 C o n A was used to effect max imal stimulation and to highlight the difference between resting and activated lymphocytes in terms of B P D uptake and susceptibil ity to B P D - mediated P D T . In our system, B P D uptake or association was evaluated between two populations of C F U - G M progenitors wh ich were much closer in their b iophysical and b iochemica l characteristics. Furthermore, Hunt et al. have shown discordance between B P D uptake and P D T susceptibil ity in purif ied D B A / 2 peritoneal macrophages subjected to different forms of cel l a c t i va t i on . 4 0 5 Spec i f ica l ly , they reported the highest B P D uptake in cel ls stimulated wi th 10 ug/ml l ipopolysaccharide ( LPS ) ; interestingly, cel ls activated wi th interferon- y ( IFN- y, . 100 U/ml for 72 h) or med ium control took up equivalent levels of the photosensitiser. 'Susceptibi l i ty to P D T , however, was highest in the I FN- y stimulated cel ls fo l lowed by resting cel ls (medium control) and lastly, L PS- stimulated cel ls. The authors postulated that the dichotomy between uptake and cytotoxicity cou ld be due to a multitude of factors inc luding quenching of reactive oxygen species, enhancement of P D T cytotoxici ty f rom nitric oxide generated v ia IFN- y stimulation, and distinct cel lular targets in the differential ly stimulated cel ls. O f the last point, F A C S analysis does not dist inguish between cel ls wi th different intracellular localisations of photosensitiser and therefore cells with s imi lar B P D M F I s cou ld have different intracellular distributions of the photosensitiser. The group o f W i l son et al. at the Ontario Cancer Institute noticed similar total cel lular uptake of 162 Photofr in by the PDT- resistant cel l l ine RTF- 8 A and the parental l ine REF- 1; they, however, found significant reduction in the mitochondrial accumulat ion of the photosensitiser in the P D T resistant cel l l i n e . 4 0 6 ' 4 0 7 W e believe that the unique intracellular mi l ieu during the S- phase of the ce l l cyc le presents novel targets for P D T . The expressions of S- phase specif ic protein products such as cyc l in D and the S- phase specif ic assembly of the D N A repl icat ion complexes are such e x a m p l e s . 4 0 8 ' 4 0 9 B e rg and M o a n , in their recent review article, postulated that microtubules are cycle- specif ic targets of P D T cytotoxic i ty 4 1 0 Another possible mechanism responsible for N - A c S D K P - mediated photoprotection is through the modulat ion of the cel lular thiol poo l , wh ich could effect defence against oxidative damage. W e could not detect any difference in the intracellular concentrations of glutathione (GSH ) between N - A c S D K P - treated cells and control cel ls using the Tietze method. Another assay technique using either the G S H - specif ic f luorescent probes monochlorobimane or monobromobimane in conjunction with F A C S analysis cou ld provide a more sensitive method of single ce l l measurement of G S H . 4 1 1 N - A c S D K P - mediated protection of D B A / 2 bone marrow progenitor cells f rom P D T cytotoxicity is correlated with the abil ity of the peptide to prevent cel l cycle progression. A r t i f i c i a l depletion of S- phase cells with ara- C resulted in an enrichment of cells wh ich are signif icantly more resistant to P D T than their prol iferating counterpart. Attempts to elucidate the mechanisms responsible for N - A c S D K P - mediated photoprotection included the examination B P D uptake and G S H content in cells after peptide incubation. However , neither of the two parameters showed signif icant difference between the N - A c S D K P and the control group. W e believe that other ce l l cycle- specif ic factors such as the expression of novel proteins and assembly of phase- specif ic protein complexes and therefore novel targets may contribute to the observed heightened P D T cytotoxicity in proliferating cells. 163 C H A P T E R 7: D I S C U S S I O N Purging of autologous haematopoietic stem cel l harvests with light- activated drugs is gaining increasing exposure in the c l in ica l setting as a result of the explosive growth o f the f ie ld of photodynamic therapy (PDT) and the need for new and effective purging modalit ies. Purging is sometimes necessary in autologous haematopoietic stem cel l transplantations because of the probable presence o f contaminating neoplastic cells in the harvest, wh ich can contribute to posttransplantation re l apses . 1 5 4 Deve lopment of the second generation photosensitiser benzoporphyr in derivative monoac id ring- A ( BPD ) , which has many biophysical and biochemical characteristics conducive to effective P D T , has signif icantly improved the eff icacy as we l l as broadened the indications of this treatment m o d a l i t y . 2 6 9 ' 2 8 4 In addit ion, B P D - mediated P D T has been shown to effect significant yet selective destructions of murine and human leukaemic cells in vitro and in an ex vivo mur ine bone marrow purg ing m o d e l . 2 9 1 ' 2 9 2 > 2 9 4 In this thesis, augmentation of B P D - mediated P D T cytotoxici ty was described using two independent approaches: combinat ion of P D T wi th doxorubic in (Dox) and selective preprotection of normal haematopoietic cel ls with the stem cel l inhibitory peptide N - A c S D K P . The f indings f rom the above experiments w i l l help to advance our understanding of the apparent selective cytotoxic i ty of B P D has on leukaemic cells. In addit ion, factors wh ich could influence in vitro P D T cytotoxic i ty w i l l be discussed. PDT COMBINATION THERAPY P D T combination therapy attempts to optimise photocytotoxicity through the interaction of the light- activated photosensitiser w i th another drug or treatment modal ity such as hyperthermia or ionis ing radiation. However , one cannot discount the possibi l i ty that P D T also can endow the other partner of the regimen with enhanced cytotoxic activity. Clear ly , combinat ion therapy requires mutual cooperativity between the different participants within the regimen and the effectiveness of a particular regimen is predicated on mult iple factors. Ch ie f among them are the type o f photosensitiser or drug and the fo rm o f 164 treatment modal i ty selected, the temporal order of drug administration i.e. simultaneous or sequenced, and the type of model system and tumour (in vitro vs in vivo, mur ine vs human) used in the experiment. Adequate del ivery of the chemotherapeutic agent or photosensitiser to the desired target organ remains one o f the chief obstacle in in vivo drug therapy. Parenteral and nonparenteral administration present two different routes of drug del ivery; in addit ion, binding to various serum protein components and hepatic metabol ism further complicate the delivery of drug in an organism. A s mentioned in a previous chapter, bone marrow purging, being an ex vivo (or in vitro) procedure, bypasses many o f the constraints encountered in vivo. In addit ion, ex vivo P D T eliminates the problems of ensuring adequate l ight del ivery to the appropriate organ. In studies performed for this thesis, the combinat ion invo lv ing 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 PDT-> D o x and simultaneous D o x / P D T treatment were not as eff icacious as the first regimen (chapter 3). The enhancement in cytotoxic i ty was dependent on the concentration of B P D ; specif ical ly , enhanced k i l l i ng of L 1 2 1 0 cells was achieved with 5.0 but not 2.5 ng/ml B P D . Add i t iona l l y , even though Dox-> P D T also effected heightened cytotoxicity against D B A / 2 haematopoietic progenitors, L l 210 cells were found to be much more susceptible to the Dox-> P D T sequence than normal haematopoietic cells. Subsequent investigations revealed that the photophysical properties of D o x and B P D are unique such that D o x d id not interfere with the photoactivation of B P D at X e x above 600 nm (chapter 4). The presence o f D o x appeared to reduce the uptake o f B P D whereas preincubation of L 1 2 1 0 cells with D o x d id not affect the subsequent uptake of B P D (Dox/PDT) . In addit ion, preincubation wi th 2.5 u M or 5 u M D o x d id not signif icantly reduCe cel lular glutathione (GSH) . The factors behind the superiority of the Dox-> P D T regimen are still not clear and mult iple mechanisms are responsible. N-ACSDKP- MEDIATED PHOTOPROTECTION FROM BPD Haematopoietic stem cel l protection attempts to selectively protect normal b lood cel l progenitors and precursors f rom treatment- related cytotoxic i ty . M a n y of mediators of 165 protection are the negative regulators of haematopoiesis wh i ch effect negative inhibit ion on the growth of haematopoietic c e l l s . 2 1 0 - 3 6 8 Fo r some of the negative mediators, speci f ica l ly the peptides N - A c S D K P and p E E D C K , the protective mechanism appeared to rely on the selective inhibit ion of cel l cyc l ing in normal haematopoietic cells wh ich therefore results in the sparing of above population f rom cycle- specif ic toxic i ty of t reatment . 3 6 7 Coutton and colleagues later demonstrated that N - A c S D K P also effected selective protection of normal human C F U - G M progenitors f rom Photofrin® - mediated P D T . 2 0 9 In chapter 4, preincubation of D B A / 2 bone marrow cel ls with 100 n M of the tetrapeptide N - A c S D K P resulted in statistically significant photoprotection f rom B P D -mediated P D T . The control peptides N - A c S D K E and S D K P , as we l l as tissue culture med ium control , d id not alter sensitivity to P D T . The protective effect, however, was restricted to the late committed progenitor populat ion wh ich was measured by the C F U -G M assay. Ce l l s o f earlier lineages, as measured by the L T B M C assay, were not protected by N - A c S D K P . The L I 2 1 0 murine leukaemic ce l l l ine was not responsive to N - A c S D K P as we l l as the two control peptides and therefore was not protected f rom P D T . F indings f rom the murine experiments were extended into human cel ls. Spec i f ica l ly , normal human late haematopoietic progenitors were photoprotected by preincubation with N - A c S D K P whereas the leukaemic cel l l ine K 5 6 2 and primary leukaemic cells f rom C M L patients were not protected by N - A c S D K P preincubation. T o further examine the mechanisms responsible, I measured B P D uptake (or association) in a subpopulat ion of D B A / 2 bone marrow cells responsible for short- term as we l l as long- term haematopoiesis. Interestingly, B P D uptake was not statistically different between C D 3 4 - expressing cells f rom the N - A c S D K P preincubated and the control sample. In addit ion, no signif icant difference in cel lular G S H between peptide- treated and control cel ls was noted. Us ing the traditional ara- C suicide assay, I showed that preincubation of D B A / 2 cells with 100 n M N - A c S D K P for 1.5 h d id prevent the progression o f ce l l cyc le into S- phase. Therefore, the observed photoprotective effect can be attributed to cel l cyc le inhibi t ion. I was able to prove this by depleting the harvested D B A / 2 bone marrow sample of S- phase cells with 50 p M ara- C fo l lowed by P D T . S- phase depletion resulted in cel ls with higher survival to P D T which showed that susceptibil ity to P D T was related to ce l l cyc le and N - A c S D K P effected its photoprotective effect v ia the inhibit ion o f cel l cyc l ing . However , the exact downstream 166 mechanisms are sti l l not clear sti l l we were not able to show significant difference in the uptake of B P D and in the amount of the cel lular antioxidant G S H . FACTORS AFFECTING PDT CYTOTOXICITY Thiols, a - tocopherol, and haeme oxygenase Cel lu lar G S H is correlated with susceptibil ity to a variety of chemotherapeutic agents, possibly v ia detoxif icat ion o f reactive oxygen species (ROIs) and l i p id p e r o x i d e s . 2 4 5 - 4 1 2 - 4 1 3 In vitro and in vivo P D T cytotoxic i ty also appeared to be modulated by the amount of 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 Another important cel lular antioxidant is a- tocopherol, a form of v i tamin E. We l l s and colleagues have demonstrated a correlation between a- tocopherol level and resistance to D o x 4 1 4 G o m e r and colleagues init ia l ly described the induction of haeme oxygenase 1 (HO I ) transcription after Photofrin® - mediated P D T 4 1 5 L i n etal recently described the induction of H O I expression and P D T resistance (MC540 ) after prolonged incubation of L 1 2 1 0 cells with haemin (ferriprotoporphyrin IX ) ; the role of H O I in the induct ion of P D T resistance is not clear, h o w e v e r . 4 1 6 N F - K B and its role in P D T cytotoxicity A variety of s t imul i , inc luding cyc lohex imide , double- stranded R N A , ca l c ium ionophore, T N F - a , active phorbol ester, interleukin- 1, l ipopolysacchar ide and lect in, can mediate the induction of the transcriptional factor NF- kappa B (NF- K p ) 4 1 7 In addit ion, Bo l and et al. showed the anthracycline daunorubic in also activates NF- K p in the H L - 60 167 promyelocyt ic and Jurkat T l ymphoma cel l l i n e s . 4 1 8 Ac t i va t ion of N F - K p involves separation f rom its cytoplasmic b ind ing partner, IKP , fo l l owed by nuclear translocation the induct ion of KP- dependent gene expression 4 1 9 Ryter and G o m e r in i t ia l ly reported that Photofrin- mediated P D T causes NF- K P activation in the L l 2 1 0 murine leukaemic ce l l l i n e . 2 6 4 . The i r observation was not surpris ing in l ight of f indings that reactive oxygen species (ROIs) are essential as s ignal l ing intermediates in the activation of NF- K p by a variety of s t i m u l i . 4 2 0 " 4 2 2 Schreck and colleagues also demonstrated that the radical scavengers N-Acetyl- cysteine ( N A C ) and the pyrrol idone derivative of dithiocarbamate ( P D T C ) effectively inhibited N F - K p activation upon cel lular st imulat ion. RO Is are also important intermediates mediat ing the activation of NF- K P in response to stimulation of the T ce l l surface receptor C D 2 8 4 2 3 Through their studies on human B ce l l l ines, Schieven et al. have impl icated tyrosine kinase in the ROI- mediated activation of N F - K P 4 2 4 Recently, several reports have demonstrated that NF- K p plays an important role in determining cel lular response to a variety of cytotoxic agents. S igni f icant ly , W a n g and colleagues found that activation of the transcription factor was paramount in the cytoprotection of the human sarcoma ce l l l ine H T 1 0 8 0 f rom T N F - a , ion is ing radiation, and the dauno rub i c i n . 3 2 7 Paradoxica l ly , several papers have reported conf l i c t ing roles o f NF- K P in in vitro exci totoxic neurodest ruc t ion . 3 2 8 W i t h respect to the role o f N F - K p in determining cel lular response to P D T , Anderson et al. init ia l ly reported that the presence of 0.1-10 m M sal icycl ic acid (SA) during M C 5 4 0 - mediated P D T signif icantly enhanced cytotoxici ty to L 1 2 1 0 and K 5 6 2 ce l l lines 4 2 5 In addit ion, S A and sod ium salicyclate ( A S A or aspirin) are known inhibitors of N F - K P a c t i v a t i o n . 4 2 6 - 4 2 7 Therefore, one can assume that the modulat ion of NF- K P activation can also alter cel lular response to P D T . Unfortunately, TrauJ et al. later showed that S A induced nonselective enhancement of P D T k i l l i ng of L 1 2 1 0 cells and normal haematopoietic stem cells but more importantly, they found that S A displaces M C 5 4 0 f rom serum albumin wh ich resulted in the increase of free photosensitiser available for b i n d i n g . 4 2 8 However , the question of the role that N F - K P plays in P D T remain opened. W e used the proteasome inhibitor M G 1 3 2 (carbobenzoxyl-leucinyl- leucinyl- leucinal) , wh ich inhibits the proteasome degradation of IKP and hence nuclear translocation of NF- K P . 3 2 7 P re l iminary results showed that 1 h preincubation of Jurkat cells with 40 u M M G 1 3 2 resulted in moderate yet consistent enhancement of B P D -mediated P D T cytotoxicity (data not shown). I found that the same experimental protocol 168 also potentiated doxorubic in k i l l i ng in Jurkat cel ls. Interestingly, M G 1 3 2 preincubation protected the human C M L cel l l ine K 5 6 2 f rom B P D - mediated P D T . I am in the process of examining the amount of IK(3 in cel l lysates f rom the above two ce l l l ines and w i l l continue to investigate the role of NF- K(3 in PDT- mediated cel l k i l l i ng . I also tried to introduce M G 1 3 2 into the P D T combinat ion experiments described in chapter 3. S ince W a n g et al. reported that M G 1 3 2 protected the ce l l l ine H T 1 0 8 0 f rom daunorubicin cytotoxici ty, I was interested in whether the peptide aldehyde could affect the k i l l i ng of L 1 2 1 0 cel ls in the Dox-> P D T c o m b i n a t i o n . 3 2 7 Ob l ique ly , I was curious o f the role of N F - K(3 activation during the D o x pretreatment phase of Dox-> P D T , wh ich resulted in significant enhancement of cytotoxicity of the regimen. Unfortunately, the L I 210 ce l l l ine was found to be extremely sensitive to M G 1 3 2 and prevented the incorporation of the inhibitor into the exist ing P D T experiments in L I 2 1 0 . S u m m a r y A s long as there is a need for the therapeutic transfusions of autologous haematopoietic stem cells in the oncologic setting, there is a need for purging. The overal l eff ic iency of the different established purging modalit ies is s imi lar to each other. Therefore, enhancements to a proven modal i ty , such as B P D - mediated P D T purging, should increase its attractiveness to the c l in ic ian. O n the other hand, new and exci t ing technologies are emerging and may offer a quantum leap in purg ing p e r f o r m a n c e . 2 1 3 ' 4 2 9 The underly ing theme of this project is the improvement of P D T cytotoxicity v i a two independent approaches. D o x treatment pr ior to P D T (Dox-> P D T ) resulted in a significant improvement in the k i l l ing of the leukaemic cel l l ine L I 210 but not of D B A / 2 haematopoietic progenitor cells. The reverse sequence (PDT-> D o x ) and simultaneous treatment (Dox/PDT) only effected moderate improvement in k i l l i ng . Therefore, one must be cautious in combin ing P D T with other drugs or treatment modalit ies in the c l in ic . Speci f ica l ly , the sequence of the combinat ion cou ld signif icantly affect the outcome of the treatment. The second approach involved the selective protection of normal haematopoietic cells with N - A c S D K P prior to P D T . Mur i ne and human leukaemic cel ls d id not respond to 169 the tetrapeptide and therefore were not afforded subsequent photoprotection. The above f indings should also aid in the cont inuing understanding of the mechanisms of P D T cytotoxici ty. 170 R E F E R E N C E S 1. Freire ich EJ , L emak N A . 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