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Effects of three radiosensitizing drugs on radiation-induced DNA damage in hypoxic mammalian cells Hohman, William Frank 1975

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EFFECTS OF THREE RADIOSENSTT.IZING DRUGS ON RADIATION-INDUCED I N HYPOXIC  DNA  DAMAGE  MAMMALIAN CELLS by  WILLIAM FRANK HOHMAN B.Sc,  U n i v e r s i t y of Waterloo,  1973  A THESIS SUBMITTED I N P A R T I A L FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF  SCIENCE  i n <• THE FACULTY OF GRADUATE STUDIES (Department o f M e d i c a l G e n e t i c s )  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the required  standard  THE UNIVERSITY OF E R I T I S H COLUMBIA January,  197 5  OF  In p r e s e n t i n g  this  thesis  an advanced degree at the I  Library shall  f u r t h e r agree  for  scholarly  by h i s of  this  written  make it  a  t  that permission  for  the requirements  Columbia,  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  for  It  i s understood that copying or  financial  gain shall  not  /77<£-&/t:/9/L.  <^g^rr/<r s  Columbia  y 9 H > ^ J  X.T  /"?7C  for  that  study. thesis  purposes may be granted by the Head of my Department  University of B r i t i s h  e  of B r i t i s h  freely available  2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  D  fulfilment of  permission.  Department of The  the U n i v e r s i t y  representatives. thesis  in p a r t i a l  or  publication  be allowed without my  RESEARCH SUPERVISOR: P r o f e s s o r L. D. Skarsgard,  Ph.D.  ABSTRACT: The m o d i f i c a t i o n of DNA  damage by 3 r a d i o s e n s i t i z i n g  drugs, p r e s e n t d u r i n g y - i r r a d i a t i o n of hypoxic Chinese hamster c e l l s , was gradients of  DNA  investigated. (ASG's) was  molecules.  The method of a l k a l i n e sucrose  used t o determine  the s i z e  distribution  Both metronidazole and Ro-07-0582 were  found t o cause l a r g e i n c r e a s e s i n the y i e l d of DNA s t r a n d breaks  single  (SSB's); triacetoneamine-N-oxyl (TAN)  was  found  to have o n l y a s m a l l e f f e c t on SSB p r o d u c t i o n . A p u l s e l a b e l and chase procedure was p o s t - i r r a d i a t i o n DNA  synthesis.  i r r a d i a t i o n under hypoxia was subsequent  DNA  synthesis.  exposure  TAN p r e s e n t d u r i n g  found t o cause i n t e r r u p t i o n s i n  M e t r o n i d a z o l e and Ro-07-0582 had  no e f f e c t on p o s t - i r r a d i a t i o n DNA In  used to examine  synthesis. .  a d d i t i o n , the e f f e c t s of pre- and  post-irradiation  to TAN were i n v e s t i g a t e d , s i n c e these treatments  have shown i n c r e a s e d c e l l k i l l i n g and Skarsgard, 1972).  TAN  i n survival studies  pre- and post-treatments were  found t o have no s i g n i f i c a n t e f f e c t on subsequent synthesis.  (Agnew  DNA  iii  TABLE OF CONTENTS  Page 1.  2.  INTRODUCTION  1  1.1  Cancer and R a d i a t i o n Therapy  1  1.2  R a d i a t i o n Damage o f P a r e n t a l DNA  2  1.3  Repair o f I r r a d i a t e d P a r e n t a l DNA  4  1.4  DNA S y n t h e s i s  5  1.5  Chemical R a d i o s e n s i t i z e r s  7  MATERIALS AND METHODS  11  2.1  C e l l Manipulations  11  2.1.1  C e l l Line  11  2.1.2  Irradiation of C e l l s  12  2.1.3  Radioactive L a b e l l i n g  13  2.1.4  Repair  14  and Chase Incubations  2.2  R a d i o s e n s i t i z i n g Drugs  16  2.3  A l k a l i n e Sucrose Gradients  18  2.3.1  Gradient  18  2.3.2  L y s i n g Procedure  19  2.3.3  C e n t r i f ugation  19  2.3.4  G r a d i e n t F r a c t i o n a t i o n and Sample  and L y s i n g S o l u t i o n s  Counting  20  2.3.5  L y s i n g Time  21  2.3.6  L y s i n g Temperature  23  2.3.7  C a l c u l a t i o n of Molecular  Weight  23  iv  Page 3.  RESULTS  26  3.1  26  3.2  4.  P a r e n t a l DNA 3.1.1  Single Strand  3.1.2  Rejoining of S i n g l e Strand  Newly S y n t h e s i z e d  Break P r o d u c t i o n  26  Breaks  . . . .  D a u g h t e r DNA  36  3.2.1  TAN  Treatment o f H y p o x i c CH2B  3.2.2  TAN  Pretreatment  43  3.2.3  TAN  Post-treatment  45  3.2.4  TAN  Treatment of A e r o b i c  3.2.5  Dose R e s p o n s e  52  3.2.6  TAN  55  3.2.7  Metronidazole Hypoxic C e l l s  CH2B  2  2  Cells  Cells  . . .  . . .  Concentration and  Ro-07-0582 T r e a t m e n t  36  49  of 57  DISCUSSION 4.1  32  61  Reliability Technique  of the A l k a l i n e  Sucrose  Gradient 61  4.2  Effects  o f H i g h Doses  4.3  E f f e c t s of I r r a d i a t i o n Template F u n c t i o n  62 and  Aerobic  Drug Treatment  on 64  4.3.1  H y p o x i c and  Irradiation  4.3.2  TAN  4.3.3  N i t r o i m i d a z o l e Treatment  Treatment  65 67 71  4.4  TAN  Pretreatment  73  4.5  TAN  Post-treatment  74  V  Page 5.  CONCLUSIONS  APPENDIX A APPENDIX B  BIBLIOGRAPHY  76  77 . . .  79  80  vi  L I S T OF TABLES  Table I  page 30  Table I I  page 71  vii  LIST OF FIGURES  Figure  Title  Page  1  Proposed Mechanisms o f R a d i o s e n s i t i z a t i o n and Radioprotection  2  C o n t i n u o u s l y L a b e l l e d and P u l s e L a b e l l e d DNA  15  3  Flow Chart R e p r e s e n t a t i o n of the V a r i o u s Experimental Procedures  17  4  E f f e c t of L y s i n g Time on Sedimentation  22  5  E f f e c t o f L y s i n g Temperature on Sedimentation Profile  24  6  P r o d u c t i o n of S i n g l e Strand Breaks under A e r o b i c and Hypoxic C o n d i t i o n s  28  7  P r o d u c t i o n of S i n g l e Strand Breaks under Hypoxia w i t h TAN or Ro-07-0582  29  8  Graph o f 1/Mw versus Dose f o r S i n g l e S t r a n d Break P r o d u c t i o n under S e v e r a l C o n d i t i o n s  31  9  R e j o i n i n g of S i n g l e Strand Breaks a f t e r Hypoxic I r r a d i a t i o n w i t h and without TAN  34  10  R e j o i n i n g of S i n g l e Strand Breaks a f t e r Hypoxic I r r a d i a t i o n w i t h and without Ro-07-0582  35  11  Sedimentation P r o f i l e s f o r Newly S y n t h e s i z e d DNA a f t e r I r r a d i a t i o n under Hypoxia w i t h and without TAN  38  12  E f f e c t of TAN Treatment d u r i n g Hypoxic I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  40  13  Sedimentation P r o f i l e s f o r P a r e n t a l and Newly S y n t h e s i z e d Daughter DNA a f t e r I r r a d i a t i o n under Hypoxia w i t h and without TAN  42  14  E f f e c t of TAN Pretreatment DNA S y n t h e s i s  44  Profile  on P o s t - I r r a d i a t i o n  9  viii  Figure  Title  Page  15  S e d i m e n t a t i o n P r o f i l e s f o r DNA L a b e l l e d w i t h and w i t h o u t TAN  16  E f f e c t o f TAN P o s t - T r e a t m e n t on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  48  17  E f f e c t o f TAN T r e a t m e n t d u r i n g A e r o b i c I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  50  18  P o s t - I r r a d i a t i o n DNA S y n t h e s i s as a F u n c t i o n o f Dose u n d e r H y p o x i a w i t h and w i t h o u t TAN  53  19  Dose R e s p o n s e o f N e w l y S y n t h e s i z e d P l o t t e d 1/Mw v e r s u s Dose  as  54  20  E f f e c t o f TAN C o n c e n t r a t i o n d u r i n g H y p o x i c I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  56  21  E f f e c t of M e t r o n i d a z o l e Treatment d u r i n g H y p o x i c I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA Synthesis  58  22  E f f e c t o f Ro-07-0582 T r e a t m e n t d u r i n g H y p o x i c I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  59  23  S c h e m a t i c D i a g r a m o f P r o p o s e d TAN I n t e r r u p t i o n s i n Newly S y n t h e s i z e d DNA  69  r  Continuously  Daughter  46  ix  ACKNOWLE DGEMENT  I wish t o express my g r a t i t u d e t o Dr. L. D. Skarsgard, under whose s u p e r v i s i o n t h i s p r o j e c t was c a r r i e d o u t .  1  1.  1.1  INTRODUCTION  CANCER AND RADIATION THERAPY  R a d i a t i o n therapy i s the treatment o f c h o i c e f o r many human tumours. effectiveness  For some types o f cancer, however, the o f t h i s mode of therapy i s thought t o be l i m i t e d  by the r e l a t i v e r a d i o r e s i s t a n c e  o f the hypoxic f r a c t i o n o f a  tumour growth.  c e l l s have a reduced oxygen  tension that  a r i s e s as the tumour outgrows i t s v a s c u l a r  I t i s known t h a t the  These r e s i s t a n t  such hypoxic c e l l s are much more r e s i s t a n t t o  l e t h a l e f f e c t s of i o n i z i n g r a d i a t i o n  oxygenated c e l l s found i n normal t i s s u e . delivered  r e l a t i v e radioresistance  any procedure t h a t  reduces t h i s  should i n c r e a s e the e f f e c t i v e n e s s  solution  chemical r a d i o s e n s i t i z e r s  of  this selective  t o t h i s problem i s the use o f  which s p e c i f i c a l l y s e n s i t i z e  c e l l s to i o n i z i n g r a d i a t i o n .  systems.  Since the dose  therapy.  One p o s s i b l e  exhibit  than the w e l l  t o a tumour volume i s l i m i t e d by the t o l e r a n c e o f  adjacent h e a l t h y t i s s u e s ,  radiation  supply.  Many compounds have been shown t o  sensitization, largely i n i n vitro  I t i s hoped t h a t  the study o f the e f f e c t s o f these  s e n s i t i z e r s a t the molecular l e v e l w i l l f a c i l i t a t e application  hypoxic  t o human r a d i o t h e r a p y .  their  2  1.2  RADIATION DAMAGE OF PARENTAL DNA  DNA  ( d e o x y r i b o n u c l e i c acid) i s u s u a l l y regarded  primary molecular  target f o r radiation-induced c e l l  s i n c e g e n e t i c i n t e g r i t y i s necessary and r e p r o d u c t i o n .  as the  death,  f o r normal c e l l u l a r  Presumably, other damaged c e l l u l a r  life  components  can be r e p l a c e d i f the r e q u i r e d g e n e t i c i n f o r m a t i o n i s present i n i n t a c t DNA  molecules.  Exposure t o r a d i a t i o n , both i o n i z i n g and n o n - i o n i z i n g , i s known t o damage DNA molecules. violet  C e l l u l a r absorption of u l t r a -  (UV) l i g h t , a n o n - i o n i z i n g r a d i a t i o n , leads mainly t o  the formation  o f p y r i m i d i n e dimers between adjacent  bases i n the same DNA s t r a n d .  pyrimidine  I o n i z i n g r a d i a t i o n , on the other  hand, produces a whole spectrum of DNA damage i n c l u d i n g both s i n g l e and double s t r a n d breaks, A s i n g l e s t r a n d break  and base and sugar damage.  (SSB) occurs when r a d i a t i o n  causes a s c i s s i o n o f only one s t r a n d o f the sugar-phosphate DNA backbone. many authors  The p r o d u c t i o n  o f SSB's has been i n v e s t i g a t e d by  i n organisms ranging  to mammalian c e l l s .  i n complexity  A l a r g e p o r t i o n o f these  employed the a l k a l i n e sucrose  gradient  i n t r o d u c e d by McGrath and W i l l i a m s  from v i r u s e s  s t u d i e s has  (ASG) technique  i n 1966.  This  was used i n t h i s work t o measure SSB p r o d u c t i o n  technique  i n Chinese  hamster c e l l s . I t should be noted t h a t the ASG assay used i n t h i s  3  work ( s e c t i o n 2.3)  cannot d i s t i n g u i s h between those bonds i n  the backbone which are broken d i r e c t l y and those which become a l k a l i l a b i l e as a r e s u l t of the r a d i a t i o n exposure.  Hence the  two types of bond r u p t u r e s are c o l l e c t i v e l y r e f e r r e d to as SSB's.  In a d d i t i o n , some of the SSB's measured w i l l be due  to  double s t r a n d breaks. Double s t r a n d breaks are produced damaged very c l o s e t o g e t h e r .  when both strands are  The appearance of double s t r a n d  breaks a f t e r i r r a d i a t i o n has a l s o been s t u d i e d i n v a r i o u s c e l l types w i t h s e v e r a l authors having r e p o r t e d y i e l d s of one s t r a n d break f o r every 10 t o 15 SSB's B u r r e l l , F e l d s c h r e i b e r and Dean, 1971; Little  (Freifelder,  1966;  Veatch and Okada, 1969).  i s known about the base and sugar damage caused  by i o n i z i n g r a d i a t i o n i n c e l l u l a r systems. R o t i R o t i and C e r u t t i produce  double  However, r e c e n t l y  (1974) have r e p o r t e d t h a t gamma-rays  thymine damage i n Chinese hamster c e l l s w i t h an  e f f i c i e n c y s i m i l a r to t h a t f o r SSB's.  In a d d i t i o n ,  of i r r a d i a t e d E. C o l i w i t h an endomiclease  treatment  has been shown t o  cause a l a r g e i n c r e a s e i n the number of s t r a n d breaks, beyond those caused by the r a d i a t i o n alone  (Setlow and C a r r i e r ,  1973).  Presumably, these e n d o n u c l e a s e - s e n s i t i v e s i t e s r e f l e c t base damage i n the i r r a d i a t e d  DNA.  4  1.3  REPAIR OF  IRRADIATED PARENTAL  DNA  Mammalian c e l l s have evolved complex enzymatic r e p a i r systems t o cope w i t h damage to t h e i r DNA. r a d i a t i o n , r e p a i r processes t h a t may  A f t e r exposure t o  occur i n c l u d e  of p y r i m i d i n e dimers or o t h e r damaged bases, and  the  the  excision  rejoining  of SSB's. Perhaps the most e x t e n s i v e l y  studied  enzymatic r e p a i r  system i s the removal of thymine dimers f o l l o w i n g irradiation.  T h i s e x c i s i o n r e p a i r system f i r s t removes a  segment c o n t a i n i n g using  the dimer and  the complementary s t r a n d  Rauth, 1970).  The  unscheduled DNA cycle  nucleotides 2 to 10  (Fox The  extensively  and  can occur throughout the  cell  a l s o been observed i n mammalian  Wheatley, 1971).  Fox,  SSB  (Painter and  Generally,  studied  produced i s s m a l l , of the order of  1973a; P a i n t e r  and  Young, 1972).  i n many l a b o r a t o r i e s .  throughout the c e l l c y c l e and  hours  Okada, 1970;  1973c).  Fox,  has  been  T h i s p r o c e s s occurs  appears t o be  l a r g e l y complete i n a few and  Cleaver,  the number of  r e j o i n i n g of SSB's i n mammalian DNA  Fox  see  C l e a v e r , 1969).  i n s e r t e d per and  region  ( f o r a review  c e l l s a f t e r exposure to i o n i z i n g r a d i a t i o n Brent and  the  small  process i s a l s o c a l l e d r e p a i r r e p l i c a t i o n or  synthesis  (Painter and  then r e s y n t h e s i z e s  as template  Repair r e p l i c a t i o n has  1967;  UV  fairly  ( L e t t e t a l . , 1967; Rejoining  r a p i d , being Sawada  i s temperature  and  5  dependent, being completely  inhibited  1970;  1972b).  P a l c i c and  Skarsgard,  a t 0°C  (Sawada and  Okada,  Mammalian c e l l s have been r e p o r t e d to be incapable repairing  double s t r a n d breaks  and Omerod, 1970)  although  (Sawada and Okada, 1970;  Lehmann  double s t r a n d break r e j o i n i n g  been r e p o r t e d i n Chinese hamster c e l l s  (Corry and  of  has  Cole, 197 3).  While t h i s disagreement i s not y e t r e s o l v e d , the double s t r a n d break has been proposed as the l e t h a l molecular ionizing radiation  1.4  DNA  event f o r  (Chadwick and Leenhouts, 1973).  SYNTHESIS  Normal semi-conservative the S phase of the c e l l c y c l e .  DNA  s y n t h e s i s occurs  In mammalian c e l l s ,  r e p l i c a t i o n appears to i n v o l v e the i n i t i a t i o n and s m a l l fragments, known as r e p l i c o n s , p o i n t s on the template DNA. subsequently  Schandl and  Rutman, 1973;  T a y l o r , 1969).  ionizing radiation  DNA  s y n t h e s i s of starting  These small fragments are  j o i n e d to form l a r g e r  ( G o l d s t e i n and  a t numerous  during  i n t a c t DNA  T a y l o r , 1973;  molecules  Ioannu,  1973;  Exposure to both i o n i z i n g and  i s known to a f f e c t DNA  non-  replication in  mammalian c e l l s . Mammalian DNA  s y n t h e s i z e d w i t h i n 1 or 2 hours a f t e r  i r r a d i a t i o n i s made i n s m a l l e r segments than t h a t  UV  synthesized  6  by u n i r r a d i a t e d c e l l s , dimers i n t e r r u p t DNA synthesized  strands.  are elongated and (Cleaver 1972).  and  suggesting t h a t unexcised  synthesis,  Upon i n c u b a t i o n ,  these s h o r t  Thomas, 1969;  DNA  Buhl e t a l . , 1972;  Chija and  DNA Rauth,  irradiation,  human c e l l s r e g a i n the a b i l i t y  i n n e a r l y the same s i z e as u n i r r a d i a t e d  (Buhl, Setlow and Kirk-Bell,  segments  j o i n e d to form high molecular weight  Chinese hamster, mouse and  and  l e a v i n g gaps i n the newly  In a d d i t i o n , at long times a f t e r UV  synthesize  pyrimidine  Regan, 1973;  Meyn and  to  cells  Humphrey, 1971;  Lehmann  1972).  I o n i z i n g r a d i a t i o n s are known to depress the r a t e of i n c o r p o r a t i o n of p r e c u r s o r s incorporation nuclear  DNA  i s generally  synthesis  i n t o mammalian DNA;  t h i s decreased  i n t e r p r e t e d as a lower r a t e  (Makino and  response of r a d i a t i o n - i n d u c e d  Okada, 1975).  depression  b i p h a s i c w i t h a r a d i o s e n s i t i v e component much more r e s i s t a n t component  The  of DNA (D  of s y n t h e s i s  3 7  - l krad) and  (D -20 k r a d s ) .  Makino and  37  i n r e p l i c o n s and  r e s i s t a n t process i s the e l o n g a t i o n synthesis  has  I t has hamster c e l l s  already  been  the  is a Okada  represents  t h a t the  radio-  i n r e p l i c o n s i n which  initiated.  a l s o been shown t h a t DNA  synthesized  i n Chinese  a f t e r X - i r r a d i a t i o n i s of lower molecular weight  than i n u n i r r a d i a t e d c e l l s . increases  dose  synthesis  have suggested t h a t the r a d i o s e n s i t i v e component the i n i t i a t i o n  of  P o s t - i r r a d i a t i o n incubation  s i z e of the newly s y n t h e s i z e d  DNA  to c o n t r o l  7  levels the  (Korner and Malz, 1973).  In the work r e p o r t e d here,  a b i l i t y o f Chinese hamster c e l l s t o s y n t h e s i z e c o n t r o l  DNA a t v a r i o u s times a f t e r gamma-irradiation under  size  different  c o n d i t i o n s was examined.  1.5  CHEMICAL RADIOSENSITIZERS  Many drugs are known t o modify the r a d i a t i o n of b a c t e r i a l  and mammalian c e l l s .  Of p a r t i c u l a r  response  relevance to  cancer therapy are those compounds which s e l e c t i v e l y  sensitize  hypoxic mammalian c e l l s t o the k i l l i n g e f f e c t s o f i o n i z i n g radiation.  The f i r s t r e p o r t s o f t h i s s e l e c t i v e  were p u b l i s h e d by Parker, Skarsgard and Emmerson and  Ashwood-Smith e t a l .  sensitization (1966, 1969)  (1967).  In 196 3, Adams and Dewey suggested t h a t the e l e c t r o n a f f i n i t y o f a compound was d i r e c t l y r e l a t e d ness as a s e n s i t i z e r ; be  this electron  a f f i n i c s e n s i t i z a t i o n can  e x p l a i n e d as f i x a t i o n or o x i d a t i o n o f f r e e r a d i c a l s ,  either  produced  d i r e c t l y i n the t a r g e t molecule, or i n d i r e c t l y by the  a t t a c k on the t a r g e t molecule of r e a c t i v e the  to i t s e f f e c t i v e -  ionizing  s p e c i e s produced by  radiation.  Recently, Chapman and co-workers have proposed a model which e x p l a i n s both r a d i o s e n s i t i z a t i o n interaction  and p r o t e c t i o n by  o f compounds w i t h f r e e r a d i c a l s produced i n DNA  8  (Chapman, Reuvers, Borsa and a generalization 1962)  and  model, DNA the  i s represented schematically  i n f i g . 1.  r a d i c a l s , produced e i t h e r by the  i o n i z i n g r a d i a t i o n or the and  f i x a t i o n processes.  H donation by  T h i s model i s  of the o x y g e n - f i x a t i o n h y p o t h e s i s  r a d i o l y s i s products OH" and  Greenstock, 1973).  d i r e c t e f f e c t of  are  subject  reduction  water  to competing  of DNA  radicals  s u l f h y d r y l compounds) leads to r e p a i r .  of these r a d i c a l s f i x e s the damage i n an a l t e r e d structure  In t h i s  i n d i r e c t e f f e c t of the  H*,  The  (Alexander,  which may  lead to c e l l death.  In the model, r a d i o p r o t e c t o r s  ( i e . by Oxidation  chemical  O x i d a t i o n may  e i t h e r by adduct formation w i t h , or e l e c t r o n t r a n s f e r chemical s p e c i e s w i t h h i g h e r e l e c t r o n  repair  occur to,  affinities. could  act e i t h e r  by  competing w i t h endogenous o x i d i z i n g compounds f o r t a r g e t r a d i c a l s and  so making r e p a i r by r e d u c t i o n  scavenging OH*  or H*  could  e i t h e r by b i n d i n g  function  radicals.  to endogenous r e d u c i n g  oxidation  by  species  free r a d i c a l s ,  the p r o b a b i l i t y of f i x a t i o n by o x i d a t i o n .  mechanism of most e l e c t r o n to i n v o l v e  more l i k e l y , or  Chemical r a d i o s e n s i t i z e r s  or by competing d i r e c t l y with them f o r DNA increasing  free  The  a f f i n i c r a d i o s e n s i t i z e r s i s thought  of t a r g e t  free r a d i c a l s .  S e v e r a l r a d i o a c t i v e l y l a b e l l e d s e n s i t i z e r s have been shown to b i n d t o i r r a d i a t e d DNA, compounds assayed i n c l u d e S i k k e l a n d and  forming s t a b l e  triacetoneamine-N-oxyl  Brustad, 1970), n i t r o f u r a z o n e  adducts; (TAN)  (Chapman,  (Nakken;  9  Direct E f f e c t  NAAA/V  Repair  by  Indirect  V  Reduction  ie. by H donation  Effect  W  W  Fixation by O x i d a t i o n ie. by adduct  formation,  electron transfer P  - Reducing  Species  S-Oxidizing  Species  F i g u r e 1.  Proposed Mechanisms o f R a d i o s e n s i t i z a t i o n and Radioprotection  (from Chapman ejt a l . , 1974; W i l l s o n and Emmerson, 1970)  10  Greenstock,  Reuvers,  metronidazole  McDonald and Dunlop, 1973), and  ( W i l l s o n , Cramp and Ings, 1974).  a c t i v i t y bound i n c r e a s e s l i n e a r l y w i t h dose.  The amount of B i n d i n g does not  occur i n the presence of oxygen, presumably due t o c o m p e t i t i o n between s e n s i t i z e r and 0 e f f e c t s of these l e s i o n s i r r a d i a t e d DNA  2  f o r the r a d i c a l s i t e . on the template  However, the  f u n c t i o n of the  are l a r g e l y unknown, although TAN  to i n t e r f e r e w i t h DNA  has been shown  synthesis i n i r r a d i a t e d bacteria  (Rupp  e t a l . , 1969). Our work has been concerned mainly w i t h the study of the s y n t h e s i s of mammalian DNA DNA  a f t e r i r r a d i a t i o n of p a r e n t a l  i n the presence of r a d i o s e n s i t i z i n g  drugs.  of t h i s r e s e a r c h has i n v o l v e d the use of TAN, shows both a p r e - and a p o s t - i r r a d i a t i o n (Agnew and Skarsgard, 1972). TAN  enhances r a d i a t i o n  adducts.  that  damage by somewhat d i f f e r e n t mechanisms by forming  stable  M e t r o n i d a z o l e and Ro-07-0582, on the  acting only i f present during i r r a d i a t i o n  sensitizers,  (Agnew, P a l c i c  Moore, P a l c i c and Skarsgard, 1975).  these three s e n s i t i z e r s , we  and  Using  c a r r i e d out a search f o r d r u g -  s p e c i f i c d i f f e r e n c e s i n the DNA under hypoxia.  effect  I t t h e r e f o r e seems l i k e l y  other hand, behave as t y p i c a l e l e c t r o n a f f i n i c  Skarsgard, 1974;  a drug which  sensitizing  than most e l e c t r o n a f f i n i c compounds, perhaps u n r e p a i r e d DNA  A large portion  synthesized a f t e r i r r a d i a t i o n  11  2.  2.1  2.1.1  MATERIALS AND  METHODS  CELL MANIPULATIONS  Cell  Line  Chinese hamster c e l l s of the CH2B  2  line, a clonal  d e r i v a t i v e of CHEF-125 ( P r e s c o t t and Bender,  1963), have been  used i n these experiments. The CH2B  2  c e l l s were grown a t t a c h e d t o p l a s t i c i n  tissue culture flasks  (Falcon P l a s t i c s , Oxnard,  minimum e s s e n t i a l medium (MEM) calf  F-16  California) in  supplemented  w i t h 10%  serum (Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d ,  York); they were i n c u b a t e d a t 37°C i n an atmosphere 5% carbon d i o x i d e and 100% humidity. 12-14  fetal New  of 95% a i r ,  T h e i r d o u b l i n g time  was  hours. The c e l l s were s u b c u l t u r e d every 3 t o 4 days u s i n g  0.1%  trypsin  (Bacto-Trypsin, D i f c o L a b o r a t o r i e s ,  Michigan) t o detach them from the p l a s t i c .  Detroit,  C e l l s were exposed  to t r y p s i n , p r e v i o u s l y warmed t o 37°C, f o r 8 minutes a t room temperature.  The a c t i o n of the t r y p s i n was  stopped by the  a d d i t i o n of an equal volume of growth medium. then c e n t r i f u g e d  The c e l l s were  (8 minutes a t 600 rpm), resuspended i n growth  medium and p l a t e d i n t o new  culture  flasks.  C e l l s t o be used f o r experiments were p l a t e d and grown  12  f o r 2 days b e f o r e h a r v e s t i n g by t r y p s i n i z a t i o n .  F r e s h growth  medium was added to the c e l l s 1 day p r i o r t o h a r v e s t i n g .  2.1.1  I r r a d i a t i o n of C e l l s C e l l suspensions  phase monolayers.  f o r experiments were prepared  The c e l l s were t r y p s i n i z e d ,  from l o g  c e n t r i f u g e d and  resuspended a t a c o n c e n t r a t i o n o f 1 x 1 0 c e l l s / m l 6  i n normal  growth medium from which the sodium b i c a r b o n a t e had been omitted. The  The pH was a d j u s t e d to 7.2 u s i n g 1 M sodium  cell  suspensions  were then p l a c e d i n s p e c i a l g l a s s  i r r a d i a t i o n v e s s e l s (Parker, Skarsgard s t i r r e d w i t h a magnetic  and Emmerson,  parts/million  0  2  1969)  and  stirrer.  Hypoxia was o b t a i n e d by f l o w i n g n i t r o g e n 10-15  hydroxide.  (less  than  p r e s e n t , Canada L i q u i d A i r , Vancouver,  B.C.) above the s t i r r e d c e l l  suspension  a t 0.7 l i t e r s / m i n u t e  f o r a t l e a s t 45 minutes p r i o r t o and d u r i n g i r r a d i a t i o n . A e r o b i c c o n d i t i o n s were obtained by f l o w i n g oxygen i n s t e a d o f nitrogen. The c e l l s were c o o l e d t o 0°C 10 t o 15 minutes p r i o r t o irradiation  ( i . e . a f t e r g a s s i n g a t room temperature f o r 3 0 - 3 5  minutes) and maintained  a t 0°C f o r the d u r a t i o n o f the  experiment, u n l e s s s p e c i f i e d otherwise. c h a r t r e p r e s e n t a t i o n o f the experimental Irradiations therapeutic  6 0  procedure.  were performed w i t h gamma-rays from a  C o source  r a t e was 5 0 0 - 5 3 5  See f i g . 3 f o r a flow  ( 1 . 1 7 and 1.33 MeV y ~ a y s ) .  rads/minute.  r  The dose  13  2.1.3  Radioactive L a b e l l i n g The  DNA of CH2B  2  c e l l s was l a b e l l e d w i t h  3  H-thymidine  ( H-TdR, s p e c i f i c a c t i v i t y 15.7 or 47-59 Ci/mmol, Amersham3  S e a r l e , Don M i l l s , Ontario)  and i n 1 experiment,  1  *C-thymidine  ( s p e c i f i c a c t i v i t y 57 mCi/mmol, Amersham-Searle). d i f f e r e n t procedures For uniform  Two  were used t o l a b e l the DNA. l a b e l l i n g o f the p a r e n t a l DNA, 50 ml o f  normal growth medium c o n t a i n i n g 0.25 y C i of H-TdR/ml was added 3  to  1 day o l d c e l l c u l t u r e s i n l a r g e p l a s t i c f l a s k s  (75 cm  2  growth area, F a l c o n P l a s t i c s ) . These c u l t u r e s were then incubated under standard c o n d i t i o n s f o r 18 t o 24 hours. l a b e l l i n g was terminated  The  by an a d d i t i o n a l 1 hour i n c u b a t i o n i n  n o n - r a d i o a c t i v e medium, i n order t o c l e a r the c e l l u l a r p o o l o f unincorporated  precursor.  To examine newly s y n t h e s i z e d daughter DNA, u s u a l l y a f t e r i r r a d i a t i o n , a p u l s e l a b e l l i n g procedure was employed. The 1x10  cell 6  suspensions  t o be l a b e l l e d , t y p i c a l l y 10 ml c o n t a i n i n g  c e l l s , were c e n t r i f u g e d a t 0°C (8 minutes a t 600 rpm) i n  17x100 mm d i s p o s a b l e t e s t tubes  (Falcon).  The supernatant  was  poured o f f and the c e l l p e l l e t resuspended i n 1 ml o f warm (37°C) medium c o n t a i n i n g 20 y C i o f H-TdR. 3  A f t e r a 6 minute  i n c u b a t i o n i n a 37°C water bath, the p u l s e l a b e l was by the a d d i t i o n o f 10 ml o f c o l d The  terminated  (0°C) n o n - r a d i o a c t i v e medium.  c e l l s were then washed f r e e o f e x t r a c e l l u l a r l a b e l by twice  c e n t r i f u g i n g and resuspending  i n 10 ml o f normal growth medium  14  at 0°C. In 1 experiment, c e l l s were c o n t i n u o u s l y l a b e l l e d 3  with  H-TdR p r i o r t o i r r a d i a t i o n and p u l s e l a b e l l e d , as d e s c r i b e d  above, with 1 ml of 10 yCi/ml  2.1.4  Repair and Chase  1  **C-TdR a f t e r  irradiation.  Incubations  A f t e r i r r a d i a t i o n a t 0°C, the c e l l s were r o u t i n e l y twice c e n t r i f u g e d and resuspended, a l l a t 0°C, t o remove the drugs present d u r i n g i r r a d i a t i o n .  In experiments examining SSB  p r o d u c t i o n , the c e l l s were then immediately  l y s e d a t the top of  ASG's (see s e c t i o n 2.3). In experiments designed  t o examine the r e j o i n i n g of SSB's  or t o examine p o s t - i r r a d i a t i o n DNA s y n t h e s i s , c e l l s were incubated under standard c o n d i t i o n s (37°C, 95% a i r , 5% C0 ) 2  f o r v a r i o u s times processes  a f t e r i r r a d i a t i o n t o allow enzymatic r e p a i r  t o occur.  T h i s r e p a i r i n c u b a t i o n was stopped  p l a c i n g the c e l l s a t 0°C. were kept a t 0°C u n t i l  by  In r e j o i n i n g experiments, the c e l l s  lysis.  P o s t - i r r a d i a t i o n DNA s y n t h e s i s was i n v e s t i g a t e d by p u l s e l a b e l l i n g f o l l o w i n g r e p a i r , and then c a r r y i n g out a f u r t h e r 3 hour i n c u b a t i o n , again under standard c o n d i t i o n s . The  l e n g t h o f t h i s chase i n c u b a t i o n was chosen as 3 hours s i n c e  a 3 hour chase f o l l o w i n g a 6 minute p u l s e l a b e l was found, i n p r e l i m i n a r y experiments, t o y i e l d DNA of very n e a r l y the same molecular weight as t h a t obtained cells  (see f i g . 2 ) .  from c o n t i n u o u s l y  labelled  15  CO  20  O U  O  8  15  o  prelabel  •  pulse  label  10  LU  O  err LU  CL  0  5  10  15  20  25  FRACTION NUMBER DIRECTION OF SEDIMENTATION  Figure  2.  Continuously  L a b e l l e d and P u l s e  Labelled  DNA  C H 2 B c e l l s were c o n t i n u o u s l y l a b e l l e d w i t h H-TdR f o r 24 h o u r s (0) o r p u l s e l a b e l l e d f o r 6 m i n u t e s and i n c u b a t e d a t 37°C ( c h a s e d ) f o r 3 h o u r s (•#) . C e l l s were t h e n l y s e d on ASG's f o r 7 h o u r s a n d s p u n a t 12,000 r.p.m. f o r 15 h o u r s . The s m a l l v e r t i c a l a r r o w r e p r e s e n t s t h e p o s i t i o n f r o m w h i c h t h e DNA s t a r t s to sediment. 3  2  16  In  g e n e r a l then,  subsequent c e n t r i f u g i n g , done a t 0°C e x c e p t  the c e l l s Figure  and  resuspending  Also', t h e s e  double  Stock  distilled  stored  label  by  labelling  used.  (TAN), a s t a b l e n i t r o x i d e  from A l d r i c h  Chemical  s o l u t i o n s were p r e p a r e d  through  a 0.2  by d i s s o l v i n g  y filter,  free  Company, Edmonton,  w a t e r t o p r o d u c e a 0.2 M s t o c k  filtered  a t 4°C.  and c h a s e  DRUGS  was p u r c h a s e d  After being  pulse  was  i n c u b a t i o n s were t e r m i n a t e d  t h a t were  Triacetoneamine-N-oxyl  Alberta.  and o t h e r h a n d l i n g  3 shows s c h e m a t i c a l l y t h e d i f f e r e n t  RADIOSENSITIZING  radical,  a t 0°C and a l l  a t 0°C.  incubation schedules  2.2  were i r r a d i a t e d  f o r periods of repair,  i n c u b a t i o n a t 37°C. placing  cells  TAN i n  solution.  the s o l u t i o n  F r e s h s t o c k s o l u t i o n s were p r e p a r e d  every  was 2  weeks. Metronidazole,  2 - m e t h y l - 5 - n i t r o i m i d a z o l e - l - e t h a n o l , was  o b t a i n e d from Poulenc  L i m i t e d , M o n t r e a l , Quebec.  prior  s o l u t i o n was p r e p a r e d  drug  t o u s e , a 15 mM i n MEM  through  F-16  a 0.2  y  lacking  Garden C i t y ,  by d i s s o l v i n g t h e  s o d i u m b i c a r b o n a t e and  filtering  filter.  Ro-07-0582, p r o p a n o l , was  A few h o u r s  1-(2-nitro-l-imidazole)-3-methoxy-2-  s y n t h e s i z e d by Roche P r o d u c t s H e r t f o r d s h i r e , England  L i m i t e d , Welwyn  and o b t a i n e d  through  17  continuous label  pretreatment  irradiate irradiate  post-treatment lyse  repair  repair lyse  pulse label  chase  Figure  3.  Flow Chart Procedures  Representation  of  the  Various  Experimental  18  Dr. C. E. Smithen.  P r e p a r a t i o n and use of t h i s drug were  i d e n t i c a l to t h a t j u s t d e s c r i b e d f o r  2.3  ALKALINE SUCROSE GRADIENTS  The ASG  technique  d e s c r i b e d here i s e s s e n t i a l l y  d e s c r i b e d p r e v i o u s l y by P a l c i c and  2.3.1  metronidazole.  Gradient  and  Skarsgard  (1972a).  Lysing Solutions  An automatic g r a d i e n t former Instrumentation  that  Specialties  (ISCO Model 57 0,  Company, L i n c o l n ,  Nebraska)  was  used to prepare l i n e a r 5 to 20% ASG's of t o t a l volume 17 ml, c e l l u l o s e n i t r a t e tubes The  gradient solutions  water and c o n t a i n e d  0.3  ( / x 4 i n c h e s , Beckman 8  (SDS)  and  was  carefully  lysing solution A l l solutions  distilled  (NaOH), 0.001  (EDTA), 0.01%  appropriate concentrations  On top of the prepared solution  with double  M sodium hydroxide  ethylenediaminetetraacetic acid sulphate  Instruments).  5  were prepared  g r a d i e n t s , 0.5  sodium of  M  dodecyl  sucrose.  ml of  lysing  l a y e r e d j u s t p r i o r to c e l l l y s i s .  contained  0.5  M NaOH, 0.01  were passed through a 0.2  in  M EDTA and  0.2%  The SDS.  y f i l t e r p r i o r to usage.  19  2.3.2  L y s i n g Procedure A p r e c o o l e d 50 y l m i c r o s y r i n g e was used t o dispense the  c e l l s onto the l y s i n g l a y e r .  The s y r i n g e was mounted i n an o l d  microscope c h a s s i s whose 2 rack and p i n i o n d r i v e s were used t o first  lower the s y r i n g e u n t i l the needle was j u s t t o u c h i n g the  l y s i n g s o l u t i o n and then, t o d e l i v e r the c e l l s onto the l y s i n g layer.  A volume o f 0.02 ml c o n t a i n i n g 2-3X10  4  c e l l s i n normal  growth medium was d e l i v e r e d over a p e r i o d of 2 minutes.  The  g r a d i e n t s were then loaded i n t o u l t r a c e n t r i f u g e buckets and l y s e d i n the dark f o r 6 o r 7 hours b e f o r e c e n t r i f u g a t i o n .  2.3.3  Centrifugation A f t e r an a p p r o p r i a t e l y s i n g p e r i o d  the  (see s e c t i o n 2.3.5),  g r a d i e n t s were c e n t r i f u g e d a t 20°C u s i n g a SW 27 r o t o r i n  a Beckman L2-65B p r e p a r a t i v e u l t r a c e n t r i f u g e .  In SSB  experiments, an angular speed of c e n t r i f u g a t i o n , w, of 16,000 r.p.m. and a time of c e n t r i f u g a t i o n , t , of 10 hours were r o u t i n e l y used, although v a l u e s of 18,000 r.p.m. and 8 hours r e s p e c t i v e l y , were used f o r 1 experiment.  In experiments  designed t o examine SSB r e j o i n i n g or p o s t - i r r a d i a t i o n  DNA  s y n t h e s i s , c e n t r i f u g a t i o n s were u s u a l l y performed a t 14,000 r.p.m. f o r 11 t o 12 hours w i t h a l t e r n a t e v a l u e s of 12,000 r.p.m. and 12 o r 15 hours being used a few times.  20  2.3.4  G r a d i e n t F r a c t i o n a t i o n and Sample Counting A f t e r c e n t r i f u g a t i o n , each g r a d i e n t was  i n t o 25 f r a c t i o n s of 0.75 fraction collector.  fractionated  ml each, u s i n g an ISCO Model D  F r a c t i o n s were c o l l e c t e d from the top by  d i s p l a c i n g the g r a d i e n t upwards with a 30% sucrose s o l u t i o n pumped i n t o the bottom of the g r a d i e n t through a h o l e punched i n the c e n t r i f u g e tube. The f r a c t i o n s were c o l l e c t e d i n t o p l a s t i c vials  (Nuclear A s s o c i a t e s Inc., Westbury, New  a c i d i c w i t h 0.2 ml of 4 M h y d r o c h l o r i c a c i d . Aquasol  scintillation  York) and made F i v e ml of  (New England N u c l e a r , Boston, Massachusetts) was  then  added t o each sample and the v i a l s were thoroughly shaken.  The  r a d i o a c t i v i t y i n each f r a c t i o n was measured i n a Beckman scintillation  system  counting e f f i c i e n c y  (model LS-330).  T h i s procedure y i e l d e d a  (the r a t i o o f number o f counts observed t o  the expected number of r a d i o a c t i v e decays) of approximately 20 to 30%.  T h i s v a l u e r e p r e s e n t s the a b s o l u t e c o u n t i n g  e f f i c i e n c y f o r t r i t i u m u s i n g t h i s method. The expected t o t a l number of counts f o r each g r a d i e n t was  determined by l y s i n g a number o f c e l l s equal t o t h a t  l a y e r e d on the g r a d i e n t i n a separate s c i n t i l l a t i o n v i a l w i t h 0.5 ml of l y s i n g s o l u t i o n . the same way  T h i s sample was  then processed i n  as were the i n d i v i d u a l f r a c t i o n s .  The  between the sum of the counts i n the g r a d i e n t ' s 25  ratio fractions  and the expected t o t a l number of counts f o r t h a t g r a d i e n t (which was  determined as d e s c r i b e d above) was  t y p i c a l l y greater  21  than 0.75  and never l e s s than  0.60.  Sedimentation p r o f i l e s were obtained by p l o t t i n g  the  number of counts i n each f r a c t i o n as a percentage of the t o t a l counts f o r the g r a d i e n t versus f r a c t i o n number from the top of the  2.3.5  gradient.  L y s i n g Time The sedimentation p r o f i l e s o b t a i n e d from u n i r r a d i a t e d ,  c o n t i n u o u s l y l a b e l l e d c e l l s were found to be s t r o n g l y dependent on the l y s i n g time used. examining d i f f e r e n t  The r e s u l t s of an  experiment  l y s i n g times are shown i n f i g . 4.  s h o r t e r l y s i n g times  (e.g. 2 h o u r s ) , the DNA  At  appears i n 2 peaks.  With i n c r e a s i n g l y s i n g time, the h i g h e r molecular weight component d i s a p p e a r s and the sedimentation p r o f i l e s appear as 1 peak, the p o s i t i o n values with s t i l l  of which s h i f t s t o lower molecular weight longer l y s i n g times  (e.g. 12 h o u r s ) .  T h i s o b s e r v a t i o n d i f f e r s from e a r l i e r r e s u l t s o b t a i n e d u s i n g t h i s g r a d i e n t technique ( P a l c i c and Skarsgard, 1972a). P r e v i o u s l y , the DNA  p r o f i l e was  r e p r o d u c i b l e peak p o s i t i o n , from 6 to 2 4 hours.  found t o appear with a  independent of l y s i n g times r a n g i n g  This reproducible p o s i t i o n occurred  between the 2 p r o f i l e s c o r r e s p o n d i n g t o 6 and 8 hours time  ( i n f i g . 4).  Hence, a l y s i n g time o f 7 hours was  lysing employed  f o r a l l experiments except those examining SSB p r o d u c t i o n . SSB experiments, c e l l s were l y s e d f o r 6 hours, s i n c e DNA  In  from  i r r a d i a t e d c e l l s has been shown t o emerge i n a r e p r o d u c i b l e  22  FRACTION NUMBER DIRECTION OF S E D I M E N T A T I O N - — - >  Figure  4.  Effect  of Lysing  Time  on S e d i m e n t a t i o n  Profile  CH2B c e l l s were c o n t i n u o u s l y l a b e l l e d f o r 18-21 h o u r s , l y s e d f o r t h e t i m e s i n d i c a t e d a t 2 1 . 5 - 2 2 . 2 ° C , a n d s p u n a t 12,000 r.p.m. f o r 12 h o u r s . 2  23  peak p o s i t i o n i n l y s i n g times as s h o r t as 3 hours (Palcic,  2.3.6  1972).  Lysing  Temperature  The p o s i t i o n o f u n i r r a d i a t e d , u n i f o r m l y l a b e l l e d  DNA  on the g r a d i e n t was a l s o found t o be dependent on l y s i n g temperature.  F i g u r e 5 shows 2 sedimentation p r o f i l e s o b t a i n e d  from c e l l s taken from the same c e l l suspension and l y s e d a t d i f f e r e n t temperatures.  The p r o f i l e from the sample which was  l y s e d a t the h i g h e r temperature i s c l e a r l y s h i f t e d  towards  lower m o l e c u l a r weight v a l u e s . In order t o minimize t h i s " l y s i n g temperature  effect",  the ambient room temperature d u r i n g l y s i s was u s u a l l y maintained a t 22.5 t o 24.5°C.  2.3.7  C a l c u l a t i o n of M o l e c u l a r Weight Both weight average and number average m o l e c u l a r  weights were c a l c u l a t e d i n e s s e n t i a l l y the same manner as d e s c r i b e d e a r l i e r by P a l c i c and Skarsgard (1972a). B r i e f l y , the weight average m o l e c u l a r weight c a l c u l a t e d u s i n g the f o l l o w i n g e x p r e s s i o n :  Mw =  I c. (d. ) x 1 u ta 2  x  I  k  (Mw) was  24  5  10  FRACTION  15  20  NUMBER  DIRECTION OF SEDIMENTATION  F i g u r e 5.  E f f e c t o f L y s i n g Temperature on S e d i m e n t a t i o n P r o f i l e  CH2B? c e l l s were c o n t i n u o u s l y l a b e l l e d f o r 21 hours and l y s e d f o r 7 hours a t 20.5-23.5°C (O) or 25.5-27.0°C (•). C e l l s were spun a t 12,000 r.p.m. f o r 12 hours,  25  where oi i s the angular speed of c e n t r i f u g a t i o n i n r.p.m., t i s the time of c e n t r i f u g a t i o n 2.5  i n hours, and a and k are c o n s t a n t s ,  and 0.0528 r e s p e c t i v e l y ,  which were determined  by S t u d i e r  th (19 65); c^ r e p r e s e n t s the number of counts i n the i fraction, c o r r e c t e d f o r background, and d^ i s the d i s t a n c e t h a t the th molecules i n the i  f r a c t i o n have sedimented.  c a l i b r a t i o n constant, 3/ was  determined  The g r a d i e n t  u s i n g 3 DNA  markers:  the bacteriophages T4 and T7, and human adenovirus 2 ( P a l c i c and Skarsgard, 1972a). Since these DNA  sedimentation p r o f i l e s have been shown  t o r e p r e s e n t randomly s i z e d d i s t r i b u t i o n s o f molecules  (Palcic  and Skarsgard, 1972a), the number average molecular weight can be c a l c u l a t e d  (Mn)  from the e x p r e s s i o n  Mn = 0.5  x Mw  (Charlesby, 1954). The c a l c u l a t i o n of Mw  and the p l o t t i n g of sedimentation  p r o f i l e s were f a c i l i t a t e d by the use of a computer University  of B.C.)  and a H e w l i t t - P a c k a r d p l o t t e r .  (IBM  370,  The  first  20 f r a c t i o n s of each g r a d i e n t were used i n the c a l c u l a t i o n of Mw,  u n l e s s c^, f o r some f r a c t i o n i f u r t h e r  g r a d i e n t than the DNA  peak, f e l l t o l e s s than 1.5%  number of counts f o r t h a t g r a d i e n t . the f i r s t  from the top of the of the t o t a l  In t h i s second case, o n l y  i f r a c t i o n s were used i n the c a l c u l a t i o n .  26  3.  3.1  PARENTAL  RESULTS  DNA  Although the bulk of t h i s work i s concerned w i t h the s y n t h e s i s of DNA  a f t e r i r r a d i a t i o n , i t i s of i n t e r e s t to  b r i e f l y examine the s t a t e of the p a r e n t a l DNA to i o n i z i n g r a d i a t i o n .  Two  after  exposure  types o f experiments were performed  u s i n g the technique of ASG's.  The f i r s t procedure  a s s a y i n g f o r the p r o d u c t i o n of DNA  involved  SSB's, or a l k a l i l a b i l e  bonds, as a f u n c t i o n of dose, immediately a f t e r exposure;  the  second examined the r e j o i n i n g of these breaks a t 37°C.  3.1.1  S i n g l e Strand Break P r o d u c t i o n The p r o d u c t i o n of SSB's i n mammalian DNA  radiation  has been e x t e n s i v e l y s t u d i e d .  by  ionizing  The presence of oxygen  d u r i n g i r r a d i a t i o n i s known t o i n c r e a s e the y i e l d of SSB's by a f a c t o r of 3 to 4 ( P a l c i c and Skarsgard, 1972a; Roots and 1974).  Smith,  In a d d i t i o n , s e v e r a l chemical s e n s i t i z e r s have been  shown t o cause i n c r e a s e d s t r a n d breakage  (Dugle e t a l . , 1972).  In a l l SSB experiments, CH2B2 c e l l s were i r r a d i a t e d a t 0°C and kept i n an ice-water bath u n t i l l y s i s a t the top of an ASG.  Hypoxia was  achieved by f l o w i n g N 2 (-10-15 ppm  O 2 ) prior  to and d u r i n g i r r a d i a t i o n ; drugs, i f p r e s e n t , were added t o the  27  c e l l suspension p r i o r  to gassing.  Sedimentation c o n d i t i o n s f o r  a l l experiments were i d e n t i c a l , t h a t i s , g r a d i e n t s were c e n t r i f u g e d a t 16,000 r.p.m. f o r 10 hours.  Typical  sedimentation p r o f i l e s o b t a i n e d f o r i r r a d i a t i o n under v a r i o u s c o n d i t i o n s are shown i n f i g u r e s 6 and 7. In f i g . 6a, the dose dependence of the DNA sedimentation p r o f i l e i s shown.  DNA from c e l l s r e c e i v i n g a h i g h e r dose  sediments more s l o w l y and hence has s u f f e r e d more SSB's. fig.  In  6b, the l a r g e r e d u c t i o n i n the y i e l d o f s t r a n d breaks  d u r i n g hypoxic i r r a d i a t i o n i s shown. hypoxia y i e l d s  DNA fragments  A dose of 15 krads under  s l i g h t l y l a r g e r than those  obtained a f t e r an a e r o b i c dose of 5 krads, i m p l y i n g an oxygen enhancement r a t i o  (OER) f o r SSB's o f somewhat more than 3.  I t has been r e p o r t e d t h a t TAN p r e s e n t d u r i n g hypoxic i r r a d i a t i o n o f phage X DNA causes, a t most, a s m a l l i n c r e a s e i n the number o f SSB's (Emmerson, 1970; Johansen,  1972).  Agnew  (1972) found no i n c r e a s e i n the number o f SSB's i n mouse L-60 c e l l DNA i n the presence o f TAN.  The 2 sedimentation p r o f i l e s  shown i n f i g . 7a are c o n s i s t e n t w i t h these r e s u l t s .  The c e l l s  from which these p r o f i l e s were o b t a i n e d were i r r a d i a t e d  t o the  same dose under hypoxia w i t h o n l y 1 suspension c o n t a i n i n g 10 mM TAN; TAN has caused o n l y a very s m a l l s h i f t towards the top  o f the g r a d i e n t . In c o n t r a s t , when CH2B  2  c e l l s were g i v e n i d e n t i c a l  doses under hypoxia i n the presence or absence  of the  28  a  FRACTION NUMBER DIRECTION  Figure  6 a,b.  OF SEDIMENTATION  Production of Single Strand and H y p o x i c C o n d i t i o n s  Breaks  r >  under Aerobic ~" r  CH2B c e l l s were c o n t i n u o u s l y l a b e l l e d and i r r a d i a t e d a t 0°C t o 5 («) o r 15 k r a d s (O) u n d e r a e r o b i c c o n d i t i o n s ( p a n e l a ) , o r t o 15 k r a d s u n d e r h y p o x i a (#) o r 5 k r a d s u n d e r a e r o b i c conditions (o) (panel b ) . C e l l s were l y s e d f o r 6 h o u r s and spun a t 1 6 , 0 0 0 r.p.m- f o r 10 h o u r s . 2  29  • N + I AN 2  5  10  FRACTION  15  20  NUMBER  •DIRECTION OF SEDIMENTATION  Figure  7 a,b,  Production of Single Strand w i t h TAN o r R o - 0 7 - 0 5 8 2  Breaks under Hypoxia ~ ' :  CH2B c e l l s were c o n t i n u o u s l y l a b e l l e d and i r r a d i a t e d a t 0°C u n d e r h y p o x i c c o n d i t i o n s (a) t o 15 k r a d s w i t h o u t d r u g (o) o r w i t h 10 mM TAN ( * ) , o r (b) t o 12 k r a d s w i t h 10 mM TAN (o) o r 15 mM R o - 0 7 - 0 5 8 2 ( ® ) . C e l l s w e r e l y s e d f o r 6 h o u r s a n d s p u n a t 1 6 , 000- r . p . m . f o r 10 h o u r s . 2  30  n i t r o i m i d a z o l e , Ro-07-0582, a l a r g e s h i f t of the p r o f i l e observed.  was  T h i s r e s u l t , shown i n f i g . 7b, i n d i c a t e s t h a t  Ro-07-0582 causes a l a r g e i n c r e a s e i n the p r o d u c t i o n  of SSB's.  F i g u r e 8 i s a summary of the SSB experiments done. Here the r e c i p r o c a l of the weight average molecular i s p l o t t e d a g a i n s t dose.  weight  (Mw)  Each p o i n t i s the r e s u l t of 1 —8  gradient.  S t r a i g h t l i n e s p a s s i n g through 1/Mw  = 0.3 8x10  daltons  were f i t t e d t o the data by the method o f l e a s t  squares.  The dose modifying  f a c t o r (DMF)  f o r any  irradiation  c o n d i t i o n i s j u s t the slope of t h a t l i n e d i v i d e d by the slope of the N  2  line.  The energy r e q u i r e d t o cause 1 SSB can be  c a l c u l a t e d d i r e c t l y from the i n v e r s e s l o p e , assuming t h a t the d i s t r i b u t i o n o f DNA s e c t i o n 2.3.7). Table  fragments i n the g r a d i e n t i s random (see  These c a l c u l a t i o n s are summarized  i n Table  I.  I:  Irradiation  Condition  Slope  (dalton "*"rad "*")  N  2  0.451  0  2  1.94  N  2  + 10 mM  TAN  0.576 x  N  2  + 15 mM  Metronidazole  1.36  x  10~  N  2  + 15 mM  1.66  x  10~  Ro-07-0582  x x  10" 10~  eV/break  DMF  115  1.0  27  =4  90  1.3  1 2  38  3.0  1 2  31  3.7  1 2  1 2  10~  1 2  31  6  I  0  L_J  I  I  I 5  I  I  I  .1,1  1 l l  10  I  I  | - '  I  15  20  DOSE (krads)  F i g u r e 8.  Graph of 1/Mw versus Dose f o r S i n g l e Strand P r o d u c t i o n under S e v e r a l C o n d i t i o n s  Break  CH2B c e l l s were c o n t i n u o u s l y l a b e l l e d and i r r a d i a t e d a t 0°C under a e r o b i c (A) o r hypoxic c o n d i t i o n s (•) i n the presence o f 15 mM Ro-07-0582 .(•) , 15 mM metronidazole (•) o r 10 mM TAN (O) . C e l l s were l y s e d f o r 6 hours on ASG's and spun a t 16,000 r.p.m. f o r 10 hours. Mw v a l u e s were c a l c u l a t e d as d e s c r i b e d i n s e c t i o n 2.3.7 and s t r a i g h t l i n e s p a s s i n g through 0.3.8xl0~ daltons were f i t t e d t o the data by the method o f l e a s t squares. 2  8  - 1  32  TAN present d u r i n g hypoxic i r r a d i a t i o n appears a s m a l l i n c r e a s e i n the number o f SSB's produced.  N  t o cause + TAN  2  p o i n t s p l o t t e d are the r e s u l t s from 2 separate experiments; N 2 p o i n t s are from 3 separate experiments.  This r e s u l t disagrees  w i t h the e a r l i e r work o f Agnew (1972) i n t h i s l a b o r a t o r y . However, t h i s i n c r e a s e i n SSB y i e l d i s q u i t e i n s i g n i f i c a n t compared t o the l a r g e changes caused by metronidazole and Ro-07-0582.  From f i g .  8 and Table I , i t i s apparent  t h a t 15 mM  Ro-07-0582 present d u r i n g hypoxic i r r a d i a t i o n produces  almost  the f u l l a e r o b i c s t r a n d break y i e l d . I t should be noted t h a t , i n a separate  experiment,  15 mM Ro-07-0582 and 10 mM TAN were found t o cause no i n c r e a s e i n the number o f SSB's produced  3.1.2  during aerobic i r r a d i a t i o n .  R e j o i n i n g o f S i n g l e Strand Breaks The r e j o i n i n g o f SSB's i n mammalian c e l l s has been  observed by many authors Okada, 1970).  (e.g. L e t t e t a l . ,  1967; Sawada and  Under f a v o u r a b l e c o n d i t i o n s the r e j o i n i n g  process i s f a i r l y r a p i d , with most o f the SSB's being r e j o i n e d w i t h i n 1-2 hours; t h i s process i s s t r o n g l y i n h i b i t e d a t 0°C, with no r e j o i n i n g d e t e c t a b l e a t times up t o 5 hours Skarsgard,  ( P a l c i c and  1972b).  The experiments  r e p o r t e d i n t h i s s e c t i o n were done t o  i n v e s t i g a t e the p o s s i b i l i t y t h a t v a r i o u s r a d i o s e n s i t i z e r s might be i n t e r f e r i n g with SSB r e j o i n i n g .  P r e v i o u s work i n t h i s  33  laboratory  (Agnew and Skarsgard,  1970)  i n d i c a t e d t h a t TAN  not i n t e r f e r e w i t h the r e j o i n i n g p r o c e s s .  The  does  r e s u l t s shown  in f i g . 9 confirm t h i s observation.  Two  suspensions,  were g i v e n a dose of 15  1 c o n t a i n i n g 10 mM  krads under hypoxia.  TAN,  i d e n t i c a l CH2B  Samples were incubated at 37°C f o r the  times i n d i c a t e d and then returned to 0°C u n t i l l y s i s . were c a l c u l a t e d and are p l o t t e d as percentages unirradiated rotor.  cell  2  Mw's  of the  c o n t r o l value obtained from a g r a d i e n t i n the same  I t i s apparent  t h a t TAN  present d u r i n g hypoxic  i r r a d i a t i o n has no e f f e c t on the r e j o i n i n g of SSB's. S i m i l a r experiments metronidazole.  were done u s i n g both Ro-07-0582 and  Here the dose d e l i v e r e d under hypoxia was  6  krads, a value chosen to give approximately  the same number of  i n i t i a l SSB's as t h a t caused by the N  2  irradiations.  The  and N  + TAN  15 krad  r e s u l t s f o r Ro-07-0582 are shown i n f i g . 10.  N e i t h e r Ro-07-0582 nor metronidazole process.  2  i n h i b i t e d the r e j o i n i n g  34  N  70  2  15 krads  N + TAN 2  15 krads  60  0 L _ J  ,  30  60  90  120  150  REPAIR TIME (minutes)  F i g u r e 9.  R e j o i n i n g o f S i n g l e Strand Breaks a f t e r I r r a d i a t i o n w i t h and without TAN ' !  Hypoxic  :  C o n t i n u o u s l y l a b e l l e d CH2B c e l l s were i r r a d i a t e d a t 0°C under hypoxic c o n d i t i o n s t o 15 krads i n the presence (®) o r absence •($) o f 10 mM TAN. C e l l s were then incubated a t 37°C f o r the times i n d i c a t e d . C e l l s were l y s e d f o r 7 hours and spun a t 14,000 r.p.m. f o r 11 hours. Mw's were c a l c u l a t e d and p l o t t e d as a percentage o f the Mw value o b t a i n e d from the u n i r r a d i a t e d c o n t r o l i n the same r o t o r . ?  35  UNIRRADIATED CONTROL N+ Ro-07-0582 2  6 krads N  30  60  90  2  120  1 5 krads  150  180  REPAIR TIME (minutes)  F i g u r e 10.  R e j o i n i n g o f S i n g l e Strand Breaks a f t e r Hypoxic I r r a d i a t i o n w i t h and without Ro-07-0582  C o n t i n u o u s l y l a b e l l e d CH2B c e l l s were i r r a d i a t e d a t 0°C under hypoxic c o n d i t i o n s t o 6 krads i n the presence o f 15 mM Ro-07-0582 (#) o r t o 15 krads i n d r u g - f r e e medium (©). The u n i r r a d i a t e d c o n t r o l v a l u e was o b t a i n e d from a g r a d i e n t i n the same r o t o r . C e l l s were l y s e d f o r 7 hours and spun a t 14,000 r.p.m. f o r 11 hours. Mw's were c a l c u l a t e d . 2  36  3.2  NEWLY SYNTHESIZED DAUGHTER DNA  Most o f t h i s work has i n v o l v e d the i n v e s t i g a t i o n of p o s t - i r r a d i a t i o n DNA s y n t h e s i s by means o f the p u l s e and chase assay d e s c r i b e d e a r l i e r  ( s e c t i o n s 2.1.3 and 2.1.4).  was used t o i n v e s t i g a t e the a b i l i t y o f CH2B  2  T h i s assay  c e l l s to regain  the c a p a c i t y t o s y n t h e s i z e f u l l - s i z e d DNA a f t e r i r r a d i a t i o n i n the presence of v a r i o u s r a d i o s e n s i t i z i n g drugs.  The drugs  examined were TAN, metronidazole and Ro-07-0582.  3.2.1  TAN Treatment  o f Hypoxic CH2B  TAN p r e s e n t d u r i n g exposure  2  Cells  o f E. c o l i t o X-rays under  hypoxic c o n d i t i o n s i s known t o cause i n t e r r u p t i o n s i n the DNA strands s y n t h e s i z e d immediately 1969).  after irradiation  (Rupp e t a l . ,  I t was suggested t h a t these i n t e r r u p t i o n s were caused  by X-ray induced TAN-DNA adducts, which were known t o be formed i n aqueous s o l u t i o n s o f DNA  (Willson and Emmerson, 1970;  Nakken, S i k k e l a n d and Brustad, 1970), and have s i n c e been observed i n v i v o i n E. c o l i K-12  (Wold and Brustad,  1974).  Although TAN s e n s i t i z e s hypoxic hamster c e l l s t o i o n i z i n g r a d i a t i o n t o a l e s s e r degree than b a c t e r i a l  cells  (Parker, Skarsgard and Emmerson, 1969; Emmerson, 1967), n e v e r t h e l e s s o f i n t e r e s t t o determine  i t was  i f the same newly  s y n t h e s i z e d s t r a n d i n t e r r u p t i o n s occur i n mammalian c e l l s . In the f o l l o w i n g experiments, TAN, i f p r e s e n t , was  37  added to the c e l l suspension 1 hour b e f o r e i r r a d i a t i o n removed, by twice resuspending after irradiation.  i n d r u g - f r e e medium,  and  immediately  C e l l suspensions were i r r a d i a t e d a t 0°C  under hypoxic c o n d i t i o n s to a dose of 15 krads.  After  resuspension i n d r u g - f r e e medium, the c e l l s were incubated f o r v a r i o u s times a t 37°C before being r e t u r n e d to an bath.  ice-water  T h i s i n c u b a t i o n p e r i o d w i l l be r e f e r r e d t o as  i n c u b a t i o n " , d u r i n g which the c e l l may  "repair  repair radiation-induced  l e s i o n s , such as SSB's. F o l l o w i n g t h i s r e p a i r i n c u b a t i o n , the c e l l s were p u l s e l a b e l l e d f o r 6 minutes a t 37°C with H-TdR (20 uCi/ml). 3  The  c e l l suspensions were then incubated f o r 3 hours a t 37°C, a chase p e r i o d d u r i n g which u n i r r a d i a t e d CH2B s y n t h e s i z e almost The DNA  " f u l l - s i z e d " DNA  2  cells  (see s e c t i o n 2.1.4).  sedimentation p r o f i l e s o b t a i n e d from  exposed t o 15 krads under hypoxia i n the presence of 10 mM  TAN  will  are shown i n f i g .  11a.  these p r o f i l e s r e p r e s e n t the DNA  cells  and absence  I t should be noted t h a t  s y n t h e s i z e d d u r i n g a 3 hour  p e r i o d w i t h no r e p a i r i n c u b a t i o n p r i o r to the p u l s e l a b e l . p r o f i l e o b t a i n e d from the TAN  treated c e l l s i s clearly  The  shifted  towards lower molecular weight v a l u e s . F i g u r e l i b i s a s i m i l a r comparison between samples irradiated in N  2  and N  2  + TAN.  Here however, the c e l l s were  incubated a t 37°C f o r 120 minutes b e f o r e being p u l s e and chased.  The  labelled  sedimentation p r o f i l e obtained from TAN t r e a t e d  38  a  FRACTION —DIRECTION  Figure  11 a , b .  NUMBER  OF SEDIMENTATION — — •  S e d i m e n t a t i o n P r o f i l e s f o r N e w l y S y n t h e s i z e d DNA a f t e r I r r a d i a t i o n under H y p o x i a ' w i t h and w i t h o u t TAN ' • ' ' ' ~ — !  r  :  :  :  CH2B c e l l s w e r e i r r a d i a t e d a t 0 ° C t o 15 k r a d s u n d e r h y p o x i c conditions i n the presence (®) o r a b s e n c e (O) o f 10 mM T A N . A f t e r t w i c e r e s u s p e n d i n g i n d r u g - f r e e medium, c e l l s were a l l o w e d (a) n o r e p a i r o r (b) 1 2 0 m i n u t e s r e p a i r a t 3 7 ° C , b e f o r e b e i n g p u l s e l a b e l l e d w i t h 20 y C i o f H - T d R / m l f o r 6 m i n u t e s . They were t h e n i n c u b a t e d a t 37°C f o r a f u r t h e r 3 hours. C e l l s were l y s e d f o r 7 h o u r s a n d s p u n a t 1 4 , 0 0 0 r . p . m . f o r 11 h o u r s . 2  3  39  cells fig.  i s essentially 11a; t h e c u r v e  hypoxia alone higher  12.  fig.  label;  from c e l l s  weight with  irradiated  under  t h i s Mw  shift to  repair incubation.  i s p l o t t e d versus Again,  fragments  obtained  t o t h e TAN t r e a t e d p r o f i l e i n  has undergone a s m a l l b u t s i g n i f i c a n t  molecular Mw  identical  time a t 37°C i n  repair incubation  represents  the size  o f t h e DNA  i n the 3 hour chase f o l l o w i n g the p u l s e  synthesized  the r e p a i r incubation  i s carried  out p r i o r  to the pulse  label. The with N  2  DNA  i n Mw  alone,  t h e 3 hour  (synthesized during  i n c r e a s i n g time o f r e p a i r i n c u b a t i o n , presumably r e f l e c t s  f i g . 9).  synthesized  r e p a i r o f damage i n t h e t e m p l a t e  and s e d i m e n t a t i o n  daltons. hypoxia,  given  damage a r e c a p a b l e If  repair  y i e l d e d Mw  sufficient  values  These v a l u e s  ±  0.2x10  a dose o f 15 k r a d s  8  under  during  DNA  fragments.  t h e 15 k r a d  dose,  f r o m t h e 3 h o u r c h a s e p e r i o d were much d i d not increase  incubation before  o f up t o 3 h o u r s .  o f =2.8  of synthesizing f u l l - s i z e d  obtained  conditions of  time t o r e p a i r t h e i r r a d i a t i o n  however, TAN was p r e s e n t  t h e Mw v a l u e s smaller.  which under s i m i l a r  I t appears t h a t even a f t e r cells  of the  t h e 3 h o u r c h a s e was c o m p a r a b l e t o t h a t  made by u n i r r a d i a t e d c e l l s , lysis  similar  A f t e r 120 minutes r e p a i r , the s i z e  during  chase)  for irradiation i n  ( f o r example, t h e r e j o i n i n g o f SSB's, w h i c h show  kinetics, DNA  increase  the pulse  significantly  and c h a s e ,  with  f o r r e p a i r times  40  Mw range for Q d o s s control CO  O  O  X  N, N  o  IJJ  2  +TAN  ^  -> cn > c  or  o +-<  ra  < ^ i  w  Z> CJ  1.0  O UJ  t 0  0  30  1  X  60  1 90  REPAIR TIME Figure  12.  E f f e c t o f TAN T r e a t m e n t o n P o s t - I r r a d i a t i o n DNA  120  150  180  (minutes)  during Hypoxic Synthesis  Irradiation  CH23 ; c e l l s w e r e i r r a d i a t e d a t 0 ° C t o 15 k r a d s u n d e r h y p o x i a w i t h (®) o r w i t h o u t (O) 10 mM TAN. After twice resuspending i n d r u g - f r e e medium, c e l l s were a l l o w e d t o r e p a i r a t 3 7 ° C f o r v a r i o u s t i m e s b e f o r e b e i n g p u l s e l a b e l l e d and c h a s e d f o r 3 h o u r s a t 37°C. T h e d a s h e d l i n e s i n d i c a t e t h e r a n g e o f Mw values o b t a i n e d from u n i r r a d i a t e d c e l l s ( u s i n g t h e p u l s e and chase assay) i n s e v e r a l experiments. C e l l s were l y s e d f o r 7 hours and s p u n a t 1 4 , 0 0 0 r . p . m . f o r 11 h o u r s . ?  41  The  r e s u l t s i l l u s t r a t e d i n f i g u r e s 11 and 12 show t h a t  TAN present d u r i n g hypoxic  i r r a d i a t i o n causes p o s t - i r r a d i a t i o n  s y n t h e s i s o f s m a l l e r DNA fragments than those made by c e l l s exposed t o the same dose i n d r u g - f r e e medium.  I t was shown  e a r l i e r t h a t TAN does not i n h i b i t the r e j o i n i n g of SSB's ( s e c t i o n 3.1.2), and hence a p o s s i b l e e x p l a n a t i o n f o r these observations forming  i s t h a t TAN binds t o i r r a d i a t e d p a r e n t a l  DNA,  s t a b l e adducts which l e a d t o i n t e r r u p t i o n s i n the  s y n t h e s i s o f the daughter s t r a n d s .  These adducts are not  r e p a i r e d t o a measurable degree d u r i n g a 3 hour p o s t i r r a d i a t i o n incubation. In an experiment designed  t o c o n f i r m t h a t these  p i e c e s were a c t u a l l y being s y n t h e s i z e d from l a r g e l y  smaller  full-sized  p a r e n t a l s t r a n d s , c e l l s were p r e l a b e l l e d f o r 25 hours with 3  H-TdR and p u l s e l a b e l l e d a f t e r a dose of 15 krads  hypoxia with  ll+  C-TdR  (10 yCi/ml) f o r 6 minutes.  under  Sedimentation  p r o f i l e s o b t a i n e d from c e l l s i r r a d i a t e d with and without  TAN  are shown i n f i g . 13; here the c e l l s were allowed a 6 hour r e p a i r i n c u b a t i o n p r i o r t o the 6 minute chase.  C  lk  p u l s e and 3 hour  For those c e l l s not exposed t o TAN, the p a r e n t a l and  newly s y n t h e s i z e d daughter DNA y i e l d e d very s i m i l a r whereas, those c e l l s t r e a t e d with the drug d u r i n g  profiles;  irradiation  were found t o show marked d i f f e r e n c e s between p r e l a b e l and p u l s e l a b e l curves. f o r the concept  T h i s experiment p r o v i d e s f u r t h e r support  of r a d i a t i o n - i n d u c e d TAN-DNA adducts which l e a d  42 CO  20  N  O O  15 krads _ c — o r prelabel —«— -©-pulse label  2  N+TAN 15 krads -o—o- prelabe! -• »- pulse label 2  15  0  5  10  15  20  25  FRACTION NUMBER -DIRECTION  Figure  13.  OF SEDIMENTATION•  S e d i m e n t a t i o n P r o f i l e s f o r P a r e n t a l and Newly S y n t h e s i z e d D a u g h t e r DNA a f t e r I r r a d i a t T o n under H y p o x i a w i t h a n d w i t h o u t TAN ~  CH2B c e l l s were c o n t i n u o u s l y p r e l a b e l l e d (O) w i t h H - T d R f o r 25 h o u r s a n d i r r a d i a t e d t o 15 k r a d s u n d e r h y p o x i a ( a ) i n d r u g f r e e m e d i u m o r (b) i n m e d i u m c o n t a i n i n g 10 mM T A N . After twice r e s u s p e n d i n g i n d r u g - f r e e medium, c e l l s w e r e a l l o w e d t o r e p a i r f o r 6 h o u r s a t 37°C b e f o r e b e i n g p u l s e l a b e l l e d (®) w i t h 10 y C i of C-TdR/ml f o r 6 minutes and chased f o r 3 hours a t 37°C. C e l l s w e r e l y s e d f o r 7 h o u r s a n d s p u n a t 1 2 , 0 0 0 r . p . m . f o r 15 hours. 3  2  1 I +  to i n t e r r u p t i o n s i n strands s y n t h e s i z e d from the TAN damaged template.  In a d d i t i o n , i t suggests t h a t these adducts  f o r a t l e a s t 6 hours a f t e r  3.2.2  persist  irradiation.  TAN Pretreatment Agnew and Skarsgard  i r r a d i a t i o n exposure  (1972) have r e p o r t e d t h a t p r e -  t o TAN s e n s i t i z e s hypoxic Chinese hamster  c e l l s t o i o n i z i n g r a d i a t i o n with a DMF of 1.3. experiments were done t o determine  The f o l l o w i n g  i f t h i s pretreatment  effect  c o u l d be e x p l a i n e d by i n t e r r u p t i o n s i n newly s y n t h e s i z e d DNA, s i m i l a r t o those found a f t e r TAN CH2B  2  c e l l s were exposed  treatment. t o 10 mM TAN f o r 1 hour a t  22°C; they were then twice c e n t r i f u g e d and resuspended f r e e medium.  i n drug-  The remainder of the experimental procedure  was  i d e n t i c a l t o t h a t d e s c r i b e d i n the p r e v i o u s s e c t i o n : c e l l s were i r r a d i a t e d t o 15 krads under hypoxia, allowed t o r e p a i r f o r v a r i o u s times a t 37°C, p u l s e l a b e l l e d and chased f o r 3 hours. R e s u l t s from 1 experiment  are shown i n f i g .  14.  Mw v a l u e s  obtained from c e l l s t h a t were p r e t r e a t e d w i t h TAN are c o n s i s t e n t l y s l i g h t l y lower than those from c e l l s t h a t were not, although the d i f f e r e n c e s are o f m a r g i n a l s i g n i f i c a n c e .  Also,  Mw v a l u e s r i s e w i t h i n c r e a s i n g r e p a i r time, independent of TAN pretreatment.  A repeat experiment y i e l d e d comparable r e s u l t s .  I t appears t h a t TAN pretreatment has l i t t l e e f f e c t on the p o s t - i r r a d i a t i o n s y n t h e s i s of DNA.  44  3.0  ©  CO  TAN PRETREATMENT  O SHAM  o  20  I O LU <  CO  h  PRETREATMENT  O  O  c o  CC to  o  1£)  LU _J  o  0  1 30  60  X  90  REPAIR TIME  F i g u r e 14.  1  -L  120  1 150  180  (minutes)  E f f e c t o f TAN Pretreatment on P o s t - I r r a d i a t i o n DNA Synthesis ' ~~ :  !  CH2B c e l l s were exposed t o 10 mM TAN (®) o r d r u g - f r e e medium (O) f o r 1 hour a t 22°C and then twice resuspended i n d r u g - f r e e medium. C e l l s were i r r a d i a t e d a t 0°C t o 15 krads under hypoxic c o n d i t i o n s , allowed t o r e p a i r a t 37°C f o r v a r i o u s t i m e s , p u l s e l a b e l l e d f o r 6 minutes, and chased f o r 3 hours a t 37°C. Cells were l y s e d f o r 7 hours and spun a t 14,000 r.p.m. f o r 11 hours. Mw's were c a l c u l a t e d . 2  45  T h i s r e s u l t i s c o n s i s t e n t with the o b s e r v a t i o n t h a t does not b i n d t o u n i r r a d i a t e d DNA 1973).  of the DNA  3  designed to assay f o r TAN  mM  TAN.  15.  b i n d i n g to u n i r r a d i a t e d  control  uCi/ml  R e s u l t s f o r the 24 hour i n c u b a t i o n  No d e t e c t a b l e d i f f e r e n c e i n  sedimentation p r o f i l e s was  observed between TAN  treated  and  cells.  TAN  Post-treatment  Agnew and Skarsgard  (1972) have a l s o r e p o r t e d t h a t  p o s t - i r r a d i a t i o n treatment w i t h TAN almost as e f f e c t i v e l y as TAN (DMF  And secondly, i n  C o n t r o l s were incubated i n the presence  of the t r i t i u m l a b e l o n l y . are shown i n f i g .  present  found t o have no e f f e c t on the s i z e  c e l l s were t r e a t e d f o r 11 or 24 hours w i t h 0.25  H-TdR and 1.0  3.2.3  F i r s t , TAN  s y n t h e s i z e d d u r i n g a 3 hour chase.  an experiment DNA,  (Brustad, Jones and Wold,  Our evidence f o r t h i s i s t w o f o l d .  i n zero dose c o n t r o l s was  TAN  = 1.5).  determine  2  cells  treatment d u r i n g i r r a d i a t i o n  Post-irradiation sensitization i s strongly  dependent on temperature few minutes  can s e n s i t i z e CH2B  and i s o n l y observed i f TAN  after irradiation.  i s added a  Experiments were performed  to  i f t h i s post-treatment e f f e c t c o u l d be r e l a t e d to  i n t e r r u p t i o n s i n p o s t - i r r a d i a t i o n DNA A hypoxic suspension of CH2B 15 krads a t 0°C.  Immediately  synthesis. 2  c e l l s was  i r r a d i a t e d to  a f t e r i r r a d i a t i o n , a sample  added to an a e r o b i c s o l u t i o n o f TAN w i t h a r e s u l t i n g  was  46  FRACTION  NUMBER  DIRECTION OF SEDIMENTATION  Figure  15.  Sedimentation P r o f i l e s w i t h a n d w i t h o u t TAN  f o r DNA  Continuously  E>  Labelled  CH2B c e l l s w e r e c o n t i n u o u s l y l a b e l l e d f o r 24 h o u r s i n t h e presence (0) o r absence (^) o f 1.0 mM T A N . C e l l s were l y s e d f o r 7 h o u r s a n d s p u n a t 14,000 r.p.m. f o r 11.75 h o u r s . 2  47  c o n c e n t r a t i o n of 10 mM,  and incubated f o r 1 hour a t 21-22°C; a  p a r a l l e l sample was added t o d r u g - f r e e medium.  Other  samples  were allowed r e p a i r i n c u b a t i o n s of 2 0 and 60 minutes,  which  were c a r r i e d out a t 37°C under standard c o n d i t i o n s b e f o r e the 1 hour TAN post-treatment post-treatment,  a t room temperature.  F o l l o w i n g the  the c e l l s were washed f r e e of TAN by twice  c e n t r i f u g i n g and resuspending  i n d r u g - f r e e medium, p u l s e  l a b e l l e d f o r 6 minutes w i t h H-TdR and incubated f o r 3 hours, 3  before being l a y e r e d on ASG's.  In f i g .  16, Mw v a l u e s o b t a i n e d  from TAN p o s t - t r e a t e d and u n t r e a t e d c e l l s are p l o t t e d as a f u n c t i o n o f r e p a i r time a t 37°C b e f o r e the 1 hour exposure t o TAN-containing  o r d r u g - f r e e medium.  C e l l s t h a t were t r e a t e d w i t h TAN f o r 1 hour a f t e r i r r a d i a t i o n under hypoxia c o n s i s t e n t l y y i e l d e d daughter  DNA o f  lower Mw v a l u e s than those t h a t were n o t exposed t o TAN, although again, as i n the TAN pretreatment  experiments, the  d i f f e r e n c e s are of marginal s i g n i f i c a n c e .  A repeat  w i t h r e p a i r i n c u b a t i o n s performed  experiment  a t 21.5°C gave s i m i l a r  r e s u l t s , t h a t i s s l i g h t l y lower Mw v a l u e s from TAN p o s t - t r e a t e d cells. daughter  As can be seen i n f i g .  16, the molecular weight of  DNA i n c r e a s e d w i t h r e p a i r time i n a s i m i l a r f a s h i o n  f o r both TAN p o s t - t r e a t e d and u n t r e a t e d c e l l s . s h a r p l y from the s i t u a t i o n f o r TAN t r e a t e d c e l l s d u r i n g i r r a d i a t i o n ) as can be seen i n f i g . TAN post-treatment irradiation.  12.  This d i f f e r s (TAN p r e s e n t I t appears  has l i t t l e e f f e c t on DNA s y n t h e s i s a f t e r  that  48  TAN  3.0  co c  POST-TREATMENT  SHAM  o  POST-TREATMENT  co  T3  CO i  o  X  (D LU  cr  .1.0  < _ J  => U  LU I  o  20  40 REPAIR  Figure  16.  E f f e c t o f TAN Synthesis  TIME  Post-Treatment  60  80  (minutes)  on P o s t - I r r a d i a t i o n  DNA  CH2B c e l l s w e r e i r r a d i a t e d a t 0 ° C t o 15 k r a d s u n d e r h y p o x i a a n d allowed t o r e p a i r f o r v a r i o u s times under a e r o b i c c o n d i t i o n s a t 37°C. C e l l s w e r e t h e n e x p o s e d t o 10 mM TAN (®) o r d r u g - f r e e medium (B) f o r 1 h o u r a t 2 1 - 2 2 ° C , t w i c e r e s u s p e n d e d i n d r u g - f r e e medium, p u l s e l a b e l l e d f o r 6 m i n u t e s and c h a s e d f o r 3 h o u r s a t 37°C. C e l l s were l y s e d f o r 7 h o u r s and spun a t 14,000 r.p.m. f o r 11 h o u r s . Mw v a l u e s w e r e d e t e r m i n e d . 2  49  3.2.4  TAN  Treatment of A e r o b i c CH2B  I t has been shown t h a t TAN  2  Cells  does not s e n s i t i z e a e r o b i c  mammalian c e l l s t o i o n i z i n g r a d i a t i o n  (Parker, Skarsgard  Emmerson, 1969). A l s o , Wold and Brustad demonstrated t h a t TAN i r r a d i a t e d i n an 0 of  (1974) have  does not b i n d t o the DNA  saturated solution.  2  i n t e r e s t t o determine  i f TAN  a e r o b i c a l l y i r r a d i a t e d CH2B  2  and  of E. c o l i  N e v e r t h e l e s s , i t was  l e s i o n s c o u l d be d e t e c t e d i n  c e l l s u s i n g the p u l s e and  chase  assay. A e r o b i c c e l l suspensions were i r r a d i a t e d , w i t h or without  10 mM  approximately  TAN,  t o 5 krads, a value chosen t o g i v e  the same amount of damage as 15 krads under  hypoxic c o n d i t i o n s . twice resuspending  The c e l l s were then washed f r e e of TAN  by  i n d r u g - f r e e medium, allowed t o r e p a i r a t  37°C f o r v a r i o u s times, p u l s e l a b e l l e d w i t h H-TdR f o r 6 3  minutes,  and chased  f o r 3 hours at 37°C.  from TAN  t r e a t e d and u n t r e a t e d c e l l s are p l o t t e d a g a i n s t r e p a i r  i n c u b a t i o n time i n f i g . 17.  The 0  2  and 0  were o b t a i n e d from separate experiments. comparing 0  2  and 0  2  + TAN  Mw  2  values obtained  + TAN  v a l u e s shown  Repeat  experiments,  t r e a t e d samples i n the same  centrifuge rotor, yielded similar r e s u l t s , that i s s l i g h t l y l a r g e r Mw DNA  v a l u e s f o r TAN  treated c e l l s .  A l s o , Mw  values f o r  s y n t h e s i z e d d u r i n g a 3 hour chase i n c r e a s e w i t h  increasing  time of r e p a i r i n c u b a t i o n f o l l o w i n g a dose of 5 krads, w i t h or without TAN  p r e s e n t , under a e r o b i c c o n d i t i o n s , again presumably  50  ao Soo  -O  0 + TAN 2  -® CL  0  0  30  60  90  120  150  180  REPAIR TIME (minutes)  F i g u r e 17.  E f f e c t o f TAN Treatment d u r i n g A e r o b i c on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  Irradiation  CH2B c e l l s were i r r a d i a t e d a t 0°C t o 5 krads under a e r o b i c c o n d i t i o n s w i t h (O) o r without (©) 10 mM TAN. A f t e r b e i n g twice resuspended i n d r u g - f r e e medium, c e l l s were allowed t o r e p a i r f o r v a r i o u s times a t 37 C, p u l s e l a b e l l e d f o r 6 minutes and chased f o r 3 hours a t 37°C. C e l l s were l y s e d f o r 7 hours and spun a t 14,000 r.p.m. f o r 11 hours. 2  51  r e f l e c t i n g template r e p a i r .  T h i s i n c r e a s e i n Mw a f t e r  TAN treatment c o n t r a s t s with the e s s e n t i a l l y r e p a i r curve  f l a t Mw  aerobic  versus  shown i n f i g . 12 f o r TAN treatment under  hypoxia.  I t would be r a t h e r d i f f i c u l t t o e x p l a i n the o b s e r v a t i o n t h a t TAN treatment d u r i n g a e r o b i c i r r a d i a t i o n causes a small i n c r e a s e i n the s i z e o f the DNA made d u r i n g the p u l s e and chase, i f indeed  this shift i s significant.  suggest t h a t TAN competes with 0 these TAN m o d i f i e d  2  T h i s r e s u l t would  f o r damaged s i t e s and t h a t  s i t e s would then be more r a p i d l y  repaired;  however, i t was shown e a r l i e r  ( s e c t i o n 3.2.1) t h a t TAN-DNA  adducts formed d u r i n g hypoxic  i r r a d i a t i o n are not r e p a i r e d f o r  i n c u b a t i o n times of up t o 3 hours.  Hence, i t seems l i k e l y  t h i s d i f f e r e n c e i s of no r e a l importance. effect  that  In any event, the  i s s m a l l , causing only a small s h i f t i n sedimentation  p r o f i l e , t h a t i s a t best o f marginal t h e r e f o r e reasonable  significance.  It is  t o conclude t h a t TAN treatment d u r i n g  a e r o b i c i r r a d i a t i o n has no demonstrable e f f e c t on p o s t i r r a d i a t i o n DNA s y n t h e s i s .  52  3.2.5  Dose Response Although most of the experiments d e s c r i b e d i n t h i s work  i n v o l v e d exposure  t o 15 krads under hypoxia, or i t s e q u i v a l e n t  under other c o n d i t i o n s , the dose dependence of the p u l s e and 3 hour chase assay was  also  investigated.  Hypoxic c e l l suspensions w e r e . i r r a d i a t e d a t 0°C, with and without TAN,  and then twice resuspended  i n d r u g - f r e e medium.  C e l l s were maintained a t 0°C u n t i l being p u l s e l a b e l l e d (6 minutes  i n 20 yCi/ml H-TdR) and chased f o r 3 hours a t 37°C. 3  R e s u l t s are shown i n f i g .  18.  For c e l l s i r r a d i a t e d under hypoxia w i t h no drug p r e s e n t , the s i z e o f DNA  s y n t h e s i z e d d u r i n g the 3 hour chase  very l i t t l e with i n c r e a s i n g dose.  TAN  decreases  treatment, however,  leads t o a s u b s t a n t i a l dose-dependent decrease i n the Mw observed.  These r e s u l t s are r e p l o t t e d on a 1/Mw  graph i n f i g . fig.  versus dose  19; the d o t t e d l i n e shown i s the SSB curve  8 f o r i r r a d i a t i o n i n N 2 + TAN.  values  from  A p p a r e n t l y , the number o f  TAN-DNA adducts d e t e c t e d by the p u l s e and chase procedure i s s i m i l a r t o the number of SSB's produced.  Since TAN  a s m a l l i n c r e a s e i n the y i e l d of SSB's (see f i g . r e s u l t s suggest t h a t TAN,  causes o n l y  8), these  i n t e r a c t i n g with i r r a d i a t e d  DNA,  leads predominantly t o the f o r m a t i o n of adducts, r a t h e r than SSB's.  53  5  10  15  20  DOSE (krads)  Figure  18.  P o s t - I r r a d i a t i o n DNA S y n t h e s i s a s a F u n c t i o n o f Dose u n d e r H y p o x i a w i t h and w i t h o u t TAN  H y p o x i c CH2B c e l l s u s p e n s i o n s were i r r a d i a t e d a t 0°C w i t h (®) and w i t h o u t 10 mM TAN ( $ ) . A f t e r b e i n g t w i c e r e s u s p e n d e d i n d r u g - f r e e medium, c e l l s were p u l s e l a b e l l e d f o r 6 m i n u t e s a n d chased f o r 3 hours a t 37°C. C e l l s were l y s e d f o r 7 h o u r s a n d spun a t 14,000 r.p.m. f o r 11 h o u r s . 2  54  Figure  19.  Dose ' i/Mw  Response o f Newly v e r s u s Dose  Synthesized  DNA ~~'  Plotted "  as  :  F i g u r e 19 i s a r e p l o t o f . t h e d a t a i n f i g . 18 o n a 1/Mw scale. T h e d a s h e d l i n e shown i s t h e SSB r e s p o n s e f o r i r r a d i a t i o n i n N + TAN f r o m f i g . 8. 2  55  3.2.6  TAN  Concentration  In s u r v i v a l s t u d i e s i n Chinese Skarsgard TAN  and Emmerson  hamster c e l l s ,  (1969) found DMF's of 1.5  c o n c e n t r a t i o n s of 10 mM  and  1.0  mM  and  Parker, 1.3  respectively.  for  Revesz  and L i t t b r a n d (1970) have r e p o r t e d a dose modifying e f f e c t f o r TAN  c o n c e n t r a t i o n s as low as 250  line.  And,  yM f o r another hamster  u s i n g a r a d i o s e n s i t i v e s t r a i n of E. c o l i  Emmerson, F i e l d e n and Johansen  (1971) found t h a t 230  gave h a l f the maximum s e n s i t i z i n g  TAN  by twice resuspending  l a b e l l e d and chased The  The  1.0  were  i n the presence  of  c e l l s were then washed f r e e  f o r 3 hours, b e f o r e being l a y e r e d on ASG's. C o n c e n t r a t i o n s below 10 to  20 yM have no d e t e c t a b l e e f f e c t on the  approximately  TAN  i n d r u g - f r e e medium, p u l s e  r e s u l t s are shown i n f i g . 20.  s y n t h e s i s of DNA.  yM  c e l l suspensions  exposed to a dose of 15 krads under hypoxia  of  K-12,  effect.  In the f o l l o w i n g experiments,  v a r i o u s c o n c e n t r a t i o n s of TAN.  cell  post-irradiation  The maximum e f f e c t i s produced by mM  TAN.  TAN  c o n c e n t r a t i o n s h i g h e r than 10  mM  were not i n v e s t i g a t e d . The r e s u l t s shown here agree q u i t e w e l l w i t h the  half  maximum value of 230  yM f o r b a c t e r i a (Emmerson e t aJL. , 1971) .  However, the reduced  s e n s i t i z a t i o n seen a t 1.0  et  mM  by  Parker  a l . (1969) u s i n g the same mammalian c e l l l i n e i s not  apparent  i n t h i s p u l s e and chase  assay.  I t should be noted t h a t the Mw  v a l u e s shown i n f i g . 20  56  T A N  Figure  20.  C O N C E N T R A T I O N  (mM)  E f f e c t o f TAN C o n c e n t r a t i o n d u r i n g H y p o x i c I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  H y p o x i c C H 2 B c e l l s u s p e n s i o n s were i r r a d i a t e d a t 0 ° C t o 15 k r a d s i n t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f TAN. C e l l s were t h e n t w i c e r e s u s p e n d e d i n d r u g - f r e e medium, p u l s e l a b e l l e d f o r 6 m i n u t e s and c h a s e d f o r 3 h o u r s a t 3 7 ° C . Cells were l y s e d f o r 7 h o u r s a n d spun a t 14,000 r.p.m. f o r 11 h o u r s . Mw v a l u e s were c a l c u l a t e d . The e r r o r b a r s shown a t 0 mM and 10 mM a r e t h e s t a n d a r d e r r o r s i n t h e mean v a l u e s o b t a i n e d f r o m 5 and 4 s e p a r a t e g r a d i e n t s r e s p e c t i v e l y . 2  57  were obtained from c e l l s allowed no r e p a i r i n c u b a t i o n before the p u l s e and chase.  P a r a l l e l samples t h a t were allowed 30  minutes r e p a i r i n c u b a t i o n showed some evidence o f r e p a i r ,  that  i s s l i g h t l y l a r g e r Mw v a l u e s , f o r TAN c o n c e n t r a t i o n s o f .500 uM or l e s s .  T h i s r e s u l t suggests t h a t the CH2B  c e l l l i n e may  2  have a l i m i t e d c a p a c i t y t o r e p a i r TAN-DNA adducts.  3.2.7  Metronidazole and Ro-07-0582 Treatment  o f Hypoxic  Cells  The n i t r o i m i d a z o l e s , metronidazole and Ro-07-0582, have been shown t o r a d i o s e n s i t i z e hypoxic mammalian c e l l s more efficiently  (Asquith e t a l . ,  1975) than TAN  1974; Moore, P a l c i c and Skarsgard,  (Parker, Skarsgard and Emmerson,  1969).  a d d i t i o n , metronidazole has been shown t o b i n d t o DNA under hypoxia  ( W i l l s o n , Cramp and Ings, 1974).  In irradiated  Hence the  e f f e c t s o f these compounds on p o s t - i r r a d i a t i o n DNA s y n t h e s i s were i n v e s t i g a t e d . C e l l s were i r r a d i a t e d under hypoxia t o 6 krads i n the presence o f 15 mM metronidazole or Ro-07-0582, o r t o 15 krads i n d r u g - f r e e medium, c o n d i t i o n s which should  produce  approximately the same amount of i n i t i a l DNA damage.  Following  i r r a d i a t i o n a t 0°C, the c e l l s were washed f r e e o f drug, allowed to r e p a i r f o r v a r i o u s times, p u l s e l a b e l l e d and incubated f o r 3 hours as d e s c r i b e d p r e v i o u s l y , b e f o r e l a y e r i n g on ASG's.  The  r e s u l t s are shown i n f i g u r e s 21 and 22, w i t h drug and no drug r e s u l t s o b t a i n e d under s i m i l a r l y s i n g c o n d i t i o n s being  58  3.0  00 i  15 krads  o  • . N + METRONIDAZOLE  T—  2  X  X  CD  6 krads  2.0  5 2 O +-»  tr co  CJ UJ  1.0  _J O  0  30  0  JL  60  90  REPAIR TIME  F i g u r e 21.  120  150  180  (minutes)  E f f e c t o f M e t r o n i d a z o l e Treatment d u r i n g Hypoxic I r r a d i a t i o n , on P o s t - I r r a d i a t i o n DNA S y n t h e s i s  Hypoxic CH2B c e l l s were i r r a d i a t e d t o 6 krads a t 0°C i n the presence o f 15 mM m e t r o n i d a z o l e . A f t e r b e i n g twice resuspended i n d r u g - f r e e medium, c e l l s were allowed t o r e p a i r a t 37°C f o r v a r i o u s times, p u l s e l a b e l l e d f o r 6 minutes and chased f o r 3 hours. C e l l s were l y s e d f o r 7 hours and spun a t .14 ,0 00 r.p.m. f o r 11 hours. The dashed l i n e i s the newly s y n t h e s i z e d DNA response a f t e r exposure t o 15 krads under hypoxia i n d r u g - f r e e medium. 2  59  O N 1 5 krads • N+Ro-07-0582  h-  3.0  2  2  6  krads  CO •  o 2.0  I  O  Q  o-  I-  o  rr to _J  O  w  1J0  1  0  30  0  60  90  120  150  180  REPAIR TIME (minutes)  Figure  22.  E f f e c t o f Ro-07-0582 T r e a t m e n t d u r i n g H y p o x i c I r r a d i a t i o n on P o s t - I r r a d i a t i o n DNA Synthesis  H y p o x i c CH2B c e l l s were i r r a d i a t e d t o 6 k r a d s i n t h e p r e s e n c e o f 15 mM R o - 0 7 - 0 5 8 2 (®) o r t o 15 k r a d s i n d r u g - f r e e m e d i u m ( O ) . A f t e r b e i n g t w i c e r e s u s p e n d e d i n d r u g - f r e e medium, c e l l s w e r e a l l o w e d t o r e p a i r f o r v a r i o u s times a t 37°C, p u l s e l a b e l l e d f o r 6 minutes and chased f o r 3 hours a t 37°C. C e l l s were l y s e d f o r 7 h o u r s a n d s p u n a t 1 4 , 0 0 0 r . p . m . f o r 11 h o u r s . 2  60  presented i n each graph.  In a separate experiment  a l l 3 treatments i n the same c e n t r i f u g e differences  rotor,  comparing  no s i g n i f i c a n t  were found i n the Mw v a l u e s o b t a i n e d from hypoxic  c e l l s i r r a d i a t e d t o 15 krads i n d r u g - f r e e medium or t o 6 krads i n the presence of e i t h e r metronidazole o r Ro-07-0582. seems t h a t  the 2 n i t r o i m i d a z o l e s t e s t e d  e f f e c t on the template f u n c t i o n are TAN.  It  have no s i g n i f i c a n t  o f i r r a d i a t e d DNA and t h e r e f o r e  l i k e l y t o s e n s i t i z e by somewhat d i f f e r e n t mechanisms than  61  4.  4.1  DISCUSSION  RELIABILITY OF THE ALKALINE SUCROSE GRADIENT TECHNIQUE  As mentioned p r e v i o u s l y  ( s e c t i o n s 2.3.5 and 2.3.6), the  Mw v a l u e s o b t a i n e d from DNA sedimentation p r o f i l e s a r e dependent on both the time and the temperature o f l y s i s . However, w i t h i n a g i v e n experiment, the temperature o f l y s i s i s the  same f o r a l l g r a d i e n t s and the time o f l y s i s v a r i e s by o n l y  a few minutes, so t h a t w i t h i n the experiment, r e s u l t s a r e highly consistent.  In f a c t , i n a few cases i n which a l i q u o t s  from the same c e l l suspension were l y s e d on 2 d i f f e r e n t g r a d i e n t s i n the same experiment, the Mw v a l u e s o b t a i n e d d i f f e r e d by l e s s than 2 o r 3%. U n f o r t u n a t e l y , i t was r a t h e r more d i f f i c u l t t o o b t a i n a c c u r a t e l y r e p r o d u c i b l e Mw v a l u e s from experiment t o experiment. In o r d e r t o minimize v a r i a t i o n s due to l y s i n g c o n d i t i o n s , the l y s i n g time used was always 7 hours ± 5 minutes  (except f o r SSB  experiments, as d i s c u s s e d i n s e c t i o n 2.3.5), the 7 hours b e i n g the of  time from the completion o f c e l l l a y e r i n g u n t i l the s t a r t centrifugation.  A l s o , the temperature d u r i n g l y s i s was  u s u a l l y maintained between 22.5 and 24.5°C.  With these  procedures, v a r i a t i o n s o f up t o 20-30% s t i l l occured between Mw v a l u e s o b t a i n e d i n d i f f e r e n t experiments from  identically  62  treated from  cells.  Consequently,  separate experiments  verified  by  repetition  lysing  earlier  of those values i n a s i n g l e started,  were n o t e x p e c t e d  work w i t h t h i s ASG  t o be  sedimentation p r o f i l e s  to  times r a n g i n g from  Skarsgard,  1972a).  the e a r l i e r l y s i n g known.  One  temperature  The  results  EFFECTS OF  The  use  necessitated (see  by  f i g . 19)  fraction  temperature since  laboratory  relatively  hours  had  insensitive  (Palcic  and  f o r t h e d i s c r e p a n c y between those i n t h i s  thesis  i s not yet  of a s l i g h t l y higher  (22.5-24.5°C) d u r i n g t h e l y s i n g  period  than  (20-22°C).  HIGH DOSES  of h i g h doses  ( e . g . 15 k r a d s u n d e r h y p o x i a )  t h e low y i e l d o f TAN-DNA l e s i o n s  and  t h e i n a b i l i t y o f t h e ASG  s m a l l numbers o f t h e s e these doses  and  and  were  experiment.  factors  in this  t o be  6 t o 24  reason  t h a t employed p r e v i o u s l y  4.2  critical  p o s s i b i l i t y i s t h e use range  the time  technique  shown DNA lysing  between v a l u e s  were n e c e s s a r y , t h e r e s u l t s  When t h i s work was of  when c o m p a r i s o n s  lesions.  Although  a r e r e p r o d u c t i v e l y dead  o f CH2B2 c e l l s i r r a d i a t e d  per  kilorad  assay to d e t e c t c e l l s exposed  ( f o r example, t h e t o 15 k r a d s u n d e r  to surviving  hypoxia  -12 w o u l d be repair,  less  than  1x10  ), they  o f s y n t h e s i z i n g DNA  was  are capable, a f t e r  with very nearly  the  2  same Mw  hours as  63  that  of u n i r r a d i a t e d The  e f f e c t s of  progression studied of  (the G  the  radiation  the  delay  phases of  Walters,  the  the  cells  cycle.  Doses  cell  k i n e t i c s of  cell  progression  c y c l e to the  observation  Dewey, 1973;  time of  cells  will  delays  of only  Gurley  i t seems l i k e l y  DNA  are  irradiation.  move i n t o t h e  of  prior  to  15  in  the  G  2  and  that, during krads  the  under  doses under d i f f e r e n t c o n d i t i o n s ,  synthesize  the  r e s u l t s , which  of r a d i a t i o n - i n d u c e d  Nevertheless,  t h a t do  delay  However, s t u d i e s  hours a f t e r exposure to doses of  p h a s e a t the  this  cell  identification  1972).  few  some Gi  f o r a review).  have y i e l d e d c o n f l i c t i n g  hypoxia or e q u i v a l e n t the  widely  of which i s a f u n c t i o n of both  ( L e e p e r , S c h n e i d e r m a n and  first  cellular  c y c l e have been  Whitmore, 1967,  length  e f f e c t s on  division  block  synthetic  p o s i t i o n i n the  range from the all  f i g . 12).  h u n d r e d r a d s have been shown t o c a u s e d i v i s i o n  d o s e and  .the  DNA  E l k i n d and  block),  2  (see  i o n i z i n g r a d i a t i o n on  through the  (see  a few  cells  most  of  t h o s e w h i c h were i n S  It i s also possible DNA  synthetic  stage  that  during  time. Radioactive  during  the  precursors  that  p o s t - i r r a d i a t i o n pulse  replication  as w e l l  However, i t c a n  be  most o n l y  per  a few  unscheduled or  as  are  label  t a k e n up can  be  S phase s e m i - c o n s e r v a t i v e  readily calculated cent  r e p a i r DNA  of the  by  used  (see A p p e n d i x A)  since  the  cells  for repair  DNA  l a b e l l e d thymidine  synthesis,  the  synthesis. that  i s used  number  of  at for  64  nucleotides inserted 1973a).  per SSB i s l e s s than 10 (Fox and Fox,  A l s o , i t has been shown t h a t the p r o p o r t i o n o f  unscheduled t o normal s y n t h e s i s a f t e r UV exposures o f 200 ergs/mm  2  i s l e s s than 0.07 f o r a number o f mammalian c e l l  lines  (Fox and Fox, 1973b). In g e n e r a l  then, H-TdR i n t r o d u c e d 3  label after a large radiation f o r semi-conservative  4.3  by a 6 minute p u l s e  dose w i l l be used predominantly  DNA s y n t h e s i s by those c e l l s i n S phase.  EFFECTS OF IRRADIATION AND DRUG TREATMENT ON TEMPLATE FUNCTION  During i n c u b a t i o n a f t e r exposure t o i o n i z i n g c e l l u l a r DNA i s the s i t e o f a c t i o n o f both r e p a i r systems and the DNA r e p l i c a t i o n enzymes. apparatus encounters an u n r e p a i r e d  altered.  enzyme  I f the r e p l i c a t i o n  radiation-induced  the s y n t h e s i s o f daughter DNA opposite For i n s t a n c e , an unrepaired  radiation,  lesion,  t h i s l e s i o n may be SSB o r damaged base i n  the template c o u l d l e a d t o a gap i n the daughter  strand.  65  4.3.1  Hypoxic and Aerobic  Irradiation  A dose of 15 krads under hypoxia i n t r o d u c e s an average o f 1.5  to 2 SSB's per DNA  s t r a n d with Mw  ( u n i r r a d i a t e d f u l l - s i z e d DNA)  o f 2.6-3.0x10  (see f i g . 8).  daltons  8  Although the  r e j o i n i n g of these SSB's i s r a p i d (see s e c t i o n 3.1.2),  DNA  s y n t h e s i z e d immediately a f t e r i r r a d i a t i o n , t h a t i s with  no  r e p a i r i n c u b a t i o n , i s of s m a l l e r Mw  than t h a t s y n t h e s i z e d  after  a l l o w i n g some r e p a i r of r a d i a t i o n damage (see f i g . 12). Apparently,  damage done to the template DNA  under hypoxia i n t e r r u p t s DNA  by  s y n t h e s i s , but can be  r e p a i r e d to allow s y n t h e s i s of c o n t r o l - s i z e d DNA (although the g e n e t i c i n t e g r i t y of these known).  irradiation readily  fragments  fragments i s not  I t should be noted t h a t the r a d i a t i o n - i n d u c e d l e s i o n s  r e s p o n s i b l e f o r i n t e r r u p t i n g DNA  s y n t h e s i s are not n e c e s s a r i l y  SSB's and c o u l d w e l l r e p r e s e n t other damage. From the data presented  i n f i g . 8, i t can be seen t h a t  a dose of 5 krads under a e r o b i c c o n d i t i o n s produces o n l y s l i g h t l y more SSB's than 15 krads under hypoxia. Palcic  (1972) has  shown t h a t the k i n e t i c s of SSB  Further, r e j o i n i n g are  independent of whether the s t r a n d breaks are produced under a e r o b i c or hypoxic  conditions.  show an i n c r e a s e i n Mw  of DNA  The  data presented  i n f i g . 17  s y n t h e s i z e d d u r i n g a 3 hour chase  w i t h i n c r e a s i n g r e p a i r i n c u b a t i o n a f t e r a dose of 5 krads i n 0 i n d i c a t i n g template r e p a i r s i m i l a r to t h a t o c c u r r i n g a f t e r i r r a d i a t i o n under hypoxia.  T h i s r e s u l t suggests t h a t , i f 0  2  2  66  r e a c t s with DNA  r a d i c a l s predominantly by b i n d i n g to the  r a d i c a l s i t e , as i n d i c a t e d by the work of Simic and Hayon (1973), the peroxy r a d i c a l s formed do not a l t e r the f u n c t i o n of the i r r a d i a t e d DNA. c o n f i r m t h i s p r e d i c t i o n was  However, f u r t h e r work to  not done.  Another p o s s i b l e e x p l a n a t i o n i n the Mw  f o r the observed i n c r e a s e  of newly s y n t h e s i z e d daughter DNA  i n c u b a t i o n i s t h a t the o v e r a l l r a t e of DNA i n i r r a d i a t e d c e l l s and incubation.  template  i n c r e a s e s with  with r e p a i r s y n t h e s i s i s slowed  post-irradiation  However, r e c e n t work by Gerner e t a l . (1974)  suggests t h a t i o n i z i n g r a d i a t i o n has d u r a t i o n of S phase.  l i t t l e e f f e c t on  the  In any event, i t seems l i k e l y t h a t , a f t e r  exposure to the l a r g e doses used i n t h i s work, any r a d i a t i o n induced  delay i n c e l l p r o g r e s s i o n through S phase t h a t  may  e x i s t would not be a l t e r e d by a 2 hour r e p a i r p e r i o d . A l s o the r e s u l t s d i s c u s s e d here are c o n s i s t e n t w i t h those  r e p o r t e d by Korner and Malz  (1973), who  found t h a t  DNA  s y n t h e s i z e d by Chinese hamster c e l l s a f t e r X-ray doses of rads had cells.  lower molecular They concluded  segments were due  weight segments than u n i r r a d i a t e d  t h a t these  lower molecular  to gaps produced i n the  weight  complementary  daughter strands opposite r a d i a t i o n - i n d u c e d l e s i o n s i n the parental  strand.  500  67  4.3.2  TAN  Treatment  A f t e r a dose of 15 krads under hypoxia i n the presence of  10 mM  TAN,  DNA  i s s y n t h e s i z e d i n much s m a l l e r segments  those made by c e l l s i r r a d i a t e d i n the absence of TAN. the  Mw  of the DNA  than  Further,  s y n t h e s i z e d d u r i n g a 3 hour chase does not  i n c r e a s e s i g n i f i c a n t l y w i t h r e p a i r i n c u b a t i o n s of up t o 3 hours b e f o r e the p u l s e and chase  (see f i g . 12).  These r e s u l t s show  t h a t TAN-DNA adducts formed i n the template s t r a n d d u r i n g hypoxic i r r a d i a t i o n produce i n t e r r u p t i o n s i n the subsequent s y n t h e s i s of daughter DNA. readily  These TAN-DNA l e s i o n s are not  repaired. T r i t i a t e d TAN has been shown t o b i n d c o v a l e n t l y t o  r a d i a t i o n - i n d u c e d DNA  r a d i c a l s i n aqueous  solution  (Nakken,  S i k k e l a n d and B r u s t a d , 1970; B r u s t a d , Jones and Wold, 1973). Wold and Brustad to TAN  DNA  (1974) have a l s o demonstrated b i n d i n g of TAN  i n E. c o l i K-12  i r r a d i a t e d under hypoxic c o n d i t i o n s .  In  s e n s i t i z e d E. c o l i , these TAN-DNA adducts r e s u l t i n  i n t e r r u p t i o n s i n the s y n t h e s i s o f daughter strands 1969).  (Rupp e t a l . ,  Rupp and co-workers f u r t h e r showed t h a t w i l d type  E. c o l i can cope w i t h t h i s damage both by e x c i s i o n of TAN m o d i f i e d bases from the template DNA  and by recombination of  daughter s t r a n d s . The r e s u l t s presented i n s e c t i o n 3.2.1  and d i s c u s s e d  here are c o n s i s t e n t with the o b s e r v a t i o n s from b a c t e r i a l systems, and suggest t h a t a major component of TAN  sensitization  68  of hypoxic  mammalian c e l l s i s due  to the formation  of TAN-DNA  adducts which l e a d t o i n t e r r u p t i o n s i n subsequent DNA A dose o f 15 krads under hypoxia produces 1.5 adducts per f u l l - s i z e d DNA assuming t h a t every  segment  synthesis.  t o 2 TAN-DNA  (2.6-3.0xl0  8  daltons),  adduct leads to a s t r a n d i n t e r r u p t i o n .  In  a d d i t i o n , the Chinese hamster c e l l s used i n t h i s work appear to have, a t b e s t , a very l i m i t e d c a p a c i t y f o r r e p a i r of  TAN  lesions. The  o b s e r v a t i o n t h a t TAN  a e r o b i c CH2B synthesis  c e l l s has  present  during i r r a d i a t i o n  of  l i t t l e e f f e c t on p o s t - i r r a d i a t i o n  DNA  ( s e c t i o n 3.2.4) i s c o n s i s t e n t with r e p o r t s t h a t  TAN  2  s e n s i t i z e s n e i t h e r a e r o b i c b a c t e r i a (Emmerson, 1967) a e r o b i c mammalian c e l l s and does not b i n d to DNA  (Parker, Skarsgard  nor  and Emmerson,  1969)  i r r a d i a t e d i n the presence of oxygen  (Nakken, S i k k e l a n d and Brustad,  1970).  compete f o r r a d i a t i o n - i n d u c e d DNA  Presumably, 0  2  and  TAN  radicals.  F i g u r e 23 shows a h y p o t h e t i c a l schematic diagram of b i n d i n g to DNA  a f t e r hypoxic  i r r a d i a t i o n and  i n t e r r u p t i o n s i n newly s y n t h e s i z e d DNA.  causing  strand  In the absence of  i r r a d i a t i o n causes SSB's and other l e s i o n s i n the p a r e n t a l (only SSB's are shown i n f i g . 23). p u l s e l a b e l , the template DNA conservative  F o l l o w i n g an  TAN, DNA  immediate  i s the s i t e of both semi-  s y n t h e s i s and r e p a i r processes,  i n t e r r u p t i o n s w i l l occur  TAN  so t h a t some  i n daughter strands due  s y n t h e t i c apparatus encountering  unrepaired  to the  l e s i o n s (e.g.  the  69  i  pulse  chase  >  -X  hVy  IV  1 pulse •  chase.  II  WITH  T A N  ° l k >  pulse  K ) ° 1 o  TAN  molecule  —  parental  radioactive  Figure  23.  1  1  Ch  H O  0  -x  CAan  D  chase  ,  CH  Ci  il  i  DNA  daughter •  U l S e  1  chase.  O-  P  0  I*  DNA label  Schematic Diagram o f Proposed TAN Newly S y n t h e s i z e d Daughter DNA  Interruptions in  For d i s c u s s i o n see pages 68 and 70 of the t e x t .  70  gap m a r k e d "X" i n c a s e A ) .  I f repair i s allowed p r i o r  p u l s e l a b e l , many o f t h e damaged s i t e s w i l l synthesis w i l l In  proceed l a r g e l y unhindered  be r e p a i r e d a n d  (case B ) .  t h e p r e s e n c e o f TAN, TAN-DNA a d d u c t s w i l l  during hypoxic i r r a d i a t i o n , d e s c r i b e d above.  to the  i n a d d i t i o n t o those  W i t h no r e p a i r , s u b s e q u e n t  be f o r m e d  lesions  DNA s y n t h e s i s  will  be i n t e r r u p t e d b o t h b y TAN a d d u c t s a n d o t h e r u n r e p a i r e d damage (e.g. are  SSB's, case C ) .  I f , as s u g g e s t e d by f i g .  1 2 , TAN l e s i o n s  n o t r e a d i l y r e p a i r e d , then i n s p i t e o f t h e r e p a i r o f other  damage, t h e s y n t h e s i s o f DNA i n TAN t r e a t e d c e l l s in  small fragments a f t e r r e p a i r i n c u b a t i o n It  still  occurs  (case D).  s h o u l d be n o t e d t h a t s i n c e Mw v a l u e s do n o t i n c r e a s e  s i g n i f i c a n t l y w i t h r e p a i r t i m e f o r TAN t r e a t e d c e l l s ( s e e fig.  1 2 ) , t h e number o f i n t e r r u p t i o n s due t o TAN-DNA a d d u c t s i s  l a r g e compared t o t h o s e caused by o t h e r l e s i o n s fig.  2 3 ) . I t i s shown i n A p p e n d i x  (marked "X" i n  B that the expected  increase  i n Mw due t o r e p a i r o f " X - t y p e " s i t e s i s s m a l l , w i t h i n t h e scatter of the experimental N  2  + TAN p o i n t s i n f i g . 1 2 .  71  4.3.3  N i t r o i m i d a z o l e Treatment U n l i k e TAN  which produces l i t t l e  or no i n c r e a s e i n the  y i e l d of SSB's ( s e c t i o n 3.1.1; Agnew, 1972), the n i t r o i m i d a z o l e s , metronidazole  and Ro-07-0582, cause l a r g e  enhancements i n SSB p r o d u c t i o n d u r i n g hypoxic ( s e c t i o n 3.1.1).  A l s o , the presence  i r r a d i a t i o n appears  t o have l i t t l e  f u n c t i o n of p a r e n t a l DNA  irradiation  of n i t r o i m i d a z o l e s d u r i n g  e f f e c t on the  template  ( s e c t i o n 3.2.7), whereas TAN  i n t e r r u p t i o n s i n newly s y n t h e s i z e d DNA.  produces  None of the 3 drugs  examined i n h i b i t e d the r e j o i n i n g of SSB's.  These o b s e r v a t i o n s  are summarized i n Table I I along w i t h s u r v i v a l DMF  values  determined  Skarsgard,  1972;  by others i n t h i s l a b o r a t o r y (Agnew and  Moore, P a l c i c and Skarsgard, 1975;  Skarsgard,  Agnew, P a l c i c  and  1974).  Table I I :  Drug  DMF f o r Survival  DMF f o r SSB's  I n h i b i t s SSB Rejoining  A l t e r s DNA Template  TAN  1.5  1.3  No  Yes  Metronidazole  1.8-1.9  3.0  No  No  Ro-07-0582  = 3.0  3.7  No  No  72  As mentioned p r e v i o u s l y  ( s e c t i o n 1.5),  electron  affinic  r a d i o s e n s i t i z e r s r e a c t with DNA f r e e r a d i c a l s e i t h e r by e l e c t r o n t r a n s f e r or by adduct formation.  The data summarized  i n Table I I suggest t h a t metronidazole and Ro-07-0582 r e a c t w i t h i r r a d i a t e d DNA p r i m a r i l y by o x i d a t i o n by e l e c t r o n t r a n s f e r . Oxidation  o f the DNA f r e e r a d i c a l s o f t e n leads t o the  formation  o f a SSB.  TAN, on the other hand, binds t o DNA.  forming s t a b l e TAN-DNA adducts which l e a d t o i n t e r r u p t i o n s i n the subsequent s y n t h e s i s of daughter  strands.  I t i s p o s s i b l e t h a t the n i t r o i m i d a z o l e s form r a d i c a l s e n s i t i z e r adducts as w e l l but t h a t f o r some reason these l e s i o n s do n o t i n t e r r u p t DNA s y n t h e s i s .  However, i r r a d i a t i o n  of hypoxic s o l u t i o n s o f DNA c o n t a i n i n g r a d i o a c t i v e l y l a b e l l e d s e n s i t i z e r has y i e l d e d G v a l u e s ,  t h a t i s , number o f s e n s i t i z e r  molecules bound p e r 100 eV d e p o s i t e d ,  o f 1.3 f o r TAN  Jones and Wold, 1973) and ^0.02 f o r metronidazole Cramp and Ings, 1974). radiation-induced  T h i s supports the n o t i o n  (Brustad,  (Willson, that  b i n d i n g o f n i t r o i m i d a z o l e s t o DNA i s n o t a  s i g n i f i c a n t component o f t h e i r mode o f a c t i o n .  73  4.4  TAN  PRETREATMENT  Results pretreatment has synthesis.  presented i n s e c t i o n 3.2.2 no  does not bind  Brustad, Jones and TAN  i s not unexpected i n view of to u n i r r a d i a t e d  DNA  i n a c t i v a t e c e r t a i n DNA  i n newly s y n t h e s i z e d  Apparently, the TAN survival  (DMF  be e x p l a i n e d  = 1.3, by the  hence to  DNA.  Skarsgard, 1972)  cell  cannot r e a d i l y  formation of TAN-DNA adducts, s i m i l a r to TAN  treatment.  However, the  observation  t h a t some n i t r o x i d e f r e e r a d i c a l s l o s e t h e i r e l e c t r o n (ESR)  ( G i o t t a and t h a t TAN during  may  Wang, 1972;  Agnew and  Skarsgard, 1972)  suggests  become bound at other c r u c i a l s i t e s i n the  cell  This component of s e n s i t i z a t i o n would  a l s o n e c e s s a r i l y be p r e s e n t d u r i n g  i n the  spin  s i g n a l i n the presence of b i o l o g i c a l m a t e r i a l s  the pretreatment.  s i n c e TAN  that  r e p a i r systems,  pretreatment e f f e c t on  Agnew and  those produced d u r i n g  resonance  the  ( s e c t i o n 3.2.2;  to u n r e p a i r e d damage i n the template and  interruptions  DNA  Wold, 1973), although i t i s c o n c e i v a b l e  pretreatment could  leading  TAN  s i g n i f i c a n t e f f e c t on p o s t - i r r a d i a t i o n  This observation  f a c t t h a t TAN  showed t h a t  a TAN  treatment experiment  i s p r e s e n t at l e a s t 45 minutes p r i o r to i r r a d i a t i o n  treatment procedure.  74  4.5  TAN POST-TREATMENT  In s e c t i o n  3.2.3, r e s u l t s were presented  indicating  t h a t TAN added a f t e r i r r a d i a t i o n has l i t t l e e f f e c t on subsequent DNA s y n t h e s i s . have shown t h a t  addition  produces almost f u l l  s u r v i v a l , and  (1973) have demonstrated t h a t  minutes i n v i t r o .  f a i l u r e t o observe a TAN post-treatment e f f e c t on  post-irradiation  DNA s y n t h e s i s c o u l d be due t o s e v e r a l  F i r s t o f a l l , Brustad e t a l . (1973) have found t h a t transients sensitive transients  some DNA  which are able t o i n t e r a c t with TAN, have a  l i f e t i m e of s e v e r a l The  (1972)  o f TAN immediately a f t e r i r r a d i a t i o n  s e n s i t i z a t i o n of c e l l  Brustad, Jones and Wold transients,  However, Agnew and Skarsgard  and t h e i r r e a c t i o n to trace that  factors.  the DNA  with TAN in. v i t r o are extremely  amounts of oxygen.  In f a c t , the l o n g - l i v e d  they r e p o r t e d were o b t a i n e d under anoxia.  Post-treatment experiments were c a r r i e d out by adding an aliquot  of i r r a d i a t e d c e l l  containing solution,  suspension t o an a e r o b i c TAN-  i n both the r e s u l t s r e p o r t e d here and  those o f Agnew and Skarsgard.  I f oxygen does compete w i t h TAN  f o r DNA s i t e s p e r s i s t i n g a f t e r i r r a d i a t i o n , as i t appears t o f o r s i t e s produced d u r i n g i r r a d i a t i o n  (see s e c t i o n  3.2.4), TAN  a l t e r a t i o n of the DNA template would not occur d u r i n g our procedure.  Hence the post-treatment e f f e c t on c e l l  would n o t be due t o t h i s mode o f a c t i o n .  survival  75  A l s o , t h e extreme  t e m p e r a t u r e d e p e n d e n c e o f t h e TAN  p o s t - t r e a t m e n t e f f e c t on c e l l the  s u r v i v a l makes i t u n l i k e l y  mechanism o f p o s t - t r e a t m e n t i n v o l v e s i n t e r a c t i o n  w i t h r a d i a t i o n - i n d u c e d DNA r a d i c a l s . produces at  almost f u l l  S k a r s g a r d , 1972).  a 1 minute  incubation  a t 35°C a n d 37°C (Agnew a n d  Mechanisms f o r t h e removal o f f r e e  f r o m c e l l u l a r DNA w o u l d n o t l i k e l y e x h i b i t temperature  o f TAN  TAN p o s t - t r e a t m e n t  sensitization after  33°C b u t much s m a l l e r e f f e c t s  radicals  this high  sensitivity.  T h u s , i t i s r e a s o n a b l e t o c o n c l u d e t h a t TAN p o s t t r e a t m e n t s e n s i t i z a t i o n o f mammalian c e l l s b i n d i n g o f TAN t o l o n g - l i v e d DNA  that  radicals.  i s n o t due t o  76  5.  In  this  (1) increases  Both  TAN  (2)  TAN,  rejoining (3)  cells  the  TAN  DNA.  during (4)  only.a  metronidazole strand  present  during  interruptions effect  strand breaks  small  treatments  and Ro-07-0582  irradiation  TAN cell  effect  mode o f TAN  do n o t  inhibit  of hypoxic  observed  both  on c e l l effect  action.  the interruptions  treatment  hypoxic  CH2B  2  synthesis of  represent a major when  component o f TAN i s  a p r e - and a survival,  these  effects  Alternatively,  i n DNA  post-  on p o s t - i r r a d i a t i o n  Hence, p r e - and p o s t - t r e a t m e n t  that  during  breaks.  exhibits  h a v e no d e m o n s t r a b l e  a different  conclude  TAN  sensitizing  synthesis.  large  increase.  i n the subsequent  may  produce  irradiation.  Although  irradiation  must one  be  DNA due  must  synthesis observed f o r  are not the e x p l a n a t i o n o f the drug's  effect  on  survival. (5)  do  causes  This  and Ro-07-0582  mechanism o f t h e s e n s i t i z a t i o n  present  to  shown:  of single  of single  produces  daughter  have  metronidazole  i n the y i e l d  irradiation;  the  w o r k we  CONCLUSIONS  not alter  interrupt  The n i t r o i m i d a z o l e s , irradiated  metronidazole  p a r e n t a l DNA  the synthesis of daughter  i n such strands.  and  Ro-07-0582,  a manner  as t o  77  APPENDIX A  C a l c u l a t i o n o f Approximate Percentage o f Pulse L a b e l Used f o r Repair R e p l i c a t i o n 1 rad = 1 0 0 ergs/gram 1  dalton  1  eV = 1 . 6 0 2 x l 0  =  1 . 6 6 0 x 1 0 g r a m -  1  2  ergs  M = number of grams of DNA  per c e l l  f = f r a c t i o n o f c e l l s i n S phase Average molecular weight of 1 n u c l e o t i d e - 3 4 0 d a l t o n s . CH2B  2  c e l l s have approximately a 6 hour S phase i n a 1 2 hour  c e l l cycle.  Hence i n 6 minutes, a c e l l  i n S phase  will  6  s y n t h e s i z e ^ •jg-g- x M grams o f  DNA.  T h e r e f o r e i n 6 minutes, the number o f n u c l e o t i d e s used f o r semic o n s e r v a t i v e DNA s y n t h e s i s by an asynchronous C H 2 B , . . 1 Mxl0 1 population i s ^ f x _ x x ^ 2 1 +  1  >  6  6  2  cell  Q  = f x M x 2.95  x  10  1  9  .  Energy r e q u i r e d t o produce 1 S S B under hypoxic c o n d i t i o n s (from Table I ) 15,000  i s 1 1 5 eV = 1 1 5 x 1 . 6 0 2 x 1 0 ~  rads w i l l  deposit 1.5 x l O  1  2  ergs/gram of  6  ergs. DNA.  Therefore the number of S S B ' s produced per c e l l by a dose of 15  krads under hypoxia i s  1.5  x  115  x 1.602  =  8.14  10  6  x  M  x 10 "  x M x  10  1  1  2  5  .  78  C e l l s not i n Gj w i l l have more than the normal complement o f M grams o f DNA but not more than 2M;  t h e r e f o r e the maximum  number of S S B ' s / c e l l w i l l be ^ 2 x 8.14 x M x 1 0 = 1.63  M  x  10  x  1 6  1 5  .  Assuming t h a t 10 n u c l e o t i d e s are i n s e r t e d p e r SSB and, as a l a r g e o v e r e s t i m a t e , t h a t a l l SSB's are r e p a i r e d d u r i n g  the 6  minute p u l s e , the number o f n u c l e o t i d e s used f o r r e p a i r r e p l i c a t i o n w i l l be ^ 1.63 x M X 1 0  1 7  .  I t f o l l o w s t h a t the percentage o f the H-TdR, i n c o r p o r a t e d 3  d u r i n g the 6 minute p u l s e l a b e l , t h a t i s used f o r r e p a i r r e p l i c a t i o n w i l l be l e s s than ^ — f  1 63 x  M  x x  M  x  2.95  1 0 1 6  i o  0.55 f * 1% f o r f = 0.5.  •j  x 100%  r  x  1  9  79  APPENDIX B  I n f i g . 1 2 , t h e Mw o f DNA, s y n t h e s i z e d  after irradiation  h y p o x i a i n d r u g - f r e e medium, i n c r e a s e s  from ^ 2.14x10  with  no r e p a i r t o ^ 2 . 7 8 x l 0  daltons  8  with  under daltons  s  a 120 m i n u t e  repair  2 78 incubation. sites  This r e s u l t implies  <v ^' ^  ( s e e f i g . 23) p e r f u l l - s i z e  - 1 = 0.30 " X - t y p e "  ('v 2 . 8 x l 0  8  d a l t o n s ) DNA  segment. After  12 0 m i n u t e s r e p a i r f o l l o w i n g h y p o x i c i r r a d i a t i o n  p r e s e n c e o f TAN, t h e Mw o f DNA s y n t h e s i z e d ^  1.02xlo  daltons  8  i n the  i n 3 hours i s  ( f i g . 1 2 ) . T h e r e f o r e , t h e number o f  m e a s u r a b l e TAN-DNA a d d u c t s p r o d u c e d p e r f u l l - s i z e d  segment  2 78 is % ^ ' - 1 - 1 . 7 ,  since  no d e t e c t a b l e  a f t e r 2 hours r e p a i r incubation f r e e medium). TAN,  remain  (after i r r a d i a t i o n i n drug-  W i t h no r e p a i r f o l l o w i n g i r r a d i a t i o n i n N  there w i l l  be ^ 1.7 + 0.30 i n t e r r u p t i o n s  adducts and u n r e p a i r e d X-type l e s i o n s ) synthesized  X-type l e s i o n s  i n the next 3 hours.  +  2  (due t o TAN-DNA  i n t h e DNA s e g m e n t s  H e n c e , one w o u l d e x p e c t a Mw  2 78 of ^ N  2  1  7  +  0  *  3  0  +  1  ~  0  '93 l° x  + TAN l i n e i n f i g . 12.  r a n g e f r o m 0.93 t o 1 . 1 2 x 1 0 increase  8  daltons,  The N 8  2  a t no r e p a i r o n t h e  + TAN v a l u e s shown i n f i g . 12  daltons,  so t h a t  the expected  i n Mw due t o r e p a i r o f X - t y p e l e s i o n s d u r i n g  incubation  falls within  TAN  i n f i g . 12, and hence i s u n d e t e c t a b l e .  points  repair  the experimental scatter of the N  2  +  80  BIBLIOGRAPHY  ADAMS, G.E. and DEWEY, D.L. (1963). 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