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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 DNA DAMAGE IN HYPOXIC MAMMALIAN CELLS by WILLIAM FRANK HOHMAN B . S c , U n i v e r s i t y o f W a t e r l o o , 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF 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 as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF ERITISH COLUMBIA J a n u a r y , 197 5 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t i on of th i s thes is for f inanc ia l gain sha l l not be allowed without my wr i t ten permission. Department of /77<£-&/t:/9/L. <^g^rr/<r s The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e y 9 H > ^ J X.T / " ? 7 C RESEARCH SUPERVISOR: Professor L. D. Skarsgard, Ph.D. ABSTRACT: The modification of DNA damage by 3 r a d i o s e n s i t i z i n g drugs, present during y - i r r a d i a t i o n of hypoxic Chinese hamster c e l l s , was investigated. The method of al k a l i n e sucrose gradients (ASG's) was used to determine the size d i s t r i b u t i o n of DNA molecules. Both metronidazole and Ro-07-0582 were found to cause large increases i n the y i e l d of DNA single strand breaks (SSB's); triacetoneamine-N-oxyl (TAN) was found to have only a small e f f e c t on SSB production. A pulse l a b e l and chase procedure was used to examine p o s t - i r r a d i a t i o n DNA synthesis. TAN present during i r r a d i a t i o n under hypoxia was found to cause interruptions i n subsequent DNA synthesis. Metronidazole 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 synthesis. . In addition, the e f f e c t s of pre- and p o s t - i r r a d i a t i o n exposure to TAN were investigated, since these treatments have shown increased c e l l k i l l i n g i n survival studies (Agnew and Skarsgard, 1972). TAN pre- and post-treatments were found to have no s i g n i f i c a n t e f f e c t on subsequent DNA synthesis. i i i TABLE OF CONTENTS Page 1. INTRODUCTION 1 1.1 Cancer and Radiation Therapy 1 1.2 Radiation Damage of Parental DNA 2 1.3 Repair of Irradiated Parental DNA 4 1.4 DNA Synthesis 5 1.5 Chemical Radiosensitizers 7 2. MATERIALS AND METHODS 11 2.1 C e l l Manipulations 11 2.1.1 C e l l Line 11 2.1.2 I r r a d i a t i o n 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 and Chase Incubations 14 2.2 Radiosensitizing Drugs 16 2.3 Alkaline Sucrose Gradients 18 2.3.1 Gradient and Lysing Solutions 18 2.3.2 Lysing Procedure 19 2.3.3 C e n t r i f ugation 19 2.3.4 Gradient Fractionation and Sample Counting 20 2.3.5 Lysing Time 21 2.3.6 Lysing Temperature 23 2.3.7 Calculation of Molecular Weight 2 3 i v Page 3. RESULTS 2 6 3.1 P a r e n t a l DNA 2 6 3.1.1 S i n g l e Strand Break P r o d u c t i o n 2 6 3.1.2 R e j o i n i n g of S i n g l e Strand Breaks . . . . 32 3.2 Newly S y n t h e s i z e d Daughter DNA 36 3.2.1 TAN Treatment of Hypoxic CH2B 2 C e l l s . . . 36 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 CH2B 2 C e l l s . . . 49 3.2.5 Dose Response 52 3.2.6 TAN C o n c e n t r a t i o n 55 3.2.7 M e t r o n i d a z o l e and Ro-07-0582 Treatment of Hypoxic C e l l s 57 4. DISCUSSION 61 4.1 R e l i a b i l i t y of the A l k a l i n e Sucrose G r a d i e n t Technique 61 4.2 E f f e c t s of High Doses 62 4.3 E f f e c t s of I r r a d i a t i o n and Drug Treatment on Template F u n c t i o n 64 4.3.1 Hypoxic and A e r o b i c I r r a d i a t i o n 65 4.3.2 TAN Treatment 67 4.3.3 N i t r o i m i d a z o l e Treatment 71 4.4 TAN Pretreatment 73 4.5 TAN Post-treatment 74 V Page 5. CONCLUSIONS 76 APPENDIX A 77 APPENDIX B . . . 79 BIBLIOGRAPHY 80 v i LIST OF TABLES T a b l e I T a b l e I I page page 30 71 v i i LIST OF FIGURES Figure T i t l e Page 1 Proposed Mechanisms of Radiosensitization and 9 Radioprotection 2 Continuously Labelled and Pulse Labelled DNA 15 3 Flow Chart Representation of the Various 17 Experimental Procedures 4 E f f e c t of Lysing Time on Sedimentation P r o f i l e 22 5 E f f e c t of Lysing Temperature on Sedimentation 24 P r o f i l e 6 Production of Single Strand Breaks under 28 Aerobic and Hypoxic Conditions 7 Production of Single Strand Breaks under 29 Hypoxia with TAN or Ro-07-0582 8 Graph of 1/Mw versus Dose for Single Strand 31 Break Production under Several Conditions 9 Rejoining of Single Strand Breaks afte r Hypoxic 34 I r r a d i a t i o n with and without TAN 10 Rejoining of Single Strand Breaks afte r Hypoxic 35 I r r a d i a t i o n with and without Ro-07-0582 11 Sedimentation P r o f i l e s for Newly Synthesized 38 DNA aft e r I r r a d i a t i o n under Hypoxia with and without TAN 12 E f f e c t of TAN Treatment during Hypoxic 40 I r r a d i a t i o n on Post-Irradiation DNA Synthesis 13 Sedimentation P r o f i l e s for Parental and Newly 42 Synthesized Daughter DNA a f t e r I r r a d i a t i o n under Hypoxia with and without TAN 14 E f f e c t of TAN Pretreatment on Post-Irradiation 44 DNA Synthesis v i i i F i g u r e T i t l e 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 C o n t i n u o u s l y 46 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 48 DNA S y n t h e s i s 17 E f f e c t o f TAN Treatment d u r i n g A e r o b i c 50 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 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 53 o f Dose under Hy p o x i a w i t h and w i t h o u t TAN 19 Dose Response o f Newly S y n t h e s i z e d P l o t t e d as 54 1/Mw v e r s u s Dose 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 56 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 21 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 58 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 22 E f f e c t o f Ro-07-0582 Treatment d u r i n g H y p o x i c 59 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 2 3 Schematic Diagram o f Proposed TAN 69 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 Daughter DNA r ix ACKNOWLE DGEMENT I wish to express my gratitude to Dr. L. D. Skarsgard, under whose supervision t h i s project was carr i e d out. 1 1. INTRODUCTION 1.1 CANCER AND RADIATION THERAPY Radiation therapy i s the treatment of choice for many human tumours. For some types of cancer, however, the effectiveness of t h i s mode of therapy i s thought to be limited by the r e l a t i v e radioresistance of the hypoxic f r a c t i o n of a tumour growth. These re s i s t a n t c e l l s have a reduced oxygen tension that arises as the tumour outgrows i t s vascular supply. It i s known that such hypoxic c e l l s are much more re s i s t a n t to the l e t h a l e f f e c t s of i o n i z i n g radiation than the well oxygenated c e l l s found i n normal tissue. Since the dose delivered to a tumour volume i s limited by the tolerance of adjacent healthy tissues, any procedure that reduces t h i s r e l a t i v e radioresistance should increase the effectiveness of radiation therapy. One possible solution to t h i s problem i s the use of chemical r a d i o s e n s i t i z e r s which s p e c i f i c a l l y s e n s i t i z e hypoxic c e l l s to i o n i z i n g radiation. Many compounds have been shown to exhibit t h i s selective s e n s i t i z a t i o n , l a r g e l y i n i n v i t r o systems. I t i s hoped that the study of the ef f e c t s of these s e n s i t i z e r s at the molecular l e v e l w i l l f a c i l i t a t e t h e i r application to human radiotherapy. 2 1.2 RADIATION DAMAGE OF PARENTAL DNA DNA (deoxyribonucleic acid) i s usually regarded as the primary molecular target for radiation-induced c e l l death, since genetic i n t e g r i t y i s necessary for normal c e l l u l a r l i f e and reproduction. Presumably, other damaged c e l l u l a r components can be replaced i f the required genetic information i s present i n i n t a c t DNA molecules. Exposure to radiation, both i o n i z i n g and non-ionizing, i s known to damage DNA molecules. C e l l u l a r absorption of u l t r a -v i o l e t (UV) l i g h t , a non-ionizing radiation, leads mainly to the formation of pyrimidine dimers between adjacent pyrimidine bases i n the same DNA strand. Ionizing radiation, on the other hand, produces a whole spectrum of DNA damage including both single and double strand breaks, and base and sugar damage. A single strand break (SSB) occurs when radiation causes a s c i s s i o n of only one strand of the sugar-phosphate DNA backbone. The production of SSB's has been investigated by many authors i n organisms ranging i n complexity from viruses to mammalian c e l l s . A large portion of these studies has employed the alkaline sucrose gradient (ASG) technique introduced by McGrath and Williams i n 1966. This technique was used i n t h i s work to measure SSB production i n Chinese hamster c e l l s . It should be noted that the ASG assay used i n t h i s 3 work (section 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 radiation exposure. Hence the two types of bond ruptures are c o l l e c t i v e l y referred to as SSB's. In addition, some of the SSB's measured w i l l be due to double strand breaks. Double strand breaks are produced when both strands are damaged very close together. The appearance of double strand breaks aft e r i r r a d i a t i o n has also been studied i n various c e l l types with several authors having reported y i e l d s of one double strand break for every 10 to 15 SSB's ( F r e i f e l d e r , 1966; B u r r e l l , Feldschreiber and Dean, 1971; Veatch and Okada, 1969). L i t t l e i s known about the base and sugar damage caused by i o n i z i n g radiation i n c e l l u l a r systems. However, recently Roti Roti and Cer u t t i (1974) have reported that gamma-rays produce thymine damage in Chinese hamster c e l l s with an e f f i c i e n c y similar to that for SSB's. In addition, treatment of i r r a d i a t e d E. C o l i with an endomiclease has been shown to cause a large increase i n the number of strand breaks, beyond those caused by the radiation alone (Setlow and Car r i e r , 1973). Presumably, these endonuclease-sensitive s i t e s r e f l e c t base damage in 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 repair systems to cope with damage to t h e i r DNA. After exposure to radiation, repair processes that may occur include the excision of pyrimidine dimers or other damaged bases, and the r e j o i n i n g of SSB's. Perhaps the most extensively studied enzymatic repair system i s the removal of thymine dimers following UV i r r a d i a t i o n . This excision repair system f i r s t removes a small segment containing the dimer and then resynthesizes the region using the complementary strand as template (for a review see Rauth, 1970). The process i s also c a l l e d repair r e p l i c a t i o n or unscheduled DNA synthesis and can occur throughout the c e l l cycle (Painter and Cleaver, 1969). Repair r e p l i c a t i o n has also been observed i n mammalian c e l l s a f t e r exposure to i o n i z i n g radiation (Painter and Cleaver, 1967; Brent and Wheatley, 1971). Generally, the number of nucleotides inserted per SSB produced i s small, of the order of 2 to 10 (Fox and Fox, 1973a; Painter and Young, 1972). The r e j o i n i n g of SSB's i n mammalian DNA has been extensively studied i n many laboratories. This process occurs throughout the c e l l cycle and appears to be f a i r l y rapid, being largely complete i n a few hours (Lett et a l . , 1967; Sawada and Okada, 1970; Fox and Fox, 1973c). Rejoining i s temperature 5 dependent, being completely i n h i b i t e d at 0°C (Sawada and Okada, 1970; P a l c i c and Skarsgard, 1972b). Mammalian c e l l s have been reported to be incapable of repairing double strand breaks (Sawada and Okada, 1970; Lehmann and Omerod, 1970) although double strand break re j o i n i n g has been reported i n Chinese hamster c e l l s (Corry and Cole, 197 3). While t h i s disagreement i s not yet resolved, the double strand break has been proposed as the l e t h a l molecular event for io n i z i n g radiation (Chadwick and Leenhouts, 1973). 1.4 DNA SYNTHESIS Normal semi-conservative DNA synthesis occurs during the S phase of the c e l l cycle. In mammalian c e l l s , DNA r e p l i c a t i o n appears to involve the i n i t i a t i o n and synthesis of small fragments, known as replicons, at numerous s t a r t i n g points on the template DNA. These small fragments are subsequently joined to form larger i n t a c t DNA molecules (Goldstein and Rutman, 1973; Taylor, 1973; Ioannu, 1973; Schandl and Taylor, 1969). Exposure to both i o n i z i n g and non-io n i z i n g radiation i s known to a f f e c t DNA r e p l i c a t i o n i n mammalian c e l l s . Mammalian DNA synthesized within 1 or 2 hours a f t e r UV i r r a d i a t i o n i s made in smaller segments than that synthesized 6 by unirradiated c e l l s , suggesting that unexcised pyrimidine dimers interrupt DNA synthesis, leaving gaps i n the newly synthesized strands. Upon incubation, these short segments are elongated and joined to form high molecular weight DNA (Cleaver and Thomas, 1969; Buhl et a l . , 1972; Chija and Rauth, 1972). In addition, at long times a f t e r UV i r r a d i a t i o n , Chinese hamster, mouse and human c e l l s regain the a b i l i t y to synthesize DNA i n nearly the same size as unirradiated c e l l s (Buhl, Setlow and Regan, 1973; Meyn and Humphrey, 1971; Lehmann and K i r k - B e l l , 1972). Ionizing radiations are known to depress the rate of incorporation of precursors into mammalian DNA; t h i s decreased incorporation i s generally interpreted as a lower rate of nuclear DNA synthesis (Makino and Okada, 1975). The dose response of radiation-induced depression of DNA synthesis i s biphasic with a radiosensitive component ( D 3 7 - l krad) and a much more r e s i s t a n t component (D 3 7-20 krads). Makino and Okada have suggested that the radiosensitive component represents the i n i t i a t i o n of synthesis i n replicons and that the radio-r e s i s t a n t process i s the elongation i n replicons i n which synthesis has already been i n i t i a t e d . I t has also been shown that DNA synthesized i n Chinese hamster c e l l s 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 unirradiated c e l l s . P o s t - i r r a d i a t i o n incubation increases the size of the newly synthesized DNA to control 7 levels (Korner and Malz, 1973). In the work reported here, the a b i l i t y of Chinese hamster c e l l s to synthesize control size DNA at various times aft e r gamma-irradiation under d i f f e r e n t conditions was examined. 1.5 CHEMICAL RADIOSENSITIZERS Many drugs are known to modify the radiation response 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 relevance to cancer therapy are those compounds which s e l e c t i v e l y s e n s i t i z e hypoxic mammalian c e l l s to the k i l l i n g e f f e c t s of i o n i z i n g radiation. The f i r s t reports of t h i s s e l e c t i v e s e n s i t i z a t i o n were published by Parker, Skarsgard and Emmerson (1966, 1969) and Ashwood-Smith et a l . (1967). In 196 3, Adams and Dewey suggested that the electron a f f i n i t y of a compound was d i r e c t l y related to i t s e f f e c t i v e -ness as a s e n s i t i z e r ; t h i s electron a f f i n i c s e n s i t i z a t i o n can be explained as f i x a t i o n or oxidation of free r a d i c a l s , produced either d i r e c t l y i n the target molecule, or i n d i r e c t l y by the attack on the target molecule of reactive species produced by the i o n i z i n g radiation. Recently, Chapman and co-workers have proposed a model which explains both r a d i o s e n s i t i z a t i o n and protection by interaction of compounds with free r a d i c a l s produced i n DNA 8 (Chapman, Reuvers, Borsa and Greenstock, 1973). This model i s a generalization of the oxygen-fixation hypothesis (Alexander, 1962) and i s represented schematically i n f i g . 1. In t h i s model, DNA r a d i c a l s , produced either by the d i r e c t e f f e c t of the i o n i z i n g radiation or the i n d i r e c t e f f e c t of the water r a d i o l y s i s products OH" and H*, are subject to competing repair and f i x a t i o n processes. The reduction of DNA r a d i c a l s (ie. by H donation by sulfhydryl compounds) leads to repair. Oxidation of these r a d i c a l s fixes the damage i n an altered chemical structure which may lead to c e l l death. Oxidation may occur either by adduct formation with, or electron transfer to, chemical species with higher electron a f f i n i t i e s . In the model, radioprotectors could act either by competing with endogenous oxidizing compounds for target free r a d i c a l s and so making repair by reduction more l i k e l y , or by scavenging OH* or H* r a d i c a l s . Chemical ra d i o s e n s i t i z e r s could function either by binding to endogenous reducing species or by competing d i r e c t l y with them for DNA free r a d i c a l s , increasing the p r o b a b i l i t y of f i x a t i o n by oxidation. The mechanism of most electron 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 to involve oxidation of target free r a d i c a l s . Several radioa 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 bind to i r r a d i a t e d DNA, forming stable adducts; compounds assayed include triacetoneamine-N-oxyl (TAN) (Nakken; Sikkeland and Brustad, 1970), nitrofurazone (Chapman, 9 Direct Effect Indirect Effect NAAA/V V W W Repair by Reduction Fixation by Oxidation ie. by H donation ie. by adduct formation, electron transfer P - Reducing Species S - O x i d i z i n g Species Figure 1. Proposed Mechanisms of Radiosensitization and  Radioprotection (from Chapman ejt a l . , 1974; Willson and Emmerson, 1970) 10 Greenstock, Reuvers, McDonald and Dunlop, 1973), and metronidazole (Willson, Cramp and Ings, 1974). The amount of a c t i v i t y bound increases l i n e a r l y with dose. Binding does not occur i n the presence of oxygen, presumably due to competition between s e n s i t i z e r and 0 2 for the r a d i c a l s i t e . However, the ef f e c t s of these lesions on the template function of the i r r a d i a t e d DNA are largely unknown, although TAN has been shown to i n t e r f e r e with DNA synthesis i n i r r a d i a t e d bacteria (Rupp et a l . , 1969). Our work has been concerned mainly with the study of the synthesis of mammalian DNA a f t e r i r r a d i a t i o n of parental DNA i n the presence of r a d i o s e n s i t i z i n g drugs. A large portion of t h i s research has involved the use of TAN, a drug which shows both a pre- and a p o s t - i r r a d i a t i o n s e n s i t i z i n g e f f e c t (Agnew and Skarsgard, 1972). I t therefore seems l i k e l y that TAN enhances radiati o n damage by somewhat d i f f e r e n t mechanisms than most electron a f f i n i c compounds, perhaps by forming stable unrepaired DNA adducts. Metronidazole and Ro-07-0582, on the other hand, behave as t y p i c a l electron a f f i n i c s e n s i t i z e r s , acting only i f present during i r r a d i a t i o n (Agnew, P a l c i c and Skarsgard, 1974; Moore, P a l c i c and Skarsgard, 1975). Using these three s e n s i t i z e r s , we ca r r i e d out a search for drug-s p e c i f i c differences i n the DNA synthesized a f t e r i r r a d i a t i o n under hypoxia. 11 2. MATERIALS AND METHODS 2.1 CELL MANIPULATIONS 2.1.1 C e l l Line Chinese hamster c e l l s of the CH2B2 l i n e , a cl o n a l derivative of CHEF-125 (Prescott and Bender, 1963), have been used i n these experiments. The CH2B2 c e l l s were grown attached to p l a s t i c i n tissue culture flasks (Falcon P l a s t i c s , Oxnard, Cal i f o r n i a ) i n minimum es s e n t i a l medium (MEM) F-16 supplemented with 10% f e t a l c a l f serum (Grand Island B i o l o g i c a l Company, Grand Island, New York); they were incubated at 37°C i n an atmosphere of 95% a i r , 5% carbon dioxide and 100% humidity. Their doubling time was 12-14 hours. The c e l l s were subcultured every 3 to 4 days using 0.1% trypsin (Bacto-Trypsin, Difco Laboratories, Detroit, Michigan) to detach them from the p l a s t i c . C e l l s were exposed to t r y p s i n , previously warmed to 37°C, for 8 minutes at room temperature. The action of the trypsin was stopped by the addition of an equal volume of growth medium. The c e l l s were then centrifuged (8 minutes at 600 rpm), resuspended i n growth medium and plated into new culture f l a s k s . C e l l s to be used for experiments were plated and grown 12 for 2 days before harvesting by t r y p s i n i z a t i o n . Fresh growth medium was added to the c e l l s 1 day p r i o r to harvesting. 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 for experiments were prepared from log phase monolayers. The c e l l s were trypsinized, centrifuged and resuspended at a concentration of 1 x 1 0 6 c e l l s / m l i n normal growth medium from which the sodium bicarbonate had been omitted. The pH was adjusted to 7.2 using 1 M sodium hydroxide. The c e l l suspensions were then placed i n special glass i r r a d i a t i o n vessels (Parker, Skarsgard and Emmerson, 1969) and s t i r r e d with a magnetic s t i r r e r . Hypoxia was obtained by flowing nitrogen (less than 10-15 p a r t s / m i l l i o n 0 2 present, Canada Liquid A i r , Vancouver, B.C.) above the s t i r r e d c e l l suspension at 0.7 liters/minute for at least 45 minutes p r i o r to and during i r r a d i a t i o n . Aerobic conditions were obtained by flowing oxygen instead of nitrogen. The c e l l s were cooled to 0°C 10 to 15 minutes p r i o r to i r r a d i a t i o n ( i . e . aft e r gassing at room temperature for 30-35 minutes) and maintained at 0°C for the duration of the experiment, unless s p e c i f i e d otherwise. See f i g . 3 for a flow chart representation of the experimental procedure. Irradiations were performed with gamma-rays from a therapeutic 6 0Co source (1.17 and 1.33 MeV y~ rays). The dose rate was 500-535 rads/minute. 13 2.1.3 Radioactive Labelling The DNA of CH2B2 c e l l s was l a b e l l e d with 3H-thymidine (3H-TdR, s p e c i f i c a c t i v i t y 15.7 or 47-59 Ci/mmol, Amersham-Searle, 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). Two d i f f e r e n t procedures were used to lab e l the DNA. For uniform l a b e l l i n g of the parental DNA, 50 ml of normal growth medium containing 0.25 yCi of 3H-TdR/ml was added to 1 day old c e l l cultures i n large p l a s t i c flasks (75 cm2 growth area, Falcon P l a s t i c s ) . These cultures were then incubated under standard conditions for 18 to 24 hours. The l a b e l l i n g was terminated by an additional 1 hour incubation i n non-radioactive medium, i n order to clear the c e l l u l a r pool of unincorporated precursor. To examine newly synthesized daughter DNA, usually aft e r i r r a d i a t i o n , a pulse l a b e l l i n g procedure was employed. The c e l l suspensions to be la b e l l e d , t y p i c a l l y 10 ml containing 1x10 6 c e l l s , were centrifuged at 0°C (8 minutes at 600 rpm) i n 17x100 mm disposable test 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 of warm (37°C) medium containing 20 yCi of 3H-TdR. After a 6 minute incubation i n a 37°C water bath, the pulse l a b e l was terminated by the addition of 10 ml of cold (0°C) non-radioactive medium. The c e l l s were then washed free of e x t r a c e l l u l a r l a b e l by twice centrifuging and resuspending i n 10 ml of normal growth medium 14 at 0°C. In 1 experiment, c e l l s were continuously l a b e l l e d with 3H-TdR p r i o r to i r r a d i a t i o n and pulse l a b e l l e d , as described above, with 1 ml of 10 yCi/ml 1 **C-TdR after i r r a d i a t i o n . 2.1.4 Repair and Chase Incubations After i r r a d i a t i o n at 0°C, the c e l l s were routinely twice centrifuged and resuspended, a l l at 0°C, to remove the drugs present during i r r a d i a t i o n . In experiments examining SSB production, the c e l l s were then immediately lysed at the top of ASG's (see section 2.3). In experiments designed to examine the re j o i n i n g of SSB's or to examine p o s t - i r r a d i a t i o n DNA synthesis, c e l l s were incubated under standard conditions (37°C, 95% a i r , 5% C0 2) for various times af t e r i r r a d i a t i o n to allow enzymatic repair processes to occur. This repair incubation was stopped by placing the c e l l s at 0°C. In re j o i n i n g experiments, the c e l l s were kept at 0°C u n t i l l y s i s . P o s t - i r r a d i a t i o n DNA synthesis was investigated by pulse l a b e l l i n g following repair, and then carrying out a further 3 hour incubation, again under standard conditions. The length of t h i s chase incubation was chosen as 3 hours since a 3 hour chase following a 6 minute pulse l a b e l was found, i n preliminary experiments, to y i e l d DNA of very nearly the same molecular weight as that obtained from continuously l a b e l l e d c e l l s (see f i g . 2). 15 CO 2 0 O 8 U 15 O L U O err L U CL 1 0 0 o p r e l a b e l • p u l s e l a b e l 5 10 15 2 0 FRACTION NUMBER DIRECTION OF SEDIMENTATION 25 F i g u r e 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 CH2B 2 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 3H-TdR f o r 24 hours (0) o r p u l s e l a b e l l e d f o r 6 minutes and i n c u b a t e d a t 37°C (chased) f o r 3 hours (•#) . C e l l s were then l y s e d on ASG's f o r 7 hours and spun a t 12,000 r.p.m. f o r 15 hours. The s m a l l v e r t i c a l arrow r e p r e s e n t s the p o s i t i o n from which the DNA s t a r t s to sediment. 16 In g e n e r a l then, c e l l s were i r r a d i a t e d a t 0°C and a l l subsequent c e n t r i f u g i n g , resuspending and other h a n d l i n g was done a t 0°C except f o r p e r i o d s of r e p a i r , p u l s e l a b e l and chase i n c u b a t i o n a t 37°C. Also', these i n c u b a t i o n s were terminated by p l a c i n g the c e l l s a t 0°C. F i g u r e 3 shows s c h e m a t i c a l l y the d i f f e r e n t l a b e l l i n g and i n c u b a t i o n schedules t h a t were used. 2.2 RADIOSENSITIZING DRUGS Triacetoneamine-N-oxyl (TAN), a s t a b l e n i t r o x i d e f r e e r a d i c a l , was purchased from A l d r i c h Chemical Company, Edmonton, A l b e r t a . Stock s o l u t i o n s were prepared by d i s s o l v i n g TAN i n double d i s t i l l e d water to produce a 0.2 M stock s o l u t i o n . A f t e r being f i l t e r e d through a 0.2 y f i l t e r , the s o l u t i o n was s t o r e d a t 4°C. Fresh stock s o l u t i o n s were prepared every 2 weeks. Metr o n i d a z o l e , 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 ob t a i n e d from Poulenc L i m i t e d , Montreal, Quebec. A few hours p r i o r to use, a 15 mM s o l u t i o n was prepared by d i s s o l v i n g the drug i n MEM F-16 l a c k i n g sodium b i c a r b o n a t e and f i l t e r i n g through a 0.2 y f i l t e r . Ro-07-0582, 1-(2-ni t r o - l - i m i d a z o l e ) - 3 - m e t h o x y - 2 -p r o p a n o l , was s y n t h e s i z e d by Roche Products L i m i t e d , Welwyn Garden C i t y , H e r t f o r d s h i r e , England and obtained through 17 c o n t i n u o u s l a b e l p r e t r e a t m e n t i r r a d i a t e l y s e r e p a i r i r r a d i a t e p o s t - t r e a t m e n t l y s e r e p a i r p u l s e l a b e l c h a s e F i g u r e 3. F l o w C h a r t R e p r e s e n t a t i o n o f t h e V a r i o u s E x p e r i m e n t a l P r o c e d u r e s 18 Dr. C. E. Smithen. Preparation and use of t h i s drug were i d e n t i c a l to that just described for metronidazole. 2.3 ALKALINE SUCROSE GRADIENTS The ASG technique described here i s e s s e n t i a l l y that described previously by P a l c i c and Skarsgard (1972a). 2.3.1 Gradient and Lysing Solutions An automatic gradient former (ISCO Model 57 0, Instrumentation Sp e c i a l t i e s Company, Lincoln, 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, i n c e l l u l o s e n i t r a t e tubes ( 5 / 8 x 4 inches, Beckman Instruments). The gradient solutions were prepared with double d i s t i l l e d water and contained 0.3 M sodium hydroxide (NaOH), 0.001 M ethylenediaminetetraacetic acid (EDTA), 0.01% sodium dodecyl sulphate (SDS) and appropriate concentrations of sucrose. On top of the prepared gradients, 0.5 ml of l y s i n g solution was c a r e f u l l y layered just p r i o r to c e l l l y s i s . The lysi n g solution contained 0.5 M NaOH, 0.01 M EDTA and 0.2% SDS. A l l solutions were passed through a 0.2 y f i l t e r p r i o r to usage. 19 2.3.2 Lysing Procedure A precooled 50 y l microsyringe was used to dispense the c e l l s onto the l y s i n g layer. The syringe was mounted i n an old microscope chassis whose 2 rack and pinion drives were used to f i r s t lower the syringe u n t i l the needle was just touching the lys i n g solution and then, to d e l i v e r the c e l l s onto the l y s i n g layer. A volume of 0.02 ml containing 2-3X10 4 c e l l s i n normal growth medium was delivered over a period of 2 minutes. The gradients were then loaded into ultracentrifuge buckets and lysed i n the dark for 6 or 7 hours before centrifugation. 2.3.3 Centrifugation After an appropriate l y s i n g period (see section 2.3.5), the gradients were centrifuged at 20°C using a SW 27 rotor i n a Beckman L2-65B preparative u l t r a c e n t r i f u g e . In SSB experiments, an angular speed of centrifugation, w, of 16,000 r.p.m. and a time of centrifugation, t, of 10 hours were routinely used, although values of 18,000 r.p.m. and 8 hours respectively, were used for 1 experiment. In experiments designed to examine SSB re j o i n i n g or p o s t - i r r a d i a t i o n DNA synthesis, centrifugations were usually performed at 14,000 r.p.m. for 11 to 12 hours with alternate values of 12,000 r.p.m. and 12 or 15 hours being used a few times. 20 2.3.4 Gradient Fractionation and Sample Counting After centrifugation, each gradient was fractionated into 25 fractions of 0.75 ml each, using an ISCO Model D f r a c t i o n c o l l e c t o r . Fractions were coll e c t e d from the top by displacing the gradient upwards with a 30% sucrose solution pumped into the bottom of the gradient through a hole punched in the centrifuge tube. The fractions were co l l e c t e d into p l a s t i c s c i n t i l l a t i o n v i a l s (Nuclear Associates Inc., Westbury, New York) and made aci d i c with 0.2 ml of 4 M hydrochloric acid. Five ml of Aquasol (New England Nuclear, Boston, Massachusetts) was then added to 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 s c i n t i l l a t i o n system (model LS-330). This procedure yielded a counting e f f i c i e n c y (the r a t i o of number of counts observed to the expected number of radioactive decays) of approximately 20 to 30%. This value represents the absolute counting e f f i c i e n c y for t r i t i u m using t h i s method. The expected t o t a l number of counts for each gradient was determined by l y s i n g a number of c e l l s equal to that layered on the gradient i n a separate s c i n t i l l a t i o n v i a l with 0.5 ml of l y s i n g solution. This sample was then processed i n the same way as were the i n d i v i d u a l f r a c t i o n s . The r a t i o between the sum of the counts i n the gradient's 25 fractions and the expected t o t a l number of counts for that gradient (which was determined as described above) was t y p i c a l l y greater 21 than 0.75 and never less 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 for the gradient versus f r a c t i o n number from the top of the gradient. 2.3.5 Lysing Time The sedimentation p r o f i l e s obtained from unirradiated, continuously l a b e l l e d c e l l s were found to be strongly dependent on the l y s i n g time used. The r e s u l t s of an experiment examining d i f f e r e n t l y s i n g times are shown i n f i g . 4. At shorter l y s i n g times (e.g. 2 hours), the DNA appears i n 2 peaks. With increasing ly s i n g time, the higher molecular weight component disappears and the sedimentation p r o f i l e s appear as 1 peak, the position of which s h i f t s to lower molecular weight values with s t i l l longer l y s i n g times (e.g. 12 hours). This observation d i f f e r s from e a r l i e r r e s u l t s obtained using t h i s gradient technique (Palcic and Skarsgard, 1972a). Previously, the DNA p r o f i l e was found to appear with a reproducible peak position, independent of l y s i n g times ranging from 6 to 2 4 hours. This reproducible position occurred between the 2 p r o f i l e s corresponding to 6 and 8 hours l y s i n g time (in f i g . 4). Hence, a l y s i n g time of 7 hours was employed for a l l experiments except those examining SSB production. In SSB experiments, c e l l s were lysed for 6 hours, since DNA from i r r a d i a t e d c e l l s has been shown to emerge i n a reproducible 22 FRACTION NUMBER DIRECTION OF SED IMENTAT ION-—-> F i g u r e 4. E f f e c t o f L y s i n g Time on S e d i m e n t a t i o n P r o f i l e CH2B 2 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 , and s p u n a t 12,000 r.p.m. f o r 12 h o u r s . 23 peak position i n lysi n g times as short as 3 hours (Palcic, 1972). 2.3.6 Lysing Temperature The position of unirradiated, uniformly l a b e l l e d DNA on the gradient was also found to be dependent on ly s i n g temperature. Figure 5 shows 2 sedimentation p r o f i l e s obtained from c e l l s taken from the same c e l l suspension and lysed at d i f f e r e n t temperatures. The p r o f i l e from the sample which was lysed at the higher temperature i s c l e a r l y s h i f t e d towards lower molecular weight values. In order to minimize t h i s " l y s i n g temperature e f f e c t " , the ambient room temperature during l y s i s was usually maintained at 22.5 to 24.5°C. 2.3.7 Calculation of Molecular Weight Both weight average and number average molecular weights were calculated i n e s s e n t i a l l y the same manner as described 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 molecular weight (Mw) was calculated using the following expression: Mw = x I c. (d. ) k x 1 I u 2 t a 24 5 10 FRACTION 15 20 NUMBER DIRECTION OF SEDIMENTATION Figure 5. E f f e c t of Lysing Temperature on Sedimentation P r o f i l e CH2B? c e l l s were continuously l a b e l l e d for 21 hours and lysed for 7 hours at 20.5-23.5°C (O) or 25.5-27.0°C (•). C e l l s were spun at 12,000 r.p.m. for 12 hours, 25 where oi i s the angular speed of c e n t r i f ugation i n r.p.m., t i s the time of centrifugation i n hours, and a and k are constants, 2.5 and 0.0528 respectively, which were determined by Studier th (19 65); c^ represents the number of counts i n the i f r a c t i o n , corrected for background, and d^ i s the distance that the th molecules i n the i f r a c t i o n have sedimented. The gradient c a l i b r a t i o n constant, 3/ was determined using 3 DNA markers: the bacteriophages T4 and T7, and human adenovirus 2 (Palcic and Skarsgard, 1972a). Since these DNA sedimentation p r o f i l e s have been shown to represent randomly sized d i s t r i b u t i o n s of molecules (Palcic and Skarsgard, 1972a), the number average molecular weight (Mn) can be calculated from the expression 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 (IBM 370, University of B.C.) and a Hewlitt-Packard p l o t t e r . The f i r s t 20 fractions of each gradient were used i n the c a l c u l a t i o n of Mw, unless c^, for some f r a c t i o n i further from the top of the gradient than the DNA peak, f e l l to less than 1.5% of the t o t a l number of counts for that gradient. In t h i s second case, only the f i r s t i fractions were used i n the c a l c u l a t i o n . 26 3. RESULTS 3.1 PARENTAL DNA Although the bulk of t h i s work i s concerned with the synthesis of DNA aft 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 state of the parental DNA after exposure to i o n i z i n g r a d i a t i o n . Two types of experiments were performed using the technique of ASG's. The f i r s t procedure involved assaying for the production of DNA SSB's, or a l k a l i l a b i l e bonds, as a function of dose, immediately aft e r exposure; the second examined the rej o i n i n g of these breaks at 37°C. 3.1.1 Single Strand Break Production The production of SSB's i n mammalian DNA by i o n i z i n g radiation has been extensively studied. The presence of oxygen during i r r a d i a t i o n i s known to increase the y i e l d of SSB's by a factor of 3 to 4 (Palcic and Skarsgard, 1972a; Roots and Smith, 1974). In addition, several chemical s e n s i t i z e r s have been shown to cause increased strand breakage (Dugle et 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 at 0°C and kept i n an ice-water bath u n t i l l y s i s at the top of an ASG. Hypoxia was achieved by flowing N 2 (-10-15 ppm O 2 ) p r i o r to and during i r r a d i a t i o n ; drugs, i f present, were added to the 27 c e l l suspension p r i o r to gassing. Sedimentation conditions for a l l experiments were i d e n t i c a l , that i s , gradients were centrifuged at 16,000 r.p.m. for 10 hours. Typical sedimentation p r o f i l e s obtained for i r r a d i a t i o n under various conditions are shown i n figures 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 receiving a higher dose sediments more slowly and hence has suffered more SSB's. In f i g . 6b, the large reduction i n the y i e l d of strand breaks during hypoxic i r r a d i a t i o n i s shown. A dose of 15 krads under hypoxia y i e l d s DNA fragments s l i g h t l y larger than those obtained a f t e r an aerobic dose of 5 krads, implying an oxygen enhancement r a t i o (OER) for SSB's of somewhat more than 3. It has been reported that TAN present during hypoxic i r r a d i a t i o n of phage X DNA causes, at most, a small increase i n the number of SSB's (Emmerson, 1970; Johansen, 1972). Agnew (1972) found no increase i n the number of SSB's i n mouse L-60 c e l l DNA i n the presence of TAN. The 2 sedimentation p r o f i l e s shown i n f i g . 7a are consistent with 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 obtained were i r r a d i a t e d to the same dose under hypoxia with only 1 suspension containing 10 mM TAN; TAN has caused only a very small s h i f t towards the top of the gradient. In contrast, when CH2B2 c e l l s were given 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 OF SEDIMENTATION r > F i g u r e 6 a,b. P r o d u c t i o n o f S i n g l e S t r a n d B r e a k s u n d e r A e r o b i c and H y p o x i c C o n d i t i o n s ~" r CH2B 2 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 c o n d i t i o n s (o) ( p a n e l 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 16,00 0 r.p.m- f o r 10 h o u r s . 29 • N2 + I AN 5 10 15 20 FRACTION NUMBER •DIRECTION OF SEDIMENTATION F i g u r e 7 a,b, P r o d u c t i o n o f S i n g l e S t r a n d B r e a k s u n d e r H y p o x i a w i t h TAN o r Ro-07-0582 ~ ' : CH2B 2 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 Ro-07-0582 ( ® ) . C e l l s were l y s e d f o r 6 h o u r s and spun a t 16, 000- r.p.m. f o r 10 h o u r s . 30 nitroimidazole, Ro-07-0582, a large s h i f t of the p r o f i l e was observed. This r e s u l t , shown i n f i g . 7b, indicates that Ro-07-0582 causes a large increase i n the production of SSB's. Figure 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 weight (Mw) i s plotted against dose. Each point i s the r e s u l t of 1 — 8 gradient. Straight l i n e s passing through 1/Mw = 0.3 8x10 daltons were f i t t e d to the data by the method of least squares. The dose modifying factor (DMF) for any i r r a d i a t i o n condition i s just the slope of that l i n e divided by the slope of the N 2 l i n e . The energy required to cause 1 SSB can be calculated d i r e c t l y from the inverse slope, assuming that the d i s t r i b u t i o n of DNA fragments i n the gradient i s random (see section 2.3.7). These calculations are summarized i n Table I. Table I: I r r a d i a t i o n Condition Slope (dalton "*"rad "*") eV/break DMF N 2 0.451 x 10" 1 2 115 1.0 0 2 1.94 x 10~ 1 2 27 = 4 N 2 + 10 mM TAN 0.576 x 10~ 1 2 90 1.3 N 2 + 15 mM Metronidazole 1.36 x 10~ 1 2 38 3.0 N 2 + 15 mM Ro-07-0582 1.66 x 10~ 1 2 31 3.7 31 6 I L_J I I I I I I .1,1 1 l l I I | - ' I 0 5 10 15 20 DOSE (krads) Figure 8. Graph of 1/Mw versus Dose for Single Strand Break  Production under Several Conditions CH2B2 c e l l s were continuously l a b e l l e d and i r r a d i a t e d at 0°C under aerobic (A) or hypoxic conditions (•) i n the presence of 15 mM Ro-07-0582 .(•) , 15 mM metronidazole (•) or 10 mM TAN (O) . C e l l s were lysed for 6 hours on ASG's and spun at 16,000 r.p.m. for 10 hours. Mw values were calculated as described i n section 2.3.7 and straight l i n e s passing through 0.3.8xl0~ 8 d a l t o n s - 1 were f i t t e d to the data by the method of l e a s t squares. 32 TAN present during hypoxic i r r a d i a t i o n appears to cause a small increase i n the number of SSB's produced. N 2 + TAN points plotted are the results from 2 separate experiments; N 2 points are from 3 separate experiments. This r e s u l t disagrees with the e a r l i e r work of Agnew (1972) i n t h i s laboratory. However, th i s increase i n SSB y i e l d i s quite i n s i g n i f i c a n t compared to the large changes caused by metronidazole and Ro-07-0582. From f i g . 8 and Table I, i t i s apparent that 15 mM Ro-07-0582 present during hypoxic i r r a d i a t i o n produces almost the f u l l aerobic strand break y i e l d . I t should be noted that, i n a separate experiment, 15 mM Ro-07-0582 and 10 mM TAN were found to cause no increase i n the number of SSB's produced during aerobic i r r a d i a t i o n . 3.1.2 Rejoining of Single Strand Breaks The r e j o i n i n g of SSB's i n mammalian c e l l s has been observed by many authors (e.g. Lett et a l . , 1967; Sawada and Okada, 1970). Under favourable conditions the re j o i n i n g process i s f a i r l y rapid, with most of the SSB's being rejoined within 1-2 hours; t h i s process i s strongly i n h i b i t e d at 0°C, with no re j o i n i n g detectable at times up to 5 hours (Palcic and Skarsgard, 1972b). The experiments reported i n t h i s section were done to investigate the p o s s i b i l i t y that various 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 rej o i n i n g . Previous work i n t h i s 33 laboratory (Agnew and Skarsgard, 1970) indicated that TAN does not i n t e r f e r e with the rejoining process. The r e s u l t s shown in f i g . 9 confirm t h i s observation. Two i d e n t i c a l CH2B2 c e l l suspensions, 1 containing 10 mM TAN, were given a dose of 15 krads under hypoxia. Samples were incubated at 37°C for the times indicated and then returned to 0°C u n t i l l y s i s . Mw's were calculated and are plotted as percentages of the unirradiated control value obtained from a gradient in the same rotor. I t i s apparent that TAN present during hypoxic i r r a d i a t i o n has no e f f e c t on the rejoining of SSB's. Similar experiments were done using both Ro-07-0582 and metronidazole. Here the dose delivered 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 that caused by the N 2 and N 2 + TAN 15 krad i r r a d i a t i o n s . The results for Ro-07-0582 are shown i n f i g . 10. Neither Ro-07-0582 nor metronidazole i n h i b i t e d the rejoining process. 34 70 60 N 2 15 krads N 2 + TAN 15 krads 0 L _ J , 30 60 90 120 150 REPAIR TIME (minutes) Figure 9. Rejoining of Single Strand Breaks a f t e r Hypoxic  I r r a d i a t i o n with and without TAN ' ! : Continuously la b e l l e d CH2B? c e l l s were i r r a d i a t e d at 0°C under hypoxic conditions to 15 krads i n the presence (®) or absence •($) of 10 mM TAN. C e l l s were then incubated at 37°C for the times indicated. C e l l s were lysed for 7 hours and spun at 14,000 r.p.m. for 11 hours. Mw's were calculated and plot t e d as a percentage of the Mw value obtained from the unirradiated control i n the same rotor. 35 UNIRRADIATED CONTROL N2+ Ro-07-0582 6 krads N2 15 krads 30 60 90 120 150 REPAIR TIME (minutes) 180 Figure 10. Rejoining of Single Strand Breaks a f t e r Hypoxic  Ir r a d i a t i o n with and without Ro-07-0582 Continuously l a b e l l e d CH2B2 c e l l s were i r r a d i a t e d at 0°C under hypoxic conditions to 6 krads i n the presence of 15 mM Ro-07-0582 (#) or to 15 krads i n drug-free medium (©). The unirradiated control value was obtained from a gradient in the same rotor. C e l l s were lysed for 7 hours and spun at 14,000 r.p.m. for 11 hours. Mw's were calculated. 36 3.2 NEWLY SYNTHESIZED DAUGHTER DNA Most of t h i s work has involved the investigation of po s t - i r r a d i a t i o n DNA synthesis by means of the pulse and chase assay described e a r l i e r (sections 2.1.3 and 2.1.4). This assay was used to investigate the a b i l i t y of CH2B2 c e l l s to regain the capacity to synthesize f u l l - s i z e d DNA afte r i r r a d i a t i o n i n the presence of various 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 of Hypoxic CH2B2 C e l l s TAN present during exposure of E. c o l i to X-rays under hypoxic conditions i s known to cause interruptions i n the DNA strands synthesized immediately aft e r i r r a d i a t i o n (Rupp et a l . , 1969). I t was suggested that these interruptions were caused by X-ray induced TAN-DNA adducts, which were known to be formed i n aqueous solutions of DNA (Willson and Emmerson, 1970; Nakken, Sikkeland and Brustad, 1970), and have since been observed i n vivo i n E. c o l i K-12 (Wold and Brustad, 1974). Although TAN sensiti z e s hypoxic hamster c e l l s to io n i z i n g r a d i a t i o n to a lesser degree than b a c t e r i a l c e l l s (Parker, Skarsgard and Emmerson, 1969; Emmerson, 1967), i t was nevertheless of i n t e r e s t to determine i f the same newly synthesized strand interruptions occur i n mammalian c e l l s . In the following experiments, TAN, i f present, was 37 added to the c e l l suspension 1 hour before i r r a d i a t i o n and removed, by twice resuspending i n drug-free medium, immediately afte r i r r a d i a t i o n . C e l l suspensions were i r r a d i a t e d at 0°C under hypoxic conditions to a dose of 15 krads. After resuspension i n drug-free medium, the c e l l s were incubated for various times at 37°C before being returned to an ice-water bath. This incubation period w i l l be referred to as "repair incubation", during which the c e l l may repair radiation-induced lesions, such as SSB's. Following t h i s repair incubation, the c e l l s were pulse l a b e l l e d for 6 minutes at 37°C with 3H-TdR (20 uCi/ml). The c e l l suspensions were then incubated for 3 hours at 37°C, a chase period during which unirradiated CH2B2 c e l l s w i l l synthesize almost " f u l l - s i z e d " DNA (see section 2.1.4). The DNA sedimentation p r o f i l e s obtained from c e l l s exposed to 15 krads under hypoxia i n the presence and absence of 10 mM TAN are shown i n f i g . 11a. It should be noted that these p r o f i l e s represent the DNA synthesized during a 3 hour period with no repair incubation p r i o r to the pulse l a b e l . The p r o f i l e obtained from the TAN treated c e l l s i s c l e a r l y s h i f t e d towards lower molecular weight values. Figure l i b i s a similar comparison between samples i r r a d i a t e d i n N 2 and N 2 + TAN. Here however, the c e l l s were incubated at 37°C for 120 minutes before being pulse l a b e l l e d and chased. The sedimentation p r o f i l e obtained from TAN treated 38 a FRACTION NUMBER — D I R E C T I O N OF SEDIMENTATION — — • F i g u r e 11 a,b. S e d i m e n t a t i o n 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 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 '• ! r : ' ' : : ' ~ — CH2B 2 c e l l s were 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 c o n d i t i o n s i n t h e p r e s e n c e (®) o r a b s e n c e (O) o f 10 mM TAN. 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 w ere a l l o w e d (a) no r e p a i r o r (b) 120 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 3H-TdR/ml f o r 6 m i n u t e s . T h e y were t h e n i n c u b a t e d a t 3 7°C f o r a f u r t h e r 3 h o u r s . 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 . 39 c e l l s i s e s s e n t i a l l y i d e n t i c a l t o the TAN t r e a t e d p r o f i l e i n f i g . 11a; the curve o b t a i n e d from c e l l s i r r a d i a t e d under hypoxia alone has undergone a smal l but s i g n i f i c a n t s h i f t t o highe r molecular weight with r e p a i r i n c u b a t i o n . Mw i s p l o t t e d versus r e p a i r i n c u b a t i o n time at 37°C i n f i g . 1 2 . Again, t h i s Mw r e p r e s e n t s the s i z e of the DNA fragments s y n t h e s i z e d i n the 3 hour chase f o l l o w i n g the pul s e l a b e l ; the r e p a i r i n c u b a t i o n i s c a r r i e d out p r i o r t o the p u l s e l a b e l . The i n c r e a s e i n Mw ( s y n t h e s i z e d d u r i n g the 3 hour chase) w i t h i n c r e a s i n g time of r e p a i r i n c u b a t i o n , f o r i r r a d i a t i o n i n N 2 alone, presumably r e f l e c t s r e p a i r of damage i n the template DNA ( f o r example, the r e j o i n i n g o f SSB's, which show s i m i l a r k i n e t i c s , f i g . 9 ) . A f t e r 120 minutes r e p a i r , the s i z e of the DNA s y n t h e s i z e d d u r i n g the 3 hour chase was comparable 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 , which under s i m i l a r c o n d i t i o n s o f l y s i s and sedimentation y i e l d e d Mw valu e s o f =2.8 ± 0 . 2 x 1 0 8 d a l t o n s . I t appears t h a t even a f t e r a dose of 15 krads under hypoxia, c e l l s g iven s u f f i c i e n t time to r e p a i r t h e i r r a d i a t i o n damage are capable of s y n t h e s i z i n g f u l l - s i z e d DNA fragments. I f however, TAN was pr e s e n t d u r i n g the 15 krad dose, the Mw valu e s obtained from the 3 hour chase p e r i o d were much sm a l l e r . These v a l u e s d i d 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 b e f o r e the p u l s e and chase, f o r r e p a i r times of up t o 3 hours. 40 CO O X o IJJ ^ -> cn > c o +-< or ra < ^ i w Z> CJ U J Mw range for Q doss control 1.0 O N, N 2 +TAN O 0 t -1 X 1 0 30 60 90 120 150 REPAIR TIME (minutes) 180 F i g u r e 12. E f f e c t o f TAN 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 CH23 ?; c e l l s were 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. 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 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 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. The 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 v a l u e s o b t a i n e d f r o m 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 c h a s e a s s a y ) i n s e v e r a l e x p e r i m e n t s . 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 . 41 The re s u l t s i l l u s t r a t e d i n figures 11 and 12 show that TAN present during 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 synthesis of smaller DNA fragments than those made by c e l l s exposed to the same dose i n drug-free medium. I t was shown e a r l i e r that TAN does not i n h i b i t the rejo i n i n g of SSB's (section 3.1.2), and hence a possible explanation for these observations i s that TAN binds to i r r a d i a t e d parental DNA, forming stable adducts which lead to interruptions i n the synthesis of the daughter strands. These adducts are not repaired to a measurable degree during a 3 hour post-i r r a d i a t i o n incubation. In an experiment designed to confirm that these smaller pieces were ac t u a l l y being synthesized from largely f u l l - s i z e d parental strands, c e l l s were prelabelled for 25 hours with 3H-TdR and pulse l a b e l l e d a f t e r a dose of 15 krads under hypoxia with ll+C-TdR (10 yCi/ml) for 6 minutes. Sedimentation p r o f i l e s obtained 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 repair incubation p r i o r to the 6 minute lkC pulse and 3 hour chase. For those c e l l s not exposed to TAN, the parental and newly synthesized daughter DNA yielded very s i m i l a r p r o f i l e s ; whereas, those c e l l s treated with the drug during i r r a d i a t i o n were found to show marked differences between prelabel and pulse l a b e l curves. This experiment provides further support for the concept of radiation-induced TAN-DNA adducts which lead 42 CO 20 N 2 15 krads _c—or prelabel —«— -©-pulse label O O 15 N2+TAN 15 krads -o—o- prelabe! -• »- pulse label 0 5 10 15 20 FRACTION NUMBER -DIRECTION OF SEDIMENTATION• 25 F i g u r e 13. 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 a n d 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 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 ~ CH2B 2 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 3H-TdR f o r 25 h o u r s and 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 medium o r (b) i n medium c o n t a i n i n g 10 mM TAN. 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 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 o f 1 I +C-TdR/ml 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 12,000 r.p.m. f o r 15 h o u r s . to interruptions i n strands synthesized from the TAN damaged template. In addition, i t suggests that these adducts p e r s i s t for at least 6 hours after i r r a d i a t i o n . 3.2.2 TAN Pretreatment Agnew and Skarsgard (1972) have reported that pre-i r r a d i a t i o n exposure to TAN sensitizes hypoxic Chinese hamster c e l l s to i o n i z i n g radiation with a DMF of 1.3. The following experiments were done to determine i f th i s pretreatment e f f e c t could be explained by interruptions i n newly synthesized DNA, similar to those found after TAN treatment. CH2B2 c e l l s were exposed to 10 mM TAN for 1 hour at 22°C; they were then twice centrifuged and resuspended i n drug-free medium. The remainder of the experimental procedure was i d e n t i c a l to that described i n the previous section: c e l l s were ir r a d i a t e d to 15 krads under hypoxia, allowed to repair for various times at 37°C, pulse l a b e l l e d and chased for 3 hours. Results from 1 experiment are shown i n f i g . 14. Mw values obtained from c e l l s that were pretreated with TAN are consis-tently s l i g h t l y lower than those from c e l l s that were not, although the differences are of marginal s i g n i f i c a n c e . Also, Mw values r i s e with increasing repair time, independent of TAN pretreatment. A repeat experiment yielded comparable r e s u l t s . I t appears that TAN pretreatment has l i t t l e e f f e c t on the po s t - i r r a d i a t i o n synthesis of DNA. 44 3.0 CO o I 20 h O LU < CO c o CC to © TAN PRETREATMENT O SHAM PRETREATMENT O O o 1£) LU _ J o 0 1 X 1 -L 1 3 0 6 0 9 0 120 150 REPAIR TIME (minutes) 1 8 0 Figure 14. E f f e c t of TAN Pretreatment on Post - I r r a d i a t i o n DNA  Synthesis : ! ' ~~ CH2B2 c e l l s were exposed to 10 mM TAN (®) or drug-free medium (O) f o r 1 hour at 22°C and then twice resuspended i n drug-free medium. C e l l s were i r r a d i a t e d at 0°C to 15 krads under hypoxic conditions, allowed to repair at 37°C for various times, pulse l a b e l l e d for 6 minutes, and chased for 3 hours at 37°C. C e l l s were lysed for 7 hours and spun at 14,000 r.p.m. for 11 hours. Mw's were calculated. 45 This r e s u l t i s consistent with the observation that TAN does not bind to unirradiated DNA (Brustad, Jones and Wold, 1973). Our evidence for t h i s i s twofold. F i r s t , TAN present in zero dose controls was found to have no e f f e c t on the size of the DNA synthesized during a 3 hour chase. And secondly, i n an experiment designed to assay for TAN binding to unirradiated DNA, c e l l s were treated for 11 or 24 hours with 0.25 uCi/ml 3H-TdR and 1.0 mM TAN. Controls were incubated i n the presence of the t r i t i u m label only. Results for the 24 hour incubation are shown i n f i g . 15. No detectable difference i n sedimentation p r o f i l e s was observed between TAN treated and control c e l l s . 3.2.3 TAN Post-treatment Agnew and Skarsgard (1972) have also reported that p o s t - i r r a d i a t i o n treatment with TAN can s e n s i t i z e CH2B2 c e l l s almost as e f f e c t i v e l y as TAN treatment during i r r a d i a t i o n (DMF = 1.5). P o s t - i r r a d i a t i o n s e n s i t i z a t i o n i s strongly dependent on temperature and i s only observed i f TAN i s added a few minutes aft e r i r r a d i a t i o n . Experiments were performed to determine i f t h i s post-treatment e f f e c t could be related to interruptions i n p o s t - i r r a d i a t i o n DNA synthesis. A hypoxic suspension of CH2B2 c e l l s was i r r a d i a t e d to 15 krads at 0°C. Immediately aft e r i r r a d i a t i o n , a sample was added to an aerobic solution of TAN with a r e s u l t i n g 46 FRACTION NUMBER DIRECTION OF SEDIMENTATION E> F i g u r e 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 C o n t i n u o u s l y L a b e l l e d w i t h and w i t h o u t TAN CH2B 2 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 24 h o u r s i n t h e p r e s e n c e (0) o r a b s e n c e (^) o f 1.0 mM TAN. C e l l s were l y s e d f o r 7 h o u r s and s p u n a t 14,000 r.p.m. f o r 11.75 h o u r s . 47 concentration of 10 mM, and incubated for 1 hour at 21-22°C; a p a r a l l e l sample was added to drug-free medium. Other samples were allowed repair incubations of 2 0 and 60 minutes, which were carr i e d out at 37°C under standard conditions before the 1 hour TAN post-treatment at room temperature. Following the post-treatment, the c e l l s were washed free of TAN by twice centrifuging and resuspending i n drug-free medium, pulse l a b e l l e d for 6 minutes with 3H-TdR and incubated for 3 hours, before being layered on ASG's. In f i g . 16, Mw values obtained from TAN post-treated and untreated c e l l s are plotted as a function of repair time at 37°C before the 1 hour exposure to TAN-containing or drug-free medium. Ce l l s that were treated with TAN for 1 hour afte r i r r a d i a t i o n under hypoxia consistently yielded daughter DNA of lower Mw values than those that were not exposed to TAN, although again, as i n the TAN pretreatment experiments, the differences are of marginal s i g n i f i c a n c e . A repeat experiment with repair incubations performed at 21.5°C gave similar r e s u l t s , that i s s l i g h t l y lower Mw values from TAN post-treated c e l l s . As can be seen i n f i g . 16, the molecular weight of daughter DNA increased with repair time i n a si m i l a r fashion for both TAN post-treated and untreated c e l l s . This d i f f e r s sharply from the s i t u a t i o n for TAN treated c e l l s (TAN present during i r r a d i a t i o n ) as can be seen i n f i g . 12. I t appears that TAN post-treatment has l i t t l e e f f e c t on DNA synthesis aft e r i r r a d i a t i o n . 48 co c o co T3 CO i o X (D LU cr < _ J => U LU I o 3.0 T A N P O S T - T R E A T M E N T S H A M P O S T - T R E A T M E N T .1.0 20 40 60 80 REPAIR TIME (minutes) F i g u r e 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 CH2B 2 c e l l s were 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 and 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 t i m e s u n d e r a e r o b i c c o n d i t i o n s a t 3 7 ° C . C e l l s were 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 were d e t e r m i n e d . 49 3.2.4 TAN Treatment of Aerobic CH2B2 C e l l s It has been shown that TAN does not s e n s i t i z e aerobic mammalian c e l l s to i o n i z i n g radiation (Parker, Skarsgard and Emmerson, 1969). Also, Wold and Brustad (1974) have demonstrated that TAN does not bind to the DNA of E. c o l i i r r a d i a t e d i n an 0 2 saturated solution. Nevertheless, i t was of i n t e r e s t to determine i f TAN lesions could be detected i n aerobically i r r a d i a t e d CH2B2 c e l l s using the pulse and chase assay. Aerobic c e l l suspensions were i r r a d i a t e d , with or without 10 mM TAN, to 5 krads, a value chosen to give approximately the same amount of damage as 15 krads under hypoxic conditions. The c e l l s were then washed free of TAN by twice resuspending i n drug-free medium, allowed to repair at 37°C for various times, pulse l a b e l l e d with 3H-TdR for 6 minutes, and chased for 3 hours at 37°C. Mw values obtained from TAN treated and untreated c e l l s are plotted against repair incubation time i n f i g . 17. The 0 2 and 0 2 + TAN values shown were obtained from separate experiments. Repeat experiments, comparing 0 2 and 0 2 + TAN treated samples i n the same centrifuge rotor, yielded s i m i l a r r e s u l t s , that i s s l i g h t l y larger Mw values for TAN treated c e l l s . Also, Mw values for DNA synthesized during a 3 hour chase increase with increasing time of repair incubation following a dose of 5 krads, with or without TAN present, under aerobic conditions, again presumably 50 ao S-oo -O 02 + TAN -® CL 0 0 30 60 90 120 150 REPAIR TIME (minutes) 180 Figure 17. E f f e c t of TAN Treatment during Aerobic I r r a d i a t i o n  on Post-Irradiation DNA Synthesis CH2B2 c e l l s were i r r a d i a t e d at 0°C to 5 krads under aerobic conditions with (O) or without (©) 10 mM TAN. After being twice resuspended i n drug-free medium, c e l l s were allowed to repa i r for various times at 37 C, pulse l a b e l l e d for 6 minutes and chased f o r 3 hours at 37°C. C e l l s were lysed for 7 hours and spun at 14,000 r.p.m. for 11 hours. 51 r e f l e c t i n g template repair. This increase i n Mw after aerobic TAN treatment contrasts with the e s s e n t i a l l y f l a t Mw versus repair curve shown i n f i g . 12 for TAN treatment under hypoxia. It would be rather d i f f i c u l t to explain the observation that TAN treatment during aerobic i r r a d i a t i o n causes a small increase i n the size of the DNA made during the pulse and chase, i f indeed t h i s s h i f t i s s i g n i f i c a n t . This r e s u l t would suggest that TAN competes with 0 2 for damaged s i t e s and that these TAN modified s i t e s would then be more rapidly repaired; however, i t was shown e a r l i e r (section 3.2.1) that TAN-DNA adducts formed during hypoxic i r r a d i a t i o n are not repaired for incubation times of up to 3 hours. Hence, i t seems l i k e l y that th i s difference i s of no r e a l importance. In any event, the ef f e c t i s small, causing only a small s h i f t i n sedimentation p r o f i l e , that i s at best of marginal s i g n i f i c a n c e . I t i s therefore reasonable to conclude that TAN treatment during aerobic i r r a d i a t i o n has no demonstrable e f f e c t on post-i r r a d i a t i o n DNA synthesis. 52 3.2.5 Dose Response Although most of the experiments described i n t h i s work involved exposure to 15 krads under hypoxia, or i t s equivalent under other conditions, the dose dependence of the pulse and 3 hour chase assay was also investigated. Hypoxic c e l l suspensions were.irradiated at 0°C, with and without TAN, and then twice resuspended i n drug-free medium. Cel l s were maintained at 0°C u n t i l being pulse l a b e l l e d (6 minutes i n 20 yCi/ml 3H-TdR) and chased for 3 hours at 37°C. Results 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 with no drug present, the size of DNA synthesized during the 3 hour chase decreases very l i t t l e with increasing dose. TAN treatment, however, leads to a substantial dose-dependent decrease i n the Mw values observed. These re s u l t s are replotted on a 1/Mw versus dose graph i n f i g . 19; the dotted l i n e shown i s the SSB curve from f i g . 8 for i r r a d i a t i o n i n N 2 + TAN. Apparently, the number of TAN-DNA adducts detected by the pulse and chase procedure i s sim i l a r to the number of SSB's produced. Since TAN causes only a small increase i n the y i e l d of SSB's (see f i g . 8), these res u l t s suggest that TAN, in t e r a c t i n g with i r r a d i a t e d DNA, leads predominantly to the formation of adducts, rather than SSB's. 53 5 10 15 20 DOSE (krads) F i g u r e 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 of Dose  under Hypoxia w i t h and without TAN Hypoxic CH2B 2 c e l l suspensions were i r r a d i a t e d a t 0°C w i t h (®) and without 10 mM TAN ($). A f t e r being twice resuspended 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 minutes and chased f o r 3 hours at 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. 54 F i g u r e 19. Dose Response o f Newly S y n t h e s i z e d DNA P l o t t e d a s ' i/Mw v e r s u s Dose ~~' : " F i g u r e 19 i s a r e p l o t of. t h e d a t a i n f i g . 18 on a 1/Mw s c a l e . The 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 2 + TAN f r o m f i g . 8. 55 3.2.6 TAN Concentration In s u r v i v a l studies i n Chinese hamster c e l l s , Parker, Skarsgard and Emmerson (1969) found DMF's of 1.5 and 1.3 for TAN concentrations of 10 mM and 1.0 mM respectively. Revesz and Littbrand (1970) have reported a dose modifying e f f e c t for TAN concentrations as low as 250 yM for another hamster c e l l l i n e . And, using a radiosensitive s t r a i n of E. c o l i K-12, Emmerson, Fielden and Johansen (1971) found that 230 yM TAN gave half the maximum s e n s i t i z i n g e f f e c t . In the following experiments, c e l l suspensions were exposed to a dose of 15 krads under hypoxia i n the presence of various concentrations of TAN. The c e l l s were then washed free of TAN by twice resuspending i n drug-free medium, pulse la b e l l e d and chased for 3 hours, before being layered on ASG's. The re s u l t s are shown i n f i g . 20. Concentrations below 10 to 20 yM have no detectable e f f e c t on the p o s t - i r r a d i a t i o n synthesis of DNA. The maximum e f f e c t i s produced by approximately 1.0 mM TAN. TAN concentrations higher than 10 mM were not investigated. The r e s u l t s shown here agree quite well with the half maximum value of 230 yM for bacteria (Emmerson et aJL. , 1971) . However, the reduced s e n s i t i z a t i o n seen at 1.0 mM by Parker et a l . (1969) using the same mammalian c e l l l i n e i s not apparent i n t h i s pulse and chase assay. It should be noted that the Mw values shown i n f i g . 20 56 T A N C O N C E N T R A T I O N (mM) F i g u r e 20. E f f e c t of TAN C o n c e n t r a t i o n 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 2 c e l l suspensions were i r r a d i a t e d a t 0°C to 15 krads i n the presence of 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 then twice resuspended 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 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. Mw v a l u e s were c a l c u l a t e d . The e r r o r bars shown a t 0 mM and 10 mM are the standard e r r o r s i n the mean va l u e s o b t a i n e d from 5 and 4 separate g r a d i e n t s r e s p e c t i v e l y . 57 were obtained from c e l l s allowed no repair incubation before the pulse and chase. P a r a l l e l samples that were allowed 30 minutes repair incubation showed some evidence of repair, that i s s l i g h t l y larger Mw values, for TAN concentrations of .500 uM or l e s s . This r e s u l t suggests that the CH2B2 c e l l l i n e may have a limited capacity to repair TAN-DNA adducts. 3.2.7 Metronidazole and Ro-07-0582 Treatment of Hypoxic C e l l s The nitroimidazoles, metronidazole and Ro-07-0582, have been shown to radiosensitize hypoxic mammalian c e l l s more e f f i c i e n t l y (Asquith et a l . , 1974; Moore, P a l c i c and Skarsgard, 1975) than TAN (Parker, Skarsgard and Emmerson, 1969). In addition, metronidazole has been shown to bind to DNA i r r a d i a t e d under hypoxia (Willson, Cramp and Ings, 1974). Hence the eff e c t s of these compounds on p o s t - i r r a d i a t i o n DNA synthesis were investigated. C e l l s were i r r a d i a t e d under hypoxia to 6 krads i n the presence of 15 mM metronidazole or Ro-07-0582, or to 15 krads in drug-free medium, conditions 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 at 0°C, the c e l l s were washed free of drug, allowed to repair for various times, pulse l a b e l l e d and incubated for 3 hours as described previously, before layering on ASG's. The results are shown i n figures 21 and 22, with drug and no drug results obtained under si m i l a r l y s i n g conditions being 58 3.0 00 i o T— X X 2.0 CD 5 2 O +-» tr co C J U J _J O 1.0 0 JL 15 krads • . N2+ METRONIDAZOLE 6 krads 0 30 60 90 120 150 REPAIR TIME (minutes) 180 Figure 21. E f f e c t of Metronidazole Treatment during Hypoxic Irradiation, on Post-Irradiation DNA Synthesis Hypoxic CH2B2 c e l l s were i r r a d i a t e d to 6 krads at 0°C i n the presence of 15 mM metronidazole. After being twice resuspended i n drug-free medium, c e l l s were allowed to repair at 37°C for various times, pulse l a b e l l e d for 6 minutes and chased for 3 hours. C e l l s were lysed for 7 hours and spun at .14 ,0 00 r.p.m. for 11 hours. The dashed l i n e i s the newly synthesized DNA response a f t e r exposure to 15 krads under hypoxia i n drug-free medium. 59 3 . 0 h - O N2 1 5 krads • N2+ R o - 0 7 - 0 5 8 2 6 krads CO • o I 2 .0 I-O Q o rr to _ J w O 1J0 o-0 1 0 3 0 6 0 9 0 REPAIR TIME 1 2 0 1 5 0 (minutes) 1 8 0 F i g u r e 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 o n 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 CH2B 2 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 Ro-07-0582 (®) o r t o 15 k r a d s i n d r u g - f r e e medium ( 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 were 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 t i m e s a t 3 7 ° C , 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 . 60 presented i n each graph. In a separate experiment comparing a l l 3 treatments i n the same centrifuge rotor, no s i g n i f i c a n t differences were found i n the Mw values obtained from hypoxic c e l l s i r r a d i a t e d to 15 krads i n drug-free medium or to 6 krads in the presence of either metronidazole or Ro-07-0582. I t seems that the 2 nitroimidazoles tested have no s i g n i f i c a n t e f f e c t on the template function of i r r a d i a t e d DNA and therefore are l i k e l y to se n s i t i z e by somewhat d i f f e r e n t mechanisms than TAN. 61 4. DISCUSSION 4.1 RELIABILITY OF THE ALKALINE SUCROSE GRADIENT TECHNIQUE As mentioned previously (sections 2.3.5 and 2.3.6), the Mw values obtained from DNA sedimentation p r o f i l e s are dependent on both the time and the temperature of l y s i s . However, within a given experiment, the temperature of l y s i s i s the same for a l l gradients and the time of l y s i s varies by only a few minutes, so that within the experiment, re s u l t s are highly consistent. In fact, i n a few cases i n which aliquots from the same c e l l suspension were lysed on 2 d i f f e r e n t gradients i n the same experiment, the Mw values obtained d i f f e r e d by less than 2 or 3%. Unfortunately, i t was rather more d i f f i c u l t to obtain accurately reproducible Mw values from experiment to experiment. In order to minimize variations due to ly s i n g conditions, the ly s i n g time used was always 7 hours ± 5 minutes (except for SSB experiments, as discussed i n section 2.3.5), the 7 hours being the time from the completion of c e l l layering u n t i l the s t a r t of centrifugation. Also, the temperature during l y s i s was usually maintained between 22.5 and 24.5°C. With these procedures, variations of up to 20-30% s t i l l occured between Mw values obtained i n d i f f e r e n t experiments from i d e n t i c a l l y 62 t r e a t e d c e l l s . Consequently, when comparisons between val u e s from separate experiments were necessary, the r e s u l t s were v e r i f i e d by r e p e t i t i o n of those v a l u e s i n a s i n g l e experiment. When t h i s work was s t a r t e d , the time and temperature of l y s i n g were not expected to be c r i t i c a l f a c t o r s s i n c e e a r l i e r work w i t h t h i s ASG technique i n t h i s l a b o r a t o r y had shown DNA sedimentation p r o f i l e s t o be r e l a t i v e l y i n s e n s i t i v e to l y s i n g times ranging from 6 to 24 hours ( P a l c i c and Skarsgard, 1972a). The reason f o r the d i s c r e p a n c y between the e a r l i e r l y s i n g r e s u l t s and those i n t h i s t h e s i s i s not y e t known. One p o s s i b i l i t y i s the use of a s l i g h t l y h i g h e r temperature range (22.5-24.5°C) d u r i n g the l y s i n g p e r i o d than t h a t employed p r e v i o u s l y (20-22°C). 4.2 EFFECTS OF HIGH DOSES The use of h i g h doses (e.g. 15 krads under hypoxia) was n e c e s s i t a t e d by the low y i e l d of TAN-DNA l e s i o n s per k i l o r a d (see f i g . 19) and the i n a b i l i t y of the ASG assay to d e t e c t sma l l numbers of these l e s i o n s . Although c e l l s exposed to these doses are r e p r o d u c t i v e l y dead ( f o r example, the s u r v i v i n g f r a c t i o n of CH2B2 c e l l s i r r a d i a t e d t o 15 krads under hypoxia -12 would be l e s s than 1x10 ), they are capable, a f t e r 2 hours r e p a i r , of s y n t h e s i z i n g DNA with very n e a r l y the same Mw as 63 t h a t of u n i r r a d i a t e d c e l l s (see f i g . 12). The 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 on c e l l u l a r p r o g r e s s i o n through the DNA s y n t h e t i c c y c l e have been widely s t u d i e d (see E l k i n d and Whitmore, 1967, f o r a review). Doses of a few hundred rads have been shown to cause d i v i s i o n d e l a y (the G 2 b l o c k ) , the l e n g t h of which i s a f u n c t i o n of both the dose and the p o s i t i o n i n the c e l l c y c l e . However, s t u d i e s of r a d i a t i o n e f f e c t s on the k i n e t i c s of c e l l p r o g r e s s i o n p r i o r to .the d i v i s i o n d e l a y have y i e l d e d c o n f l i c t i n g r e s u l t s , which range from the i d e n t i f i c a t i o n of r a d i a t i o n - i n d u c e d delays i n a l l phases of the c e l l c y c l e to the o b s e r v a t i o n of o n l y the G 2 block (Leeper, Schneiderman and Dewey, 1973; Gurley and W a lters, 1972). N e v e r t h e l e s s , i t seems l i k e l y t h a t , d u r i n g the f i r s t few hours a f t e r exposure to doses of 15 krads under hypoxia or e q u i v a l e n t doses under d i f f e r e n t c o n d i t i o n s , most of the c e l l s t h a t do s y n t h e s i z e DNA are those which were i n S phase at the time of i r r a d i a t i o n . I t i s a l s o p o s s i b l e t h a t some Gi c e l l s w i l l move i n t o the DNA s y n t h e t i c stage d u r i n g t h i s time. R a d i o a c t i v e p r e c u r s o r s t h a t are taken up by the c e l l s d u r i n g the p o s t - i r r a d i a t i o n p u l s e l a b e l can be used f o r r e p a i r r e p l i c a t i o n as w e l l as S phase semi-conservative DNA s y n t h e s i s . However, i t can be r e a d i l y c a l c u l a t e d (see Appendix A) t h a t a t most onl y a few per cent of the l a b e l l e d thymidine i s used f o r unscheduled or r e p a i r DNA s y n t h e s i s , s i n c e the number of 64 nucleotides inserted per SSB i s less than 10 (Fox and Fox, 1973a). Also, i t has been shown that the proportion of unscheduled to normal synthesis af t e r UV exposures of 200 ergs/mm2 i s less than 0.07 for a number of mammalian c e l l l i n e s (Fox and Fox, 1973b). In general then, 3H-TdR introduced by a 6 minute pulse la b e l a f t e r a large radiation dose w i l l be used predominantly for semi-conservative DNA synthesis by those c e l l s i n S phase. 4.3 EFFECTS OF IRRADIATION AND DRUG TREATMENT ON TEMPLATE FUNCTION During incubation af t e r exposure to i o n i z i n g radiation, c e l l u l a r DNA i s the s i t e of action of both repair enzyme systems and the DNA r e p l i c a t i o n enzymes. If the r e p l i c a t i o n apparatus encounters an unrepaired radiation-induced l e s i o n , the synthesis of daughter DNA opposite t h i s l e s i o n may be altered. For instance, an unrepaired SSB or damaged base i n the template could lead to a gap i n the daughter strand. 65 4.3.1 Hypoxic and Aerobic I r r a d i a t i o n A dose of 15 krads under hypoxia introduces an average of 1.5 to 2 SSB's per DNA strand with Mw of 2.6-3.0x108 daltons (unirradiated f u l l - s i z e d DNA) (see f i g . 8). Although the rej o i n i n g of these SSB's i s rapid (see section 3.1.2), DNA synthesized immediately aft e r i r r a d i a t i o n , that i s with no repair incubation, i s of smaller Mw than that synthesized af t e r allowing some repair of radiation damage (see f i g . 12). Apparently, damage done to the template DNA by i r r a d i a t i o n under hypoxia interrupts DNA synthesis, but can be r e a d i l y repaired to allow synthesis of control-sized DNA fragments (although the genetic i n t e g r i t y of these fragments i s not known). It should be noted that the radiation-induced lesions responsible for interrupting DNA synthesis are not necessarily SSB's and could well represent other damage. From the data presented i n f i g . 8, i t can be seen that a dose of 5 krads under aerobic conditions produces only s l i g h t l y more SSB's than 15 krads under hypoxia. Further, P a l c i c (1972) has shown that the k i n e t i c s of SSB r e j o i n i n g are independent of whether the strand breaks are produced under aerobic or hypoxic conditions. The data presented i n f i g . 17 show an increase i n Mw of DNA synthesized during a 3 hour chase with increasing repair incubation af t e r a dose of 5 krads i n 0 2 i n d i c a t i n g template repair s i m i l a r to that occurring af t e r i r r a d i a t i o n under hypoxia. This r e s u l t suggests that, i f 0 2 66 reacts with DNA r a d i c a l s predominantly by binding to the r a d i c a l s i t e , as indicated 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 template function of the i r r a d i a t e d DNA. However, further work to confirm t h i s prediction was not done. Another possible explanation for the observed increase in the Mw of newly synthesized daughter DNA with repair incubation i s that the o v e r a l l rate of DNA synthesis i s slowed in i r r a d i a t e d c e l l s and increases with p o s t - i r r a d i a t i o n incubation. However, recent work by Gerner et a l . (1974) suggests that i o n i z i n g radiation has l i t t l e e f f e c t on the duration of S phase. In any event, i t seems l i k e l y that, a f t e r exposure to the large doses used i n t h i s work, any r a d i a t i o n -induced delay in c e l l progression through S phase that may exi s t would not be altered by a 2 hour repair period. Also the r e s u l t s discussed here are consistent with those reported by Korner and Malz (1973), who found that DNA synthesized by Chinese hamster c e l l s a f t e r X-ray doses of 500 rads had lower molecular weight segments than unirradiated c e l l s . They concluded that these lower molecular weight segments were due to gaps produced i n the complementary daughter strands opposite radiation-induced lesions i n the parental strand. 67 4.3.2 TAN Treatment After a dose of 15 krads under hypoxia i n the presence of 10 mM TAN, DNA i s synthesized i n much smaller segments than those made by c e l l s i r r a d i a t e d i n the absence of TAN. Further, the Mw of the DNA synthesized during a 3 hour chase does not increase s i g n i f i c a n t l y with repair incubations of up to 3 hours before the pulse and chase (see f i g . 12). These re s u l t s show that TAN-DNA adducts formed i n the template strand during hypoxic i r r a d i a t i o n produce interruptions i n the subsequent synthesis of daughter DNA. These TAN-DNA lesions are not re a d i l y repaired. T r i t i a t e d TAN has been shown to bind covalently to radiation-induced DNA rad i c a l s i n aqueous solution (Nakken, Sikkeland and Brustad, 1970; Brustad, Jones and Wold, 1973). Wold and Brustad (1974) have also demonstrated binding of TAN to DNA i n E. c o l i K-12 i r r a d i a t e d under hypoxic conditions. In TAN sensitized E. c o l i , these TAN-DNA adducts r e s u l t i n interruptions i n the synthesis of daughter strands (Rupp et a l . , 1969). Rupp and co-workers further showed that wild type E. c o l i can cope with t h i s damage both by excision of TAN modified bases from the template DNA and by recombination of daughter strands. The re s u l t s presented i n section 3.2.1 and discussed here are consistent with the observations from b a c t e r i a l systems, and suggest that a major component of TAN s e n s i t i z a t i o n 68 of hypoxic mammalian c e l l s i s due to the formation of TAN-DNA adducts which lead to interruptions i n subsequent DNA synthesis. A dose of 15 krads under hypoxia produces 1.5 to 2 TAN-DNA adducts per f u l l - s i z e d DNA segment (2.6-3.0xl0 8 daltons), assuming that every adduct leads to a strand interruption. In addition, the Chinese hamster c e l l s used i n t h i s work appear to have, at best, a very limited capacity for repair of TAN lesions. The observation that TAN present during i r r a d i a t i o n of aerobic CH2B2 c e l l s has 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 synthesis (section 3.2.4) i s consistent with reports that TAN sensi t i z e s neither aerobic bacteria (Emmerson, 1967) nor aerobic mammalian c e l l s (Parker, Skarsgard and Emmerson, 1969) and does not bind to DNA i r r a d i a t e d i n the presence of oxygen (Nakken, Sikkeland and Brustad, 1970). Presumably, 0 2 and TAN compete for radiation-induced DNA r a d i c a l s . Figure 23 shows a hypothetical schematic diagram of TAN binding to DNA a f t e r hypoxic i r r a d i a t i o n and causing strand interruptions i n newly synthesized DNA. In the absence of TAN, i r r a d i a t i o n causes SSB's and other lesions i n the parental DNA (only SSB's are shown in f i g . 23). Following an immediate pulse l a b e l , the template DNA i s the s i t e of both semi-conservative synthesis and repair processes, so that some interruptions w i l l occur i n daughter strands due to the synthetic apparatus encountering unrepaired lesions (e.g. the hVy W I T H T A N 6 9 i pulse chase > IV -X 1 pulse • chase. II pulse ° l k > 0 1 1 1 0 chase. O - Ch -x K ) ° 1 P U l S e , CH H O chase CA an D Ci il i I* o T A N molecule — parental D N A daughter D N A • rad i o a c t i v e label Figure 23. Schematic Diagram of Proposed TAN Interruptions i n  Newly Synthesized Daughter DNA For discussion see pages 68 and 70 of the text. 70 gap marked "X" i n case A ) . I f r e p a i r i s a l l o w e d p r i o r t o the p u l s e l a b e l , many o f the damaged s i t e s w i l l be r e p a i r e d and s y n t h e s i s w i l l p r o c e e d l a r g e l y u n h i n d e r e d (case B ) . In t he pr e s e n c e o f TAN, TAN-DNA adducts w i l l be formed d u r i n g h y p o x i c i r r a d i a t i o n , i n a d d i t i o n t o tho s e l e s i o n s d e s c r i b e d above. W i t h no r e p a i r , subsequent DNA s y n t h e s i s w i l l be i n t e r r u p t e d b o t h by TAN adducts and o t h e r u n r e p a i r e d damage (e.g. SSB's, case C ) . I f , as suggested by f i g . 12, TAN l e s i o n s ar e 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 o t h e r damage, the 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 s t i l l o c c u r s i n s m a l l fragments a f t e r r e p a i r i n c u b a t i o n (case D). I t s h o u l d be noted 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 ime f o r TAN t r e a t e d c e l l s (see f i g . 1 2 ) , the number o f i n t e r r u p t i o n s due t o TAN-DNA adducts i s l a r g e compared t o tho s e caused by o t h e r l e s i o n s (marked "X" i n f i g . 2 3 ) . I t i s shown i n Appendix B t h a t the e x p e c t e d i n c r e a s e i n Mw due t o r e p a i r o f "X-type" s i t e s i s s m a l l , w i t h i n the s c a t t e r o f the e x p e r i m e n t a l N 2 + TAN p o i n t s i n f i g . 12. 71 4.3.3 Nitroimidazole Treatment Unlike TAN which produces l i t t l e or no increase i n the y i e l d of SSB's (section 3.1.1; Agnew, 1972), the nitroimidazoles, metronidazole and Ro-07-0582, cause large enhancements i n SSB production during hypoxic i r r a d i a t i o n (section 3.1.1). Also, the presence of nitroimidazoles during i r r a d i a t i o n appears to have l i t t l e e f f e c t on the template function of parental DNA (section 3.2.7), whereas TAN produces interruptions i n newly synthesized DNA. None of the 3 drugs examined i n h i b i t e d the re j o i n i n g of SSB's. These observations are summarized i n Table II along with s u r v i v a l DMF values determined by others i n t h i s laboratory (Agnew and Skarsgard, 1972; Moore, P a l c i c and Skarsgard, 1975; Agnew, Pa l c i c and Skarsgard, 1974). Table I I : Drug DMF for Survival DMF for SSB's Inhibits 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 previously (section 1.5), electron a f f i n i c r a d i o s e n s i t i z e r s react with DNA free r a d i c a l s either by electron transfer or by adduct formation. The data summarized i n Table II suggest that metronidazole and Ro-07-0582 react with i r r a d i a t e d DNA primarily by oxidation by electron transfer. Oxidation of the DNA free r a d i c a l s often leads to the formation of a SSB. TAN, on the other hand, binds to DNA. forming stable TAN-DNA adducts which lead to interruptions in the subsequent synthesis of daughter strands. I t i s possible that the nitroimidazoles form r a d i c a l -s e n s i t i z e r adducts as well but that for some reason these lesions do not interrupt DNA synthesis. However, i r r a d i a t i o n of hypoxic solutions of DNA containing 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 yielded G values, that i s , number of s e n s i t i z e r molecules bound per 100 eV deposited, of 1.3 for TAN (Brustad, Jones and Wold, 1973) and ^ 0.02 for metronidazole (Willson, Cramp and Ings, 1974). This supports the notion that radiation-induced binding of nitroimidazoles to DNA i s not a s i g n i f i c a n t component of the i r mode of action. 73 4.4 TAN PRETREATMENT Results presented i n section 3.2.2 showed that TAN pretreatment has no 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 DNA synthesis. This observation i s not unexpected i n view of the fact that TAN does not bind to unirradiated DNA (section 3.2.2; Brustad, Jones and Wold, 1973), although i t i s conceivable that TAN pretreatment could inactivate c e r t a i n DNA repair systems, leading to unrepaired damage i n the template and hence to interruptions i n newly synthesized DNA. Apparently, the TAN pretreatment e f f e c t on c e l l s u r v i v a l (DMF = 1.3, Agnew and Skarsgard, 1972) cannot r e a d i l y be explained by the formation of TAN-DNA adducts, si m i l a r to those produced during TAN treatment. However, the observation that some nitroxide free r a d i c a l s lose t h e i r electron spin resonance (ESR) signal i n the presence of b i o l o g i c a l materials (Giotta and Wang, 1972; Agnew and Skarsgard, 1972) suggests that TAN may become bound at other c r u c i a l s i t e s i n the c e l l during the pretreatment. This component of s e n s i t i z a t i o n would also necessarily be present during a TAN treatment experiment since TAN i s present at least 45 minutes p r i o r to i r r a d i a t i o n i n the treatment procedure. 74 4.5 TAN POST-TREATMENT In section 3.2.3, results were presented i n d i c a t i n g that TAN added after 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 synthesis. However, Agnew and Skarsgard (1972) have shown that addition of TAN immediately after i r r a d i a t i o n produces almost f u l l s e n s i t i z a t i o n of c e l l s u r v i v a l , and Brustad, Jones and Wold (1973) have demonstrated that some DNA transients, which are able to intera c t with TAN, have a li f e t i m e of several minutes i n v i t r o . The f a i l u r e to observe a TAN post-treatment e f f e c t on po s t - i r r a d i a t i o n DNA synthesis could be due to several factors. F i r s t of a l l , Brustad et a l . (1973) have found that the DNA transients and t h e i r reaction with TAN in. v i t r o are extremely sensitive to trace amounts of oxygen. In fact, the long-lived transients that they reported were obtained under anoxia. Post-treatment experiments were carr i e d out by adding an aliquot of i r r a d i a t e d c e l l suspension to an aerobic TAN-containing solution, i n both the res u l t s reported here and those of Agnew and Skarsgard. I f oxygen does compete with TAN for 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 to for s i t e s produced during i r r a d i a t i o n (see section 3.2.4), TAN al t e r a t i o n of the DNA template would not occur during our procedure. Hence the post-treatment e f f e c t on c e l l s u r v i v a l would not be due to t h i s mode of action. 75 A l s o , the extreme t e m p e r a t u r e dependence 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 s u r v i v a l makes i t u n l i k e l y t h a t the 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 o f TAN 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 . TAN p o s t - t r e a t m e n t produces a l m o s t f u l l s e n s i t i z a t i o n a f t e r a 1 minute i n c u b a t i o n a t 33°C but much s m a l l e r e f f e c t s a t 35°C and 37°C (Agnew and S k a r s g a r d , 1972). Mechanisms f o r t h e removal o f f r e e r a d i c a l s from c e l l u l a r DNA would n ot l i k e l y e x h i b i t t h i s h i g h temperature s e n s i t i v i t y . Thus, 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 i s not due t o b i n d i n g o f TAN t o l o n g - l i v e d DNA r a d i c a l s . 76 5. CONCLUSIONS In t h i s work we have shown: (1) B o t h m e t r o n i d a z o l e and Ro-07-0582 p r o d u c e l a r g e i n c r e a s e s i n t h e y i e l d o f s i n g l e s t r a n d b r e a k s d u r i n g h y p o x i c i r r a d i a t i o n ; TAN c a u s e s o n l y . a s m a l l i n c r e a s e . (2) TAN, m e t r o n i d a z o l e and Ro-07-0582 do n o t i n h i b i t t h e r e j o i n i n g o f s i n g l e s t r a n d b r e a k s . (3) TAN p r e s e n t d u r i n g i r r a d i a t i o n o f h y p o x i c CH2B 2 c e l l s p r o d u c e s i n t e r r u p t i o n s i n t h e s u b s e q u e n t s y n t h e s i s o f d a u g h t e r DNA. T h i s e f f e c t may r e p r e s e n t a m a j o r component o f t h e mechanism o f t h e s e n s i t i z a t i o n o b s e r v e d when TAN i s p r e s e n t d u r i n g i r r a d i a t i o n . (4) A l t h o u g h TAN e x h i b i t s b o t h a p r e - and a p o s t -i r r a d i a t i o n s e n s i t i z i n g e f f e c t on c e l l s u r v i v a l , t h e s e t r e a t m e n t s have no d e m o n s t r a b 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 y n t h e s i s . Hence, p r e - and p o s t - t r e a t m e n t e f f e c t s must be due t o a d i f f e r e n t mode o f TAN a c t i o n . A l t e r n a t i v e l y , one must c o n c l u d e t h a t t h e i n t e r r u p t i o n s i n DNA s y n t h e s i s o b s e r v e d f o r TAN t r e a t m e n t a r e n o t t h e e x p l a n a t i o n o f t h e d r u g ' s e f f e c t on c e l l s u r v i v a l . (5) The n i t r o i m i d a z o l e s , m e t r o n i d a z o l e and Ro-07-0582, do n o t a l t e r i r r a d i a t e d p a r e n t a l DNA i n s u c h a manner as t o i n t e r r u p t t h e s y n t h e s i s o f d a u g h t e r s t r a n d s . 77 APPENDIX A Calculation of Approximate Percentage of Pulse Label Used for  Repair Replication 1 rad = 1 0 0 ergs/gram 1 dalton = 1 . 6 6 0 x 1 0 g r a m 1 eV = 1 . 6 0 2 x l 0 - 1 2 ergs M = number of grams of DNA per c e l l f = f r a c t i o n of c e l l s i n S phase Average molecular weight of 1 nucleotide - 3 4 0 daltons. C H 2 B 2 c e l l s have approximately a 6 hour S phase i n a 12 hour c e l l cycle. Hence i n 6 minutes, a c e l l i n S phase w i l l 6 synthesize ^ •jg-g- x M grams of DNA. Therefore i n 6 minutes, the number of nucleotides used for semi-conservative DNA synthesis by an asynchronous C H 2 B 2 c e l l , . . - 1 M x l 0 2 1 + 1 population i s ^ f x _ x 1 > 6 6 Q x ^ = f x M x 2 . 9 5 x 1 0 1 9 . Energy required to produce 1 S S B under hypoxic conditions (from Table I ) i s 1 1 5 eV = 1 1 5 x 1 . 6 0 2 x 1 0 ~ 1 2 ergs. 1 5 , 0 0 0 rads w i l l deposit 1 . 5 x l O 6 ergs/gram of DNA. Therefore the number of S SB's produced per c e l l by a dose of 1 . 5 x 1 0 6 x M 15 krads under hypoxia i s 1 1 5 x 1 . 6 0 2 x 1 0 " 1 2 = 8 . 1 4 x M x 1 0 1 5 . 78 Cel l s not i n Gj w i l l have more than the normal complement of M grams of DNA but not more than 2M; therefore the maximum number of SSB's/cell w i l l be ^ 2 x 8.14 x M x 1 0 1 5 = 1.63 x M x 10 1 6. Assuming that 10 nucleotides are inserted per SSB and, as a large overestimate, that a l l SSB's are repaired during the 6 minute pulse, the number of nucleotides used for repair r e p l i c a t i o n w i l l be ^ 1.63 x M X 10 1 7. It follows that the percentage of the 3H-TdR, incorporated during the 6 minute pulse l a b e l , that i s used for repair 1 63 x M x 1 0 1 6 r e p l i c a t i o n w i l l be less than ^ — r x 100% f x M x 2.95 x i o 1 9 0.55 f * 1% for f = 0.5. •j 7 9 APPENDIX B I n f i g . 12, the Mw o f DNA, s y n t h e s i z e d a f t e r i r r a d i a t i o n under 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 s d a l t o n s w i t h no r e p a i r t o ^  2 . 7 8 x l 0 8 d a l t o n s w i t h a 120 minute r e p a i r 2 78 i n c u b a t i o n . T h i s r e s u l t i m p l i e s <v ^' ^  - 1 = 0.30 "X-type" s i t e s (see f i g . 23) p e r f u l l - s i z e ('v 2 . 8 x l 0 8 d a l t o n s ) DNA segment. A f t e r 12 0 minutes 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 i n the pre 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 i n 3 hours i s ^ 1 . 0 2 x l o 8 d a l t o n s ( f i g . 1 2). T h e r e f o r e , t h e number o f measurable TAN-DNA adducts produced p e r f u l l - s i z e d segment 2 78 i s % ^ ' - 1 - 1 . 7 , s i n c e no d e t e c t a b l e X-type l e s i o n s remain a f t e r 2 hours r e p a i r i n c u b a t i o n ( a f t e r i r r a d i a t i o n i n dr u g -f r e e medium). 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 2 + TAN, t h e r e w i l l be ^ 1.7 + 0.30 i n t e r r u p t i o n s (due t o TAN-DNA adducts and u n r e p a i r e d X-type l e s i o n s ) i n t h e DNA segments s y n t h e s i z e d i n t h e n e x t 3 hou r s . Hence, one would e x p e c t a Mw 2 78 of ^ 1 7 + 0 * 3 0 + 1 ~ 0 ' 9 3 x l ° 8 d a l t o n s , a t no r e p a i r on the N 2 + TAN l i n e i n f i g . 12. 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