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The response of cultured normal and DNA repair deficient human cells to Adenovirus type 12 : UV-impaired… Lam, Paul Pong-Shing 1975

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THE RESPONSE OF CULTURED NORMAL AND DNA REPAIR DEFICIENT HUMAN CELLS TO ADENOVIRUS TYPE 12, UV- IMPAIRED VIRUS OR THE COMBINED EFFECT OF VIRUS AND CHEMICAL CARCINOGENS by PAUL PONG-SHING LAM B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t o f Z o o l o g y 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 t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF . A p r i I , BRIT ISH COLUMBIA 1975 In presenting th is thes is in p a 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 shal l make it f r ee ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The a c t i o n of UV and c h e m i c a l c a r c i n o g e n s , on t h e r e p l i c a t i o n of A d e n o v i r u s t y p e 12 (Ad 12) i n c u l t u r e d normal and DNA r e p a i r d e f i c i e n t human c u l t u r e s vfas*?4 examf ned. F i b r o b l a s t s o b t a i n e d from s k i n b i o p s i e s o f a normal p e r s o n and 3 p a t i e n t s w i t h Xeroderma pigmentosum (XP-E, XP-C, XP-K) were grown i n m onolayer c u l t u r e s f e d w i t h E a g l e ' s Minimum E s s e n t i a l Medium (MEM) supplemented w i t h f e t a l c a l f serum. C e l l s were exposed t o U V - i r r a d i a t e d Ad 12 o r t h e combined t r e a t m e n t w i t h Ad 12 and c h e m i c a l c a r c i n o g e n s . The c h e m i c a l s which have been examined i n c l u d e 4 - n i t r o q u i n o I I n e-N-oxide (4NQ0) and i t s d e r i v a t i v e s , N - m e t h y I - N ' n i t r o - N - n i t r o s o - g u a n i d i n e (MNNG), a f l a -t o x i n B l , and s t e r i g m a t o c y s t i n . The c e l l s were sampled a t 72 hours p o s t - i n f e c t i o n and c e l l s w i t h r e p l i c a t i n g v i r u s were i d e n t i f i e d by t h e p r e s e n c e of i n t r a n u c l e a r i n c l u s i o n b o d i e s ( I B ) . When normal and XP c u l t u r e s were exposed t o U V - i r r a d i a t e d Ad 12, t h e DNA r e p a i r d e f i c i e n t XP c u l t u r e s showed a reduced c a p a c i t y t o form IB. The a b i l i t y t o r e a c t i v a t e t h e i r r a d i a t e d v i r u s a p p e a r e d t o -depend on t h e r e p a i r c a p a c i t y of t h e c e l l s . F o r example, XP-K which has a h i g h e r r e p a i r t h a n XP-C and XP-E r e a c t i v a t e d U V - i m p a i r e d Ad 12 more e f f i c i e n t l y . • A lower f r e q u e n c y of c e l l s w i t h IB i n XP c u l t u r e s was a l s o o b s e r -ved f o l l o w i n g t h e combined t r e a t m e n t w i t h Ad 12 and c e r t a i n c h e m i c a l c a r c i n o g e n s . For example, t h e r e was a marked d i f f e r e n c e between XP and c o n t r o l c e l l s f o l l o w i n g e x p o s u r e t o Ad 12 and 4NQ0. The c a p a c i t y t o form IB was a b o l i s h e d f a s t e r i n XP c e l l s t h a n i n c o n t r o l c e l l s . I t i s p o s t u l a t e d t h a t 4NQ0 i n t e r a c t e d w i t h v i r a l DNA and t h e damage was u n r e p a i r e d o r o n l y p a r t i a l l y r e p a i r e d i n XP c e l l s r e s u l t i n g i n an a b o r t i v e i n f e c t i o n . On t h e o t h e r hand i n c o n t r o l c e l l s , t h e damaged v i r a l DNA was r e p a i r e d more e f f i c i e n t l y r e s u l t i n g i n a. s u c c e s s f u l v i r a l r e p l i c a t i o n . : Exposure o f c e l l s i n f e c t e d w i t h Ad 12 t o MNNG, however, showed no d i f f e r e n c e i n t h e c a p a c i t y t o form IB between c o n t r o l and XP c e l l s . I t i s p o s t u l a t e d t h a t MNNG i n t e r a c t e d w i t h t h e v i r a l DNA but t h e damage was r e p a i r e d e f f i c i e n t l y i n c o n t r o l and'XP c e l l s t h e r e b y r e s u l t i n g i n a s u c c e s s f u l v i r a l r e p l i c a t i o n i n both c e l l t y p e s . T h i s a g r e e s w i t h p r e v i o u s r e p o r t s t h a t DNA r e p a i r s y n t h e s i s as shown by u n s c h e d u l e d ^HTdR uptake was o b s e r v e d i n both normal and XP c e l l s f o l l o w i n g MNNG t r e a t m e n t . An rn_ v i t r o t e c h n ique t o s imu I a t e j_n_ v i vo metaboI i sm of p r e -c a r c i n o g e n s was used t o " a c t i v a t e " a f l a t o x i n Bl and s t e r i g m a t o -c y s t i n . I t was r e p o r t e d t h a t when a f l a t o x i n B l o r s t e r i g m a t o c y s t i n was combined w i t h NADP-dependent a c t i v a t i o n systems c o n t a i n i n g t h e p o s t - m i t o c h o n d r i a I s u p e r n a t a n t (9S) f r a c t i o n of mouse l i v e r homo-g e n a t e , l e v e l s of DNA r e p a i r s y n t h e s i s , chromosome a b e r r a t i o n s and c e l l l e t h a l i t y was i n c r e a s e d . In t h e p r e s e n t s t u d y , c e l l s i n f e c t e d w i t h Ad 12 were exposed t o " a c t i v a t e d " a f l a t o x i n Bl o r s t e r i g m a t o -c y s t i n . W i t h o u t any " a c t i v a t i o n " , a f l a t o x i n Bl and s t e r i g m a t o c y s t i n showed l i t t l e e f f e c t s on IB f o r m a t i o n . However,- XP c e l l s exposed t o " a c t i v a t e d " a f l a t o x i n Bl o r s t e r i g m a t o c y s t i n showed a marked. d e c r e a s e i n IB f o r m a t i o n as compared t o c o n t r o l c e l l s . I t i s p o s t u l a t e d t h a t " a c t i v a t e d " a f l a t o x i n Bl and s t e r i g m a t o c y s t i n a l s o i n t e r a c t e d w i t h v i r a l DNA as i n t h e case o f 4NQ0 and t h e damage'was u n r e p a i r e d o r p a r t i a l l y r e p a i r e d i n XP ceI Is r e s u I t i n g i n an a b o r t i v e i n f e c t i o n . The consequences o f an a b o r t i v e l y i n f e c t e d c e l l t o become a neo-p l a s t i c c e l l and t h e p o s s i b l e use of t h i s Ad 12 and c a r c i n o g e n combined t r e a t m e n t as a cheap and e f f i c i e n t way of s c r e e n i n g e n v i r o n m e n t a l c h e m i c a l c a r c i n o g e n s and D N A - r e p a i r d e f i c i e n t human p o p u l a t i o n s a r e a I so d i s c u s s e d . i v. ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o . e x p r e s s h i s s i n c e r e g r a t i t u d e t o Dr. H.F. S t i c h , P r o f e s s o r o f Z o o l o g y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , whose a d v i c e , g u i d a n c e and encouragement have made t h i s t h e s i s p o s s i b l e . He i s a l s o i n d e b t e d t o Mrs. W. S t i c h , Mr. R.H.C. San, M i s s C. Yo s h i z a w a and Dr. L.'Lo f o r t h e i r e x p e r t t e c h n i c a l a s s i s t a n c e t h r o u g h o u t . He i s a l s o g r a t e f u l t o . D r . S. Mak of McMaster U n i v e r s i t y f o r h i s i n s t r u c t i o n s i n p r e p a r i n g Adeno-v i rus I 2. F i n a n c i a l a s s i s t a n c e from t h e N a t i o n a l R e s e a r c h C o u n c i l of Canada and t h e N a t i o n a l C a n c er I n s t i t u t e o f Canada ( g r a n t s t o Dr. H.F. S t i c h ) i s g r a t e f u l l y acknowledged. TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS ' v LIST OF FIGURES. v i i i INTRODUCTION I OBJECTIVES 6 MATERIALS AND METHODS 7 1. C e l l C u l t u r e s and Media 7 2. Chemi ca I s 7 3. P r e p a r a t i o n o f Ad 12 8 4. U V - i r r a d i a t i o n o f Ad 12 9 5. Treatment o f C e l l s w i t h Ad 12 9 6. Treatment o f C e l l s w i t h Chemical C a r c i n o g e n s . .. 10 7. In V i t r o A c t i v a t i o n of P r e c a r c i n o g e n s 10 (a) P r e p a r a t i o n o f P o s t - M i t o c h o n d r i a I F r a c t i o n (9S) of Mouse L i v e r Homogenate 10* (b) Treatment o f C e l l s w i t h A c t i v a t i o n M i x t u r e .... II 8. F i x a t i o n and S t a i n i n g I I -RESULTS .. -. .. 12 1. -The Input M u l t i p l i c i t y o f Ad 12 .... 12 2. R e a c t i v a t i o n o f U V - i m p a i r e d Ad 12 i n Normal and XP ' . Cel Is 17 3. The E f f e c t s o f 4NQ0 on Ad 12 R e p l i c a t i o n 18 4. The E f f e c t s of MNNG on Ad I 2 Rep I i c a t i o n 25 5. The E f f e c t s o f A c t i v a t e d P r e c a r c i n o g e n s on Ad 12 Rep I i c a t i o n 29 - v l . Page 6. S t a t i s t i c s 32 DISCUSSION • 38 1. R e a c t i v a t i o n of U V - i m p a i r e d Ad 12 i n Norma] and XP Cel Is 38 2. ' Combined A c t i o n o f Chemical C a r c i n o g e n s and Ad 12 on Normal and XP Ce I I s 39 3. P o s s i b l e F a t e o f C e l l s f o l l o w i n g an A b o r t i v e I n f e c -t i o n 41 4. I d e n t i f i c a t i o n of DNA R e p a i r D e f i c i e n t M u t a n t s i n t h e Human P o p u l a t i o n 44 5. I d e n t i f i c a t i o n o f Chemical C a r c i n o g e n s and P r e c a r -c i n o g e n s 45 6. O u t l o o k 46 REFERENCES 47 v i i . LIST OF FIGURES F i g u r e - Page 1 A model f o r e x c i s i o n r e p a i r ' 3 2 (A) N u c l e i c o n t a i n i n g i n t r a n u c l e a r i n c l u s i o n b o d i e s ( I B ) 14 (B) I n h i b i t i o n o f IB f o r m a t i o n i n XP c e l l s by 4N00 t r e a t m e n t ' 14 3 E f f e c t of s a m p l i n g t i m e on t h e f r e q e n c y of c e l l s w i t h IB i n normal c u l t u r e s ..- 16 4 E f f e c t of m u l t i p l i c i t y of i n f e c t i o n (M.O.I.) on t h e f r e q e n c y o f ' c e l l ' s w i t h IB i n normal and XP • c u l t u r e s .- 16 5 E f f e c t o f U V - i r r a d i a t i o n on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l ceI Is and t h r e e XP e e l I cu I t u r e s • • • 19 6 E f f e c t of U V - i r r a d i a t i o n on t h e c a p a c i t y of Ad 12 t o form IB i n t h e h e t e r o z y g o u s p a r e n t s of XP-E 21 7 E f f e c t of U V - i r r a d i a t i o n on t h e c a p a c i t y of Ad 12 t o form IB i n t h e h e t e r o z y g o u s p a r e n t s of XP-C .. 21 8. , E x p e r i m e n t a l D e s i g n . C o n t r o l and XP c u l t u r e s were t r e a t e d w i t h 4NO0 a t 2 h o u r s , 10 hours o r 14 hours f o l l o w i n g Ad 12 i n f e c t i o n 24 9. E f f e c t of 4N00 t r e a t m e n t on t h e c a p a c i t y of Ad 12 t o form IB i n n o r m a l , XP-E, XP-E f a t h e r and XP-E mother c u l t u r e s 24 1 v i i i . F i gure Page 10 E f f e c t o f 4NQ0 t r e a t m e n t g i v e n a t v a r i o u s i n t e r -•' va I s a f t e r Ad 12 i n f e c t i o n on IB f o r m a t i o n i n c o n t r o l and XP-E c u l t u r e s 27 I I E f f e c t o f d e r i v a t i v e s o f 4N00 on t h e c a p a c i t y o f Ad 12 t o form IB i n normal and XP-E c u l t u r e s .... 28 12 E x p e r i m e n t a l D e s i g n . C o n t r o l and XP c e l l c u l t u r e s were t r e a t e d w i t h MNNG a t 2 hours f o l l o w i n g Ad 12 i n f e c t i o n 31 13 E f f e c t o f MNNG on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l and XP-E c u l t u r e s 31 14 E x p e r i m e n t a l D e s i g n . C o n t r o l and XP c u l t u r e s were exposed t o p r e c a r c i n o g e n s a c t i v a t e d by a 9S mouse l i v e r f r a c t i o n a t 2 hours f o l l o w i n g Ad 12 i n f e c t i o n ;. 34 ^ 15 E f f e c t o f n o n a c t i v a t e d a f l a t o x i n Bl on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l and XP-E c u l t u r e s . . 34 { 16 E f f e c t o f a c t i v a t e d a f l a t o x i n Bl on t h e c a p a c i t y o f Ad l'2 t o form IB i n c o n t r o l and XP-E cu ltur.es .... 34 17 E f f e c t o f n o n a c t i v a t e d s t e r i g m a t o c y s t i n on t h e . c a p a c i t y of Ad 12 t o form IB i n contro'l and XP-E cu I t u r e s .. . 36 18 E f f e c t of a c t i v a t e d s t e r i g m a t o c y s t i n on t h e c a p a -c i t y o f Ad 12 t o form IB i n c o n t r o l and XP-E cu I t u r e s 36 i x. F i g u re Page 19 Proposed model f o r t h e f a t e o f Ad 12 i n normal and XP c e l l s t r e a t e d w i t h 4NQ0 o r MNNG 43 x. INTRODUCTION The f i r s t e v i d e n c e t h a t c a n c e r can be caused by e n v i r o n -mental f a c t o r s came i n 1775 when P e r c i v a l P o t t p u b l i s h e d a paper on t h e f o r m a t i o n of s k i n c a n c e r o f t h e s c r o t u m i n chim-ney sweeps due t o ' p r o l o n g e d e x p o s u r e t o s o o t ( P o t t , r e p r i n t e d 1963). F u r t h e r e v i d e n c e came a t t h e t u r n of t h e 20th c e n t u r y when a h i g h i n c i d e n c e of c a n c e r was noted among w o r k e r s i n dye f a c t o r i e s and i n gas f a c t o r i e s who were exposed t o c e r t a i n d y e s t u f f s and t a r ( r e v i e w e d by H i e g e r , 1961). Today i t i s e v i -dent t h a t e n v i r o n m e n t a l f a c t o r s such as c h e m i c a l c a r c i n o g e n s and o n c o g e n i c v i r u s e s a r e t h e major cause of c a n c e r ( H i e g e r , 1961; Andrewes,1970). The mode o f a c t i o n o f c a r c i n o g e n i c chemi-c a l s and v i r u s e s on mammalian c e l l s a r e e n t i r e l y d i f f e r e n t . Chemi-c a l c a r c i n o g e n s i n t e r a c t w i t h c e l l u l a r DNA i n such .a way t h a t t h e g e n e t i c e x p r e s s i o n i s a l t e r e d ( M i l l e r , 1970), w h i l e o n c o g e n i c v i r u s e s add a d d i t i o n a l g e n e t i c i n f o r m a t i o n t o t h e h o s t c e l l t h e r e b y a l t e r i n g normal c e l l f u n c t i o n s . B a s i c a l l y t h e r e a r e two t y p e s of c h e m i c a l c a r c i n o g e n s : (a) c h e m i c a l s t h a t i n t e r a c t d i r e c t l y w i t h t h e DNA, and (b) c h e m i c a l s t h a t a r e f i r s t t r a n s f o r m e d i n t o h i g h l y r e a c t i v e compounds c a p a b l e pf i n t e r a c t i n g w i t h t h e DNA. The l a t t e r t y p e of c h e m i c a l s a r e termed " p r e c a r c i n o g e n s " and i t i s p o s t u l a t e d t h a t t h e i n t e r m e d i a t e r e a c t i v e compounds formed a r e u s u a l l y e l e c t r o p h i I i c s p e c i e s of t h e p r e c a r c i nogens (Mi I I e r e t a I . , 1969a,b; _M.i>l her?, 1970). The major s i t e o f r e a c t i o n i n t h e DNA w i t h c a r c i n o g e n s o r a c t i v e s p e c i e s o f t h e p r e c a r c i n o g e n s i s a t t h e 7 - p o s i t i o n o f g u a n i n e (Bardos e t a j _ . , 1965; Boy I and, I 9 6 9 ) . Under a v a r i e t y o f c o n d i t i o n s , o t h e r s i t e s of r e a c t i o n have a l s o been r e p o r t e d (Law l e y , 1966; Mi I l e r and Mi I l e r , 1971; I r v i n g , 1973; Sarma e t aj_. , 1974). A p a r t from t h e s e c o v a l e n t b o n d i n g s , n o n - c o v a l e n t b o n d i n g s a r e a l s o p o s s i b l e by i n t e r c a l a t i o n ( i n s e r t i o n o f a c a r c i n o g e n i n between base p a i r s i n t h e DNA d o u b l e h e l i x ) o r by a d l i n e a t i o n ( b i n d i n g of t h e c a r c i n o g e n e x t e r n a l l y t o t h e bases p e r p e n d i c u l a r t o t h e p l a n e s of t h e base p a i r s ) , ( A r c o s and A r g u s , 1968). A l t e r a t i o n of t h e DNA s t r u c t u r e e i t h e r by c o v a l e n t o r non-c o v a l e n t i n t e r a c t i o n s w i l l e l i c i t DNA r e p a i r s y n t h e s i s (Gaudin e t aj_. , I97|': Hei de I b e r g e r , 1973; H o w a r d - F l a n d e r s , 1968; P a i n t e r , 1971). F i g u r e I shows t h e e s s e n t i a l f e a t u r e s of t h e e x c i s i o n -r e p a i r system which can be used t o e x p l a i n t h e r e p a i r of c h e m i c a l -induced DNA damage. The b i n d i n g of c a r c i n o g e n s t o t h e DNA r e s u l t s i n a s t r u c t u r a l d i s t o r t i o n of t h e DNA h e l i x (Paul et-'a I.-. , 1971) which s e r v e s as a r e c o g n i t i o n s i t e f o r e n d o n u c l e a s e a t t a c k . DNA p o lymerase t h e n r e s y n t h e s i z e s t h e DNA a t t h e n i c k e d s i t e u s i n g t h e complementary s t r a n d as a t e m p l a t e . F i n a l l y a - T i g a s e j o i n s t h e r e s y n t h e s i z e d DNA w i t h t h e o r i g i n a l DNA, I t must be noted a t t h i s p o i n t t h a t w i t h c e r t a i n c h e m i c a l c a r c i n o g e n s , spontaneous h y d r o l y t i c c h a i n f i s s i o n of t h e DNA i s p o s s i b l e and t h e e n d o n u c l e a s e s t e p i s not r e q u i r e d . C e r t a i n l y DNA r e p a i r r e s t o r e s t h e c e l l t o i t s o r i g i n a l f u n c t i o n i n g s t a t e ' m o s t of t h e t i m e , but o c c a s i o n a l l y , t h e DNA may be u n r e p a i r e d o r f a l s e l y r e p a i r e d so t h a t an a b n o r m a l l y f u n c t i o n -ing c e l l may a r i s e . There i s now ample c i r c u m s t a n t i a l e v i d e n c e base damage E1 chain breakage 1 n E2 E4 E3 Possible enzymes: E1 endonuclease E 2 exonuclease E 3 DNA polymerase E<4 ligase Figure I: A model for excision repair. (Adapted from Cleaver, I974) 4. t h a t DNA r e p a i r p l a y s an i m p o r t a n t r o l e i n c a r c i n o g e n e s i s ( C l e a v e r , 1973; H e i d e I b e r g e r , 1973). When n u c l e a r DNA i s a l t e r e d by a c h e m i c a l c a r c i n o g e n , one of t h e f o l l o w i n g t h i n g s may t a k e p l a c e : (a) t h e DNA damage i s f a i t h f u l l y r e p a i r e d and t h e , e e l I f u n c t i o n s n o r m a l l y , (b) t h e DNA damage i s t o o s e v e r e t o be r e p a i r e d and t h e c e l l d i e s , o r ( c ) some of t h e damage i s u n r e p a i r e d o r f a l s e l y r e p a i r e d and t h e c e l l f u n c t i o n s a b n o r m a l l y . A p h y s i o l o g i c a l l y t r a n s f o r m e d c e l l such as a c a n c e r c e l l may a r i s e i n t h e l a s t i n s t a n c e . S u p port o f t h i s h y p o t h e s i s comes from t h e r e p o r t s t h a t p a t i e n t s w i t h a r a r e h e r e d i t a r y d i s e a s e known as Xeroderma p i g -mentosum (XP) d e v e l o p e d tumours on UV-exposed a r e a s o f t h e i r s k i n (Montgomery, 1967; C l e n d e n n i n g , 1971; El-Hefnaw'-, 1972; Reed et_ aj_. , 1969; Robb i ns e t aj_. , 1974). Cu I tured. XP e e l I s showed, " a reduced l e v e l o f DNA r e p a i r ( C l e a v e r , - 1968; C l e a v e r , 1973; S t i c h and San, 1971; S t i c h e t a L , 1973, 1974b) which i s p r o b a b l y caused by a l a c k of e n d o n u c l e a s e a c t i v i t y ( C l e a v e r , 1969; S e t I ow e t a j _ . , 1969). An i n c r e a s e d s e n s i t i v i t y of XP c e l l s t o some c h e m i c a l c a r -c i n o g e n s was r e p o r t e d r e c e n t l y ( C l e a v e r , 1971.; S t i c h and San, 1971; S t i c h e t a j _ . , 1972b; S t i c h and San, 1973; S t i c h e t a_N, 1973) For example, when XP c e l l s were t r e a t e d w i t h 4-n-i t r o q u i no I i ne-N-oxide (4N00), a reduced l e v e l o f DNA r e p a i r as i n d i c a t e d by t h e un s c h e d u l e d uptake o f ^HTdR was o b s e r v e d . Thus t h e d i s e a s e Xeroderma pigmentosum d e f i n e s a d e f i n i t e r e l a t i o n s h i p between 5. DNA damage by c a r c i n o g e n i c a g e n t s "such as UV and 4NQ0, DNA r e p a i r and c a r c i n o g e n e s i s . The mechanism of t r a n s f o r m a t i o n by u n r e p a i r e d DNA damage i s a t p r e s e n t p o o r l y u n d e r s t o o d . The second major group o f c a r c i n o g e n s , t h e o n c o g e n i c v i r u s e s , i n -duces c a n c e r by a c o m p l e t e l y d i f f e r e n t mechanism. Oncogenic DNA v i r u s e s such as human A d e n o v i r u s 12 i n f e c t mammalian c e l l s by a mechanism known as " v i r o p e x i s " which i s s i m i l a r t o p i n o c y t o s i s (Fazekas de S t . G r o t h , 1948). The v i r a l DNA i s r e l e a s e d i n t h e c y t o p l a s m by the. removal o f t h e p r o t e i n c o a t ( P h i l i p s o n , 1967; Sussenbach, 1967), and i t i s t h e n t r a n s -p o r t e d t o t h e n u c l e u s . I t i s g e n e r a l l y b e l i e v e d t h a t t h e v i r a l DNA i n t e -g r a t e s i n t o t h e h o s t c e l l DNA accompanied by p a r t i a l e x p r e s s i o n o f t h e v i r a l genome ( C a m p b e l l , 1962; N i c h o l s e t a j _ . , 1968; Zur Hausen, 1968; Oxman and B l a c k , 1966; Oxman e t a l . , 1967). Oncogenic v i r u s e s . s u c h as polyoma v i r u s , SV 40 and Ad 12 induce t h e s y n t h e s i s o f an a n t i g e n known as t h e tumour o r T a n t i g e n which can be l o c a l i z e d by f e r r i t i n - t a g g e d a n t i body I abe I I i ng (Ka I n i ns e_t a j _ . , I 9 6 7 ) . The T a n t i gen a s s o c i a t e s w i t h t h e e e l I ' s s u r f a c e and c o u l d a c c o u n t f o r t h e changed r e s p o n s e o f t h e e e l I t o c o n t a c t w i t h o t h e r c e l l s . The i n t e g r a t i o n o f v i r a l DNA i n t o c e l l u l a r genome r e q u i r e s DNA breakage and r e p a i r . A p o s s i b l e l i n k between DNA b r e a k a g e , DNA r e p a i r . and c a r c i n o g e n e s i s i s un c o v e r e d by s t u d i e s w i t h combined v i r u s and c h e -m i c a l c a r c i n o g e n t r e a t m e n t . F o r example, t h e combined a p p l i c a t i o n o f Ad 12 and 4NQ0 which i n d u c e s DNA breakage r e s u l t s i n an enhanced t r a n s f o r m a t i o n f r e q u e n c y i n hamster c e l l c u l t u r e s ( S t i c h e t a I . , -I 972a; C a s t o e t a I ., 1973; C a s t o , 1973). I t i s assumed t h a t DNA breakage by 4NQ0 f a c i l i t a t e s t h e i n c o r p o r a t i o n of v i r a l DNA i n t o t h e h o s t genome which i n t u r n en-hances t r a n s f o r m a t i o n . OBJECTIVES The p r e s e n t s t u d y a t t e m p t s t o i n v e s t i g a t e t h e i n v o l v e m e n t of 'DNA r e p a i r i n t h e e x p r e s s i o n o f v i r a l f u n c t i o n . I t i s b a s i -caI Iy d i v i ded i nto two p o r t i ons: ( I ) i n t h e f i r s t p a r t t h e capa-c i t y o f D N A - r e p a i r d e f i c i e n t XP c u l t u r e s t o s u p p o r t t h e r e p l i c a t i o n of U V - i m p a i r e d .Ad 12 w i l l be examined; and (2) i n t h e second p a r t t h e r esponse of XP c u l t u r e s t o t h e combined t r e a t m e n t w i t h Ad 12 and c h e m i c a l c a r c i n o g e n s i s i n v e s t i g a t e d . Ad 12 was used because i t r e a d i l y r e p l i c a t e s i n human c e l l s and forms i n t r a n u c l e a r i n c l u -s i o n b o d i e s ( I B) which a r e t h e r e p l i c a t i o n s i t e f o r a d e n o v i r u s and a r e t h e i n d i c a t i o n o f a s u c c e s s f u l v i r a l r e p l i c a t i o n . The human p o p u l a t i o n i s a f f e c t e d by i n f e c t i o u s and d e f e c t i v e v i r u s e s , c h e m i c a l c a r c i n o g e n s , and i s o f t e n exposed s i m u l t a n e o u s l y t o both c h e m i c a l and v i r a l a g e n t s . I t i s t h e purpose o f t h i s s t u d y t o examine t h e re s p o n s e o f D N A - r e p a i r d e f i c i e n t mutants such as XP p a t i e n t s t o d e f e c t i v e v i r u s e s ( e . g . U V - i m p a i r e d Ad 12) and t o t h e combined a c t i o n of v i r u s e s and c h e m i c a l s , and t o d e v e l o p a q u i c k a s s ay f o r DNA r e p a i r d e f i c i e n c y i n man based on t h e r e a c t i v a t i o n of i n a c t i v a t e d v i r u s e s i n XP c e l l s . 7. MATERIALS AND METHODS 1. C e l l C u l t u r e s and Media •> C u l t u r e d human f i b r o b l a s t s were o b t a i n e d from s k i n b i o p s i e s of a normal C a u c a s i a n f e m a l e , t h r e e p a t i e n t s w i t h Xeroderma pigmentosum , (XP-E, XP-C and XP-K) and p a r e n t s of t h e XP p a t i e n t s . S t o c k c u l t u r e s were grown i n m o n olayers i n 10 cm. d i a m e t e r . p e t r i p l a t e s ( F a l c o n p l a s t i c ) , and m a i n t a i n e d i n E a g l e ' s Minimum E s s e n -t i a l Medium (MEM) supplemented w i t h \0% f e t a l c a l f serum and t h e f o l l o w i n g a n t i b i o t i c s : p e n i c i l l i n (204 u n i t s / m l . ) , s t r e p t o m y c i n s u l f a t e (29.6 mg./ml.), f u n g i z o n e i \ % ) and kanamycin (\%). The pH of t h e c e l l c u l t u r e s was m a i n t a i n e d a t 7.4 by t h e a d d i t i o n of 1.8% sodium b i c a r b o n a t e (16 ml./800 ml. MEM) t o t h e c u l t u r e medium, and t h e c u l t u r e s were kept i n a h u m i d i f i e d i n c u b a t o r (37°C) s t a f f e d w i t h 5% C 0 2 and 95% a i r . For e x p e r i m e n t s , t h e c e l l s were seeded o n t o g l a s s c o v e r s l i p s (20x20 mm.) kept i n s m a l l 35 mm. d i a m e t e r p e t r i p l a t e s a t a p p r o -4 x i m a t e l y 5x10 c e l l s p e r p l a t e . T r e a t m e n t s were s t a r t e d b e f o r e c o n t a c t i n h i b i t i o n t o a l l o w b e t t e r c e l l e x p o s u r e t o v i r u s and c a r -c i n o g e n s . . 2. ChemicaIs 4 - n i t r o q u i n o l i n e - N - o x i d e (4NQ0) was o b t a i n e d from D a i i c h i Pure C h e m i c a l s Co. L t d . , Tokyo, Japan. 3 - m e t h y I — 4 - n i t r o q u i n o I i n e -N-oxide (3Me4NQ0) and 6 - n i t r o q u i n o l i n e - N - o x i d e (6NQ0) were s u p p l i e d by Dr. Y. Kawazoe, N a t i o n a l C a n cer C e n t e r R e s e a r c h I n s t i t u t e , Tokyo, Japan. N - m e t h y I - N ' - n i t r o - N - n i t r o s o - g u a n i d i n e (MNNG) was p u r c h a s e d from A l d r i c h Chemical Co. I n c . , M i l w a u k e e , Wis., U.S.A., and a f l a t o x i n Bl and s t e r i g m a t o c y s t i n were p u r c h a s e d from Makor Chemi- • c a l s , J e r u s a l e m , I s r a e l . F o r t h e a c t i v a t i o n of a f l a t o x i n Bl and s t e r i g m a t o c y s t i n , t h e c o f a c t o r s n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e phosphate (NADP) and g I u c o s e - 6 - p h o s p h a t e (G-6-P) were o b t a i n e d from Sigma Chemical Company, S t . L o u i s , Mo. Magnesium c h l o r i d e (MgCI 2-61^0) was o b t a i n e d from F i s h e r C h e m i c a l , Vancouver, B.C. 3. P r e p a r a t i o n o f A d e n o v i r u s 12 A d e n o v i r u s 12 was p r e p a r e d by t h e i n f e c t i o n o f KB c e l l s (Mak, p e r s o n a l c o m m u n i c a t i o n ) . S t o c k KB c e l l s were grown i n monolayers i n 250ml. t i s s u e c u l t u r e f l a s k s ( F a l c o n p l a s t i c ) , and m a i n t a i n e d a t 37°C 8 i n MEM supplemented w i t h \0% f e t a l c a l f serum. A p p r o x i m a t e l y 5x10 c e l l s were c o n c e n t r a t e d by c e n t r i f u g a t i o n t o Ix10^ c e l l s / m l . i n MEM (no serum). The removal of serum i n t h e medium i s i m p o r t a n t because t h e serum b i n d s w i t h t h e v i r u s p a r t i c l e s r e n d e r i n g them n o n i n f e c t i v e . Ad 12 was added t o t h e c o n c e n t r a t e d c e l l s u s p e n s i o n a t an i n p u t mul-t i p l i c i t y o f 10^ p a r t i c l e s / c e l l . D u r i n g t h e 3 hour a b s o r p t i o n p e r i o d , t h e KB c e l l s were kept i n s u s p e n s i o n by c o n s t a n t s t i r r i n g . A t t h e end of t h e a b s o r p t i o n p e r i o d , t h e i n f e c t e d c e l l s were d i l u t e d w i t h ME/^ I ( w i t h \0% f e t a l c a l f serum) and i n c u b a t e d i n s u s p e n s i o n c u l t u r e s a t 37°C f o r 3 days. To h a r v e s t t h e v i r u s , t h e KB c e l l s were resuspended i n T r i s b u f f e r (pH 8.1) and s o n i c a t e d w i t h a B i o s o n i c s o n i c a t o r a t 30$ o u t -put f o r 2 min. In o r d e r t o f r e e t h e v i r u s p a r t i c l e s from c e l l mem-b r a n e s , an equal volume of Freon 113 ( o b t a i n e d from Matheson of Canada L t d . W h i t b y , O n t a r i o ) was added t o t h e s o n i c a t e d c e l l s u s -p e n s i o n , which was t h e n homogenized w i t h a S e r v a l I- Omni-Mixer f o r I min. and c e n t r i f u g e d a t 2000 rpm. f o r 2 min. The s u p e r -n a t a n t c o n t a i n i n g t h e v i r u s was c o l l e c t e d , and r e d u c t i o n i n volume was a c h i e v e d by c e n t r i f u g a t i o n i n CsCI (p=I.43) f o r 90 min. a t 20,000 rpm. To f u r t h e r p u r i f y t h e v i r u s and s e p a r a t e i t from t h e A d e n o - a s s o c i a t e d v i r u s (AAV), t h e v i r u s f r a c t i o n was banded i n CsCI (p=I.34) by c e n t r i f u g i n g f o r 20 hours a t 33,000 rpm. a t 5°C. The Ad 12 o b t a i n e d was t h e n d i l u t e d w i t h T r i s b u f f e r ( w i t h 20% g l y c e r i n ) and s t o r e d a t -70°C. 4. UV-i r r a d i a t i o n of Ad 12 A p p r o x i m a t e l y I ml. of t h e s t o c k Ad 12 was t r a n s f e r r e d t o a 60 mm. d i a m e t e r p e t r i p l a t e ( F a l c o n p l a s t i c ) w i t h t h e t o p r e -moved, and UV was a d m i n i s t e r e d by i r r a d i a t i o n w i t h a S y l v a n i a g e r m i c i d a l lamp ( G ^ T g ) a t a d i s t a n c e of 7 i n c h e s . T h i s produced -2 a d o s e - r a t e of 30 ergs/mm / s e c . as measured by a U V - l i g h t meter ( S c i e n t i f i c A p p a r a t u s , Thomas Co., P h i l a d e l p h i a ) . . 5. Treatment of C e l l s w i t h Ad 12 F o r e x p e r i m e n t s , f i b r o b l a s t s seeded o n t o g l a s s c o v e r s l i p s (see s e c t i o n I) were washed w i t h MEM (no serum) b e f o r e i n f e c t i n g w i t h Ad I2>. UV-i r r a d i a t e d o r noni r r a d i a t e d Ad 12 was added a t an i nput mu1 1i pI i c i t y of I 0^ v i raI p a r t i c I e s / c e I I, and an a b s o r p t ion p e r i o d of 3 hours was a l l o w e d b e f o r e MEM w i t h 10$ f e t a l c a l f , serum was added t o t h e c u l t u r e s . D u r i n g t h e a b s o r p t i o n p e r i o d , t h e p e t r i 10. p l a t e s were r o c k e d e v e r y 15 min. t o e n s u r e an even d i s t r i b u t i o n o f t h e v i r u s a c r o s s t h e c o v e r s l i p . 6. Treatment of C e l l s w i t h Chemical C a r c i n o g e n s A l l c h e m i c a l s were d i s s o l v e d i n MEM w i t h 5% f e t a l c a l f serum i m m e d i a t e l y b e f o r e t r e a t m e n t . I ml. o f c h e m i c a l s o l u t i o n was added t o each p e t r i p l a t e and t h e c e l l s were i n c u b a t e d i n t h e c h e m i c a l s o l u t i o n a t 37°C. At t h e end of t h e t r e a t m e n t , t h e c e l l s were washed t w i c e w i t h MEM (no serum) b e f o r e norma I c u l t u r e medium (MEM w i t h 10$. f e t a l c a l f serum) was r e p l a c e d . 7. In V i t r o A c t i v a t i o n o f P r e c a r c i n o g e n s (a) P r e p a r a t i o n of P o s t - m i t o c h o n d r i a I F r a c t i o n (9S) of Mouse L i v e r Homogenate Young a d u l t s w i s s mice (males w i t h a v e r a g e body w e i g h t 20 gm.) o b t a i n e d from t h e Animal U n i t , F a c u l t y of M e d i c i n e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , were k i l l e d by c e r v i c a l d i s l o c a t i o n and were i m m e d i a t e l y d i s s e c t e d . A p p r o x i m a t e l y 6 gm. of l i v e r s were removed and p l a c e d i n 10 ml. of P B S / s u c r o s e b u f f e r . . The o r g a n s were f i r s t minced w i t h a p a i r o f s c i s s o r s b e f o r e t r a n s f e r r i n g t o h o m o g e n i z a t i o n v e s s e l s . They were homogenized by a P o t t e r - E l v e j h e m Homogenizer o p e r a t i n g a t 1000 r e v . / m i n . and t h e homogenate was c e n t r i f u g e d a t 9000 xg f o r 10 min. a t 4°C. The p o s t - m i t o c h o n d r i a l s u p e r n a t a n t (9S) f r a c t i o n o b t a i n e d was t h e n f r o z e n i m m e d i a t e l y i n l i q u i d n i t r o g e n 11. and s t o r e d a t -70°C ( L a i s h e s , 1974; S t i c h and L a i s h e s , i n p r e p a r a t i o n ) . (b) Treatment of C e l l s w i t h A c t i v a t i o n M i x t u r e The' a c t i v a t i o n m i x t u r e was p r e p a r e d by d i s s o l v i n g NADP (4.0 Vi m o l e s ) , M g C I 2 (25 y moles) and G-6-P (20 y moles) i n 0.4 ml. of t h e 9S f r a c t i o n (thawed i m m e d i a t e l y b e f o r e u s e ) . A 1:1 m i x t u r e o f t h e a c t i v a t i o n m i x t u r e and a s o l u t i o n of t h e p r e c a r c i n o g e n was added t o t h e e e l I c u l t u r e s and t h e e e l Is were i n c u b a t e d a t 37°C ( L a i s h e s , 1974; S t i c h and L a i s h e s , i n p r e p a r a t i o n ) . A t t h e end o f t h e t r e a t m e n t , t h e c e l l s were washed t h r e e t i m e s w i t h MEM b e f o r e normal c u l t u r e medium was r e p l a c e d . 8. F i x a t i o n and S t a i n i n g To sample t h e c e l l s , t h e c o v e r s l i p s were t r a n s f e r r e d t o p e t r i d i s h e s c o n t a i n i n g a h y p o t o n i c s o l u t i o n o f \% sodium c i t r a t e f o r 3 min. The c i t r a t e s o l u t i o n a l l o w s t h e c e l l s t o s w e l l . The c e l l s , were th e n f i x e d w i t h Carnoy's (I a c e t i c a c i d : 3 e t h a n o l ) and a i r d r i e d . F o l l o w i n g f i x a t i o n , t h e c e l l s were s t a i n e d w i t h 2% a c e t o - o r c e i n f o r 10 min. and d e h y d r a t e d t h r o u g h e t h a n o l , b u t a n o l , b u t a n o l - x y l o l , x y l o l (30 s e c . i n e a c h ) . The c o v e r s l i p s were t h e n mounted w i t h Permount on gl.ass s l i d e s w i t h t h e c e l l s f a c i n g down. 12. RESULTS I . The Input M u l t i p l i c i t y o f Ad 12 Ad 12 p r o d u c t i v e l y i n f e c t s c u l t u r e s o f human f i b r o b l a s t s . V i r a l DNA s y n t h e s i s b e g i n s a t 9 o r 10 hours a f t e r i n f e c t i o n and c e l l l y s i s o c c u r s i n a p p r o x i m a t e l y 4 days (Green e_t a_l_., 1970). C e l l s w i t h rep I i c a t i ng- vi. r us may be i d e n t i f i e d by t h e p r e s e n c e of i n t r a n u c l e a r i n c l u s i o n b o d i e s ( I B ) ( F i g u r e 2A) which a r e t h e r e p l i c a -t i o n s i t e o f A d e n o v i r u s (Zur Hausen, 1967; S t i c h e t a I . , 1967, 1968; M a r t i nez-Pa I omo e_t a j _ . , 1967). By c o u n t i n g t h e f r a c t i o n o f c e l l s w i t h IB, i t i s p o s s i b l e t o d e t e r m i n e t h e number of c e l l s t h a t s u p p o r t a. s u c c e s s f u l v i r a l r e p l i c a t i o n ( S t i c h e t a I . , 1974b). B e f o r e a t t e m p t i n g any e x p e r i m e n t s , i t was n e c e s s a r y t o d e t e r -mine two f a c t o r s which were v e r y i m p o r t a n t i n a s c e r t a i n i n g a h i g h p e r c e n t a g e of c e l l s w i t h IB. These a r e (a) t h e t i m e of s a m p l i n g and (b) t h e mu 11 i.p I i c i t y of i n f e c t i o n (M.O. I . ), F i g u r e 3 shows t h e e f f e c t s o f s a m p l i n g t i m e on IB f o r m a t i o n . The c e l l s were i n f e c t e d w i t h a M.O.I, o f 10^ v i r a l p a r t i c l e s / c e l l , and were sampled a t 30 h r s . , 48 h r s . , 72 h r s . and 96 h r s . p o s t - i n f e c t i o n . An i n c r e a s e of IB w i t h t i m e was o b s e r v e d i n t h e f i r s t 72 h o u r s , and' t h e h i g h e s t f r a c t i o n o f c e l l s w i t h IB was o b s e r v e d a t 72 h o u r s . There was a d e c r e a s e , of IB a t 96 h o u r s , presumably due t o c e l l l y s i s ' . F i g u r e 4 shows t h e e f f e c t s o f M.O.I, on IB f o r m a t i o n . D i f f e r -e n t d i l u t i o n s of t h e s t o c k Ad 12 were used t o ' i n f e c t normal and XP f i b r o b l a s t s . The c e l l s were sampled a t 72 hours a f t e r i n f e c t i o n . 13. F i g u r e 2: (A) N u c l e i c o n t a i n i n g i n t r a n u c l e a r i n c l u s i o n b o d i e s (IB) sampled a t 72 hours a f t e r Ad 12 i n f e c t i o n and s t a i n e d w i t h 2% a c e t o -o r c e i n . (B) I n h i b i t i o n o f IB f o r m a t i o n i n XP c e l l s by 4NQ0 t r e a t -ment f o l l o w i n g Ad 12 i n f e c t i o n . i » 15:. F i g u r e 3: E f f e c t o f s a m p l i n g t i m e on t h e f r e q u e n c y of c e l l s w i t h IB i n norma I c u I t u r e s . F i g u r e 4: E f f e c t of m u I t i p I i c i t y of i n f e c t i o n (M.0.I.) on t h e f r e q u e n c y of ce I I s w i t h I B i n normal (O) and XP (•) c u l t u r e s . 16. IOO-i 80-H ' 1 96 H O U R S P O S T - I N F E C T I O N F i g u r e 3 IOO-i so H 1 I ~ ~ I I 1 I 1 .. 1 2 3 < 5 6 7 8 9 10 « 10 1 / D I L U T I O N Of- A O 1 2 S T O C K F i g u r e 4 17. No d i f f e r e n c e between normal and XP c e l l s was o b s e r v e d and i t was found t h a t w i t h a 100 f o l d d i l u t i o n , which was e q u i v a I e n t t o a p p r o x i m a t e l y 10^ v i r a l p a r t i c l e s / c e l l , IB was formed i n o v e r 60% o f t h e c e l l s . Thus i t was d e c i d e d t o use a M.O.I, of IO 3 v i r a l p a r t i c l e s / c e l l and a l l samples were t a k e n a t 72 hours t o e n s u r e a maximum y i e l d o f IB. 2. R e a c t i v a t i o n o f U V - i m p a i r e d Ad 12 i n Normal and XP C e l | s C u l t u r e s of t h r e e XP c e l l s (XP-E, XP-C and XP-K) and c o n t r o l c e l l s were i n f e c t e d w i t h Ad 12 p r e v i o u s l y i r r a d i a t e d w i t h UV l i g h t . Samples were t a k e n a t 72 hours p o s t - i n f e c t i o n and t h e f r a c t i o n of c e l l s w i t h IB was e s t i m a t e d . The e x t e n t o f r e c o v e r y o f t h e r e p l i c a t i v e c a p a c i t y o f UV-i m p a i r e d Ad 12 depends on t h e degree o f r e p a i r d e f i c i e n c y o f t h e h o s t c e l l . The l e v e l o f DNA r e p a i r s y n t h e s i s , which was e s t i m a t e d by m e a s u r i n g t h e un s c h e d u l e d uptake o f ^HTdR ( S t i c h and San, 1970), was about 21$ f o r XP-E, 32% f o r XP-C and 56% f o r XP-K as compared t o t h a t found i n c o n t r o l c e l l s f o l l o w i n g U V - i r r a d i a t i o n . In t h e p r e s e n t s t u d y , t h e r e c o v e r y o f IB f o r m a t i o n of U V - i r r a d i a t e d Ad J2 Was a l s o i n t h e o r d e r XP-E < XP-C < XP-K. F o r example, UV-doses o f more tha n "f-20> e r g s mm. d e s t r o y e d t h e c a p a c i t y o f t h e Ad 12 t o r e c o v e r i t s r e p l i c a t i v e p o t e n t i a l i n XP-E c e l l s , whereas a s l i g h t r e c o v e r y o c c u r r e d i n t h e l e s s r e p a i r d e f i c i e n t XP-C and XP-K c e l l s and a r e s t o r a t i o n o f about 10% t o o k p l a c e i n c o n t r o l c e l l s ( F i g u r e 5 ) . The r e a c t i v a t i o n o f U V - i m p a i r e d Ad 12 i n t h e c e l l s o f t h e h e t e r o z y g o u s XP p a r e n t s was a l s o d e t e r m i n e d . F i b r o b l a s t s o f XP-E and XP-C p a r e n t s were exposed t o U V - i r r a d i a t e d Ad 12. These c e l l s showed a normal l e v e l o f " W d R i n c o r p o r a t i o n f o l l o w i n g U V - i r r a d i a -t i o n ( S t i c h e t a I . , 1974a), and t h e e x t e n t o f r e c o v e r y of i r r a d i a t e d Ad 12 was a I so s i m i l a r t o t h a t o f c o n t r o I ceI Is ( F i g u r e s 6,7).. 3. The E f f e c t s o f 4NQ0 on Ad 12 R e p l i c a t i o n An a t t e m p t was made t o s t u d y t h e e f f e c t s o f c h e m i c a l c a r c i n o -gens on Ad 12 r e p l i c a t i o n . Two problems a r e of i n t e r e s t h e r e : (a) do t h e c h e m i c a l c a r c i n o g e n s i n t e r a c t w i t h t h e v i r a l DNA as i n t h e case o f U V - i r r a d i a t e d v i r u s , and (b) i f t h e y do, w i l l v i r a l r e p l i -c a t i o n be r e a c t i v a t e d i n t h e h o s t c e l l ? If v i r a l DNA i s damaged, two o f t h e c i r c u m s t a n c e s t h a t may happen a r e : (a) t h e damage i s r e p a i r e d by t h e h o s t r e p a i r enzymes and v i r a l r e p l i c a t i o n c o n t i n u e s , and (b) t h e damage i s u n r e p a i r e d o r o n l y p a r t i a l l y r e p a i r e d and v i r a l r e p l i c a t i o n c e a s e s . If v i r a l r e p l i c a t i o n c o n t i n u e s , IB w i l l be formed; but i f v i r a l r e p l i c a t i o n c e a s e s , IB w i l l ' n o t be formed. ! The f i r s t c a r c i n o g e n i n v e s t i g a t e d was 4-n i t r o q u i no I i,ne-Nj-ox i de (4NQ0). | t was r e p o r t e d t h a t XP c e l l s c a n n o t r e p a i r t h e DNA damage induced by 4NQ0 ( S t i c h et_ a j _ . , 1973), and i n t h e p r e s e n t s t u d y , i t . 19. UV DOSE TO.AD 12 (ERG MM - 2) F i g u r e 5: E f f e c t of UV i r r a d i a t i o n on t h e c a p a c i t y o f Ad 12 t o form i n t r a n u c l e a r i n c l u s i o n b o d i e s ( I B ) i n c o n t r o l c e l l s (O) and t h r e e XP c e l l c u l t u r e s : XP-E ( • ) , XP-C ( A ) and XP-K ( B ) . 20 F i g u r e 6: E f f e c t o f UV i r r a d i a t i o n on t h e c a p a c i t y of Ad 12 t o form IB in t h e h e t e r o z y g o u s p a r e n t s of XP-E. O c o n t r o l c e l l s ; , 6 XP-E c e l l s ; A XP-E f a t h e r ; • XP-E mother. F i g u r e 7: E f f e c t o f UV i r r a d i a t i o n on t h e c a p a c i t y of Ad 12 t o form IB i n t h e h e t e r o z y g o u s p a r e n t s of XP-C. O c o n t r o l c e l l s ; • XP-C c e l l s ; A XP-C f a t h e r ; • XP-C mother. F i q u re 6 F i g u re 7 was hoped t h a t XP c e l l s c a n n o t r e a c t i v a t e 4NQ0-damaged Ad 12 as i n t h e c a s e of U V - i r r a d i a t e d v i r u s . The d e s i g n of t h e e x p e r i m e n t i s shown i n F i g u r e 8. 4NQ0 was a p p l i e d t o t h e c e l l a f t e r Ad 12 i n f e c t i o n . S i n c e t h e v i r u s i s d e p r i v e d of i t s p r o t e c t i v e p r o t e i n c o a t once i t i s i n s i d e t h e h o s t c e l l , i t was hoped t h a t by e x p o s i n g t h e c e l l t o 4NQ0, t h e c h e m i c a l might i n t e r a c t w i t h t h e "naked" v i r a l DNA. The e x p e r i m e n t was b a s i c a l l y d i v i d e d i n t o 3 p o r t i o n s : (a) Some c e l l s were exposed t o 4NQ0 a l m o s t i m m e d i a t e l y a f t e r Ad 12 i n f e c t i o n ( F i g u r e 8 A ) . S i n c e v i r a l DNA s y n t h e s i s b e g i n s o n l y a t 9 t o 10 hours p o s t - i n f e c t i o n , t h e c e l l s were o n l y a t t h e i r l a t e n t p e r i o d when t h e y were exposed t o 4NQ0. (b) Some c e l l s were exposed t o 4NQ0 a t 10 hours a f t e r i n f e c t i o n ( F i g u r e 8 B ) . In t h i s c a s e , v i r a l DNA s y n t h e s i s had j u s t s t a r t e d when t h e c e l l s were exposed t o t h e c h e m i c a l . ( c ) F i n a l l y , some c e l l s were exposed a t 14 hours a f t e r i n f e c t i o n ( F i g u r e 8 C ) . Here, v i r a l DNA s y n t h e s i s was a I most compIete when t h e c e l l s were exposed t o 4NQ0. When c u l t u r e s of normal o r XP e e l Is were exposed t o 4NQ0 im m e d i a t e l y a f t e r Ad 12 i n f e c t i o n , IB f o r m a t i o n was a b o l i s h e d ( F i g u r e 2 B ) ; and i t was a b o l i s h e d f a s t e r i n XP c e l l s t h a n i n normal c e l l s ( F i g u r e 9 ) . A complete i n h i b i t i o n o f IB f o r m a t i o n was o b s e r v e d a t a 4NQ0 c o n c e n t r a t i o n o f 2x10 6M w i t h XP c e l l s , w h i l e a c o m p l e t e i n h i b i t i o n was o b s e r v e d o n l y a t 4x10 w i t h normal c e l l s . 23. F i g u r e 8: E x p e r i m e n t a l d e s i g n . C o n t r o l and XP c u l t u r e s were t r e a t e d w i t h 4N00' >at (A) 2 h o u r s , (B) 10 h o u r s , o r (C) 14 hours f o l l o w i n g Ad 12 i n f e c t i o n . F i g u r e 9: E f f e c t of 4N00 t r e a t m e n t on t h e c a p a c i t y of Ad 12 t o form IB i n normal c u l t u r e s ( O ) , XP-E c u l t u r e s . ( • ) , XP-E f a t h e r c u l t u r e s ( A ) and XP-E mother c u l t u r e s ( • ) . 25. Exposure o f c e l l s a t 10 hours o r 14 hours a f t e r i n f e c t i o n a l s o showed an i n h i b i t i o n o f IB f o r m a t i o n ; I n h i b i t i o n i n XP c e l l s was a l s o f a s t e r t h a n i n normal c e l l s . However t h e degree o f i n h i b i t i o n was f a r l e s s as compared t o t h e c a s e where c e l l s were exposed t o 4NQ0 i m m e d i a t e l y a f t e r Ad 12 i n f e c t i o n ( F i g u r e 1 0 ) . I t i s i n t e r e s t i n g t o note t h a t i f 4NQ0 was g i v e n a t 14 h o u r s , LB f o r m a t i o n was a l m o s t n o r m a l . The i n h i b i t i o n o f IB f o r m a t i o n by 4NQ0 was a l s o examined i n c e l l s of t h e h e t e r o z y g o u s XP p a r e n t s . F i b r o b l a s t s of XP-E p a r e n t s were exposed t o 4NQ0 i m m e d i a t e l y f o l l o w i n g Ad 12 i n f e c t i o n and t h e degree o f i n h i b i t i o n o f IB f o r m a t i o n c l o s e l y r e s embled t h a t i n c o n t r o l s ( F i g u r e 9 ) . The e f f e c t s o f d e r i v a t i v e s o f 4NQ0 on Ad 12 r e p l i c a t i o n were a l s o i n v e s t i g a t e d . Normal and XP c e l l s were exposed t o 3- m e t h y l -4-n i t r o q u i ho I i ne-N-oxi de (3Me4NQ0) and 6-n i troq.i.noI i ne-N-oxi de (6NQ0) i m m e d i a t e l y a f t e r Ad 12 i n f e c t i o n . The e f f e c t s o f 3Me4NQ0 resembled t h a t o f 4NQ0. An i n h i b i t i o n o f IB was o b s e r v e d and IB f o r m a t i o n was a b o l i s h e d f a s t e r i n XP c e l l s t h a n i n normal c e l l s . However, t h e i n h i b i t i o n by 3Me4NQ0, which i s a weaker c a r c i n o g e n t h a n 4NQ0, o c c u r r e d o n l y a t a c o n s i d e r a b l y h i g h e r c o n c e n t r a t i o n t h a n t h a t of 4NQ0. On t h e o t h e r hand, 6NQ0, which i s a n o n c a r c i n o g e n , showed no e f f e c t s on IB f o r m a t i o n even a t a h i g h c o n c e n t r a t i o n of 1x10 ( F i g u r e I I ) . 4. The E f f e c t s o f MNNG on Ad 12 R e p l i c a t i o n The second major c a r c i n o g e n o f i n t e r e s t was N - m e t h y I - N ' n i t r o -26. F i g u r e 10: E f f e c t of 4N00 t r e a t m e n t g i v e n a t v a r i o u s i n t e r v a l s a f t e r Ad 12 i n f e c t i o n on IB f o r m a t i o n i n c o n t r o l ( O ) and XP-E (•) c u I t u r e s . . C e l I s were t r e a t e d w i t h 4NQ0 a t (A) 2 h o u r s , (B) 10 hours o r (C) 14 hours p o s t - i n f e c t i o n . 27. j 4 U LU I u • z LL • 0 s CD. I I-LO _l _J LU u • Z • I— u CL LL too-, 9 0 i 8 0 ' 70 6 0 -5 0 -4 0 -3 0 -2 0 -10-J 1 0 0 9 0 8 0 -7 0 -6 0 -5 0 -4 0 -3 0 -2 0 -10 100-1 9 0 -8 0 -7 0 -6 0 -5 0 -4 0 -3 0 -2 0 -0»/^=.0»r=r.o» : : 0 « . — C A ) (B) ( C : 10-1 // , r — , , 1 r / / 1 A Q i n '"IO 2 4 6 8 10 M O c h e m i c a l 4 N Q Q ( M ) 28. d N Q O 100 io--/f ,o o I h <; in U Z 0 u < (I Li. 60 .40 20 H 3 M r : 4 M Q O B M Q O N O R M A L C E L L S C '. • 1( » f Tt|( 10"' I O - 6 100 80 -i 60 _i L U u 0 z O H U <; a 40H 20 • -// <3 N a o • • IO"5 3 M e a N Q O 111 -> IO"4 10" C O N C E N T R A T I O N 1 IVI ) B N Q O X R-E C E L L S •-» \ " n . 10"' 10" 10' IO"4 10" C O N C E N T R A H a N l W F i g u r e i 1: E f f e c t o f d e r i v a t i v e s of 4NQO on t h e c a p a c i t y of Ad 12 t o form IB f n normal and XP-E c u l t u r e s . 29. N - n i t r o s o - g u a n i d i n e (MNNG). I t was r e p o r t e d t h a t MNNG induced normal DNA r e p a i r i n XP c e l l s ( S t i c h e_f aj_. , 1973), and i t was hoped t h a t t h e r e a c t i v a t i o n o f MNNG-damaged Ad 12 i n XP c e l l s would a l s o be normal. H a v i n g e s t a b l i s h e d t h e f a c t t h a t t h e most s e n s i t i v e p e r i o d t o a c a r c i n o g e n d u r i n g v i r a l r e p l i c a t i o n was b e f o r e v i r a l DNA s y n t h e s i s , i t was de t e r m i n e d t o expose t h e eel Is t o MNNG imme-d i a t e l y a f t e r i n f e c t i o n . The d e s i g n o f t h e e x p e r i m e n t i s shown i n F i g u r e 12 and t h e r e s u l t s a r e summarized i n F i g u r e 13. An i n h i b i t i o n o f IB f o r m a t i o n was a l s o o b s e r v e d w i t h MNNG as i n t h e ca s e o f 4NQ0, but IB f o r m a t i o n was a b o l i s h e d a t t h e same r a t e i n normal and. XP e e l I s . A co m p l e t e i n h i b i t i o n o f IB was o b s e r v e d a t -4 a c o n c e n t r a t i o n o f 2x10 M i n both c e l l t y p e s . •5. The E f f e c t s o f A c t i v a t e d P r e c a r c i n o g e n s on Ad 12 R e p l i c a t i o n In v i v o DNA damage and r e p a i r by c h e m i c a l p r e c a r c i n o g e n s may be s i m u l a t e d i n v i t r o by m i x i n g p r e c a r c i n o g e n s such as a f l a t o x i n Bl and s t e r i g m a t o c y s t i n w i t h a mouse l i v e r p o s t - m i t o c h o n d r i a I (9S) s u p e r n a t a n t f r a c t i o n ( L a i s h e s , 1974; S t i c h and L a i s h e s , i n p r e -p a r a t i o n ) . I t was assumed t h a t t h e l i v e r c o n t a i n e d t h e enzymes r e -q u i r e d t o " a c t i v a t e " t h e p r e c a r c i n o g e n s , and by e x p o s i n g c e l l s t o p r e c a r c i n o g e n s a c t i v a t e d i n v i t r o by t h e 9S l i v e r f r a c t i o n , an i n c r e a s e ' i n DNA damage and r e p a i r , chromosome a b e r r a t i o n s and c e l l l e t h a l i t y was r e p o r t e d . In t h e p r e s e n t study., an atte m p t was made t o examine t h e r e a c t i v a t i o n of Ad 12 damaged by " a c t i v a t e d " p r e c a r c i n o g e n s . Normal 50. F i g u r e 12: E x p e r i m e n t a l D e s i g n . C o n t r o l and XP c e l l c u l t u r e s were t r e a t e d a t 2 hours w i t h MNNG f o l l o w i n o ' A d 12 i n f e c t i o n . F i g u r e 13:. E f f e c t o f MNNG on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l (O) and XP-E c u l t u r e s ( # ) . F i g u r e 15 32. and XP c u l t u r e s were t r e a t e d w i t h an a c t i v a t i o n m i x t u r e c o n t a i n -i n g t h e p r e c a r c i n o g e n and t h e 9S l i v e r f r a c t i o n f o l l o w i n g Ad 12 i n f e c t i o n . The d e s i g n of t h e e x p e r i m e n t i s shown i n F i g u r e 14. Two s e t s of c o n t r o l s were run s i m u l t a n e o u s l y . One s e t was exposed o n l y t o t h e p r e c a r c i n o g e n and t h e second s e t o n l y t o the'9S f r a c t -i o n o f t h e I i v e r . Two p r e c a r c i n o g e n s , namely a f l a t o x i n B l and s t e r i g m a t o c y s t i n , were examined i n t h e p r e s e n t s t u d y , and t h e r e s u l t s a r e summarized i n F i g u r e s 15-18. When t h e i n f e c t e d c e l l s were exposed t o o n l y t h e p r e c a r c i n o g e n o r t o o n l y t h e l i v e r f r a c t i o n , l i t t l e o r no e f f e c t s on IB f o r m a t i o n were o b s e r v e d . The f r a c t i o n of c e l l s w i t h IB i n t h e s e c a s e s resembled t h a t i n c u l t u r e s unexposed t o any c h e m i c a l s . However i f t h e i n f e c t e d c e l l s were exposed t o both t h e p r e c a r c i n o -gen and t h e l i v e r f r a c t i o n , t h e f o r m a t i o n of IB was a b o l i s h e d . A d i f f e r e n c e between normal and XP c u l t u r e s as s i m i l a r t o t h e case of 4NQ0 was o b s e r v e d . 6. S t a t i s t i cs The s t a n d a r d d e v i a t i o n o f t h e mean (a) was c a l c u l a t e d i n one e x p e r i m e n t t o de m o n s t r a t e t h e e r r o r made by t e c h n i q u e s and c o u n t i n g . Ten p e t r i p l a t e s o f c e l l s (XP) were i n f e c t e d w i t h Ad 12 a t a m u l t i p l i c i t y of I 0 3 v i r a l p a r t i c l e s / c e l l (see S e c t i o n I ) . Samples were t a k e n a t 72 hours and t h e p e r c e n t a g e o f eel Is w i t h IB was c o u n t e d f o r each p l a t e . A p p r o x i m a t e l y 2000 c e l l s p e r p l a t e were c o u n t e d as i n a l l t h e o t h e r e x p e r i m e n t s . The sample mean (u) 33 F i g u r e 14: E x p e r i m e n t a l D e s i g n . C o n t r o l and XP c u l t u r e s were exposed t o p r e c a r c i n o g e n s a c t i v a t e d by a 9S mouse l i v e r f r a c t i o n a t 2 hours f o l l o w i n g Ad 12 i n f e c t i o n . Two s e t s o f c o n t r o l s were run s i m u l t a n e o u s l y one s e t was exposed t o o n l y t h e p r e c a r c i n o g e n ( a f l a t o x i n BT o r s t e r i g -m a t o c y s t i n) and t h e o t h e r s e t t o o n l y t h e 9S f r a c t i o n . F i g u r e 15 E f f e c t of n o n a c t i v a t e d a f l a t o x i n B l on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l (O) and XP-E (•) c u l t u r e s . F i g u r e ! 6 : E f f e c t o f a c t i v a t e d a f l a t o x i n Bl on t h e c a p a c i t y o f Ad 12 t o form IB i n c o n t r o l (O) and XP-E ( 6 ) c u l t u r e s . 34. Ad I 2 A f l a t o x i n BI+9S F r a c t i o n -3 0 2 3 Ad I 2 A f I a t o x i n Bl ( C o n t r o -3 0 2 3 Ad I 2 9S F r a c t i o n ( C o n t r o l ) -3 0 2 3 Ad I 2 S t e r i g m a t o c y s t i n + 9 S F r a c t i o n .3 0 2 4 Ad I 2 S t e r i g n a t o c y s t i n ( C o n t r o I ) -3 0 2 4 •Ad 12 9S F r a c t i o n ( C o n t r o l ) -3 0 2 4 -II--II--II--II--II--II-SampIi ng 72 hours Samp I i ng 72 hours Samp I i ng ' 72 hours Samp I i ng 72 hours Samp I i ng 72 hours Samp I i ng 72 hours F i g u r e 14 A F L A T O X I M B M N O N A C T I V A T E O H M ) A f L 4 T D X I N B 1 I « C T I V 4 T E D I I M I F i g u r e I 5 < F i g u r e 16 35. F i g u r e 17: E f f e c t of n o n a c t i v a t e d s t e r i g m a t o c y s t i n on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l (O) and XP-E (•) c u l t u r e s . .. F i g u r e 18: E f f e c t of a c t i v a t e d s t e r i g m a t o c y s t i n on t h e c a p a c i t y of Ad 12 t o form IB i n c o n t r o l (O) and XP-E ' ( • ) c u l t u r e s . 36. IOO-i 20H n o c h e m i c a l , - 5 S T E R I G M A T O C Y S T I N ( M O M A C T I V A T E D ) I M 1 F i g u re 17 J S T E R I G M A T Q C Y S T I N ( A C T I V A T E D ) ! M •) F i g u r e 18 37. was found t o be 90.08$ and t h e s t a n d a r d d e v i a t i o n o f t h e mean (a) was c a l c u l a t e d t o be 2.67. The s t a n d a r d e r r o r (S.E.) was 0.84 and t h e c o e f f i c i e n t o f v a r i a t i o n was 2.96$. 38. DISCUSSION I. R e a c t i v a t i o n of U V - i m p a i r e d Ad 12 i n Normal and XP C e l l s A s i m i l a r f r e q u e n c y of c e l l s w i t h IB i n normal and XP c u l t u r e s f o l l o w i n g t h e i n f e c t i o n w i t h n o n i r r a d i a t e d Ad 12 s u g g e s t s t h a t t h e c a p a c i t y of t h e v i r u s t o p e n e t r a t e and r e -p l i c a t e i s comparable i n both c e l l t y p e s . However IB f o r m a t i o n was reduced i n D N A - r e p a i r d e f i c i e n t XP c u l t u r e s f o l l o w i n g t h e i n f e c t i o n w i t h U V - i m p a i r e d Ad 12. The s i m p l e s t i n t e r p r e t a t i o n i s t o assume t h a t t h e XP c e l l s have a d e c r e a s e d c a p a c i t y t o r e p a i r t h e UV damage on t h e v i r a l genome, and t h e v i r u s f a i l s t o r e p l i c a t e and form IB i f t h e damage i s not r e p a i r e d . In s u p p o r t o f t h i s a r e t h e f o l l o w i n g o b s e r v a t i o n s : (a) The r e a c t i v a t i o n o f U V - i m p a i r e d Ad 12 appears t o depend on t h e D N A - r e p a i r c a p a c i t y of t h e c e l l c u l t u r e s . F o r example, t h e r e p a i r c a p a c i t y of t h e v a r i o u s XP c u l t u r e s employed i n t h i s s t u d y i s i n t h e o r d e r o f XP-K -> XP-C > XP-E. IB f o r m a t i o n f o l l o w i n g t h e i n f e c t i o n w i t h U V - i r r a d i a t e d Ad 12 was a l s o i n t h e o r d e r o f XP-K > XP-C > XP-E. • ' (b) In a d d i t i o n t o t h e XP c u l t u r e s , IB f o r m a t i o n i n t h e h e t e r o -zygous XP p a r e n t c e l l s which have normal DNA r e p a i r was s i m i l a r t o t h a t , i n c o n t r o l c u l t u r e s . The p r e s e n t r e s u l t s can be compared w i t h t h e reduced T-a n t i g e n f o r m a t i o n and c e l l t r a n s f o r m a t i o n i n XP c e l l s i n f e c t e d w i t h U V - i m p a i r e d SV 40 (Aaronson and L y t l e , ,1970) and t h e d i m i n i s h e d v i r u s p r o d u c t i o n f o l l o w i n g i n f e c t i o n w i t h U V - i r r a d i a t e d herpes 39. s i m p l e x v i r u s ( L y t l e erf aj_. , 1972). T h e r e f o r e U V - i m p a i r e d v i r u s e s such as Ad 12, SV 40 and herpes s i m p l e x v i r u s a r e r e a c t i v a t e d i n normal human c u l t u r e s r e s u l t i n g i n v i r a l r e p l i c a t i o n and c e l l l y s i s , but a r e u n r e p a i r e d i n D N A - r e p a i r d e f i c i e n t XP c u l t u r e s l e a d i n g t o a b o r t i v e i n f e c t i o n and no l y s i s . 2. Combined A c t i o n o f Chemical C a r c i n o g e n s and Ad 12 on Normal and  XP C e l I s A s t u d y has been c o n d u c t e d t o examine t h e i n t e r a c t i o n between v i r u s e s and c h e m i c a l c a r c i n o g e n s i n c u l t u r e d human e e l I s . . I t was . o b s e r v e d t h a t IB f o r m a t i o n was a b o l i s h e d i n normal and XP c u l t u r e s i f t h e c e l l s were t r e a t e d w i t h c h e m i c a l c a r c i n o g e n s f o l l o w i n g Ad 12 i n f e c t i o n ; and i t was a b o l i s h e d f a s t e r i n D N A - r e p a i r d e f i c i e n t XP c e l l s w i t h c e r t a i n c a r c i n o g e n s such as 4NQ0. The s i m p l e s t i n t e r -p r e t a t i o n i s t o assume t h a t t h e c h e m i c a l s i n t e r a c t w i t h t h e v i r a l genome and i f t h e damage i s u n r e p a i r e d , v i r a l r e p l i c a t i o n and IB f o r m a t i o n w i l l be i n h i b i t e d . In t h e c a s e o f 4NQ0, t h e c h e m i c a l presumably b i n d s t o t h e v i r a l - D N A and e l i c i t s e n d o n u c l e a s e a t t a c k which i s d e f i c i e n t i n t h e XP c e l l s . Thus t h e r e a c t i v a t i o n o f 4NQ0 damaged v i r a l . j f u n c t i o n i s more e f f i c i e n t i n normal c u l t u r e s t h a n i n XP c u l t u r e s . I n ' s u p p o r t o f t h i s i s t h e p r e v i o u s r e p o r t t h a t DNA r e p a i r as i n-d i c a t e d by u n s c h e d u l e d ~WdR uptake was o b s e r v e d i n normal but. not in XP c u l t u r e s f o l l o w i n g 4NQ0 t r e a t m e n t ( S t i c h e t aj_. , 1972b, 1974a), 40. On t h e o t h e r hand w i t h t h e c a r c i n o g e n MNNG, no d i f f e r e n c e between normal and XP c u l t u r e s was n o t i c e a b I e . PresumabIy' MNNG in d u c e s l e s i o n s i n t h e DNA s t r a n d which can be r e p a i r e d d i r e c t l y by DNA poly m e r a s e w i t h o u t e m p l o y i n g any e n d o n u c l e a s e a c t i v i t y . Thus t h e r e a c t i v a t i o n of MNNG damaoed v i r a l f u n c t i o n i s comparable i n normal and XP c e l l s . In s u p p o r t o f t h i s view i s t h e r e p o r t t h a t DNA r e p a i r as i n -d i c a t e d by t h e i n c o r p o r a t i o n o f ^ HTdR was o b s e r v e d i n XP c u l t u r e s f o l l o w i n g MNNG t r e a t m e n t ( S t i c h e t a l _ . , I 972b, I 9 7 4 a ) . F i n a l l y w i t h t h e p r e c a r c i n o g e n s a f l a t o x i n Bl and s t e r i g m a t o c y s t i n , t h e n o n a c t i v a t e d p r e c a r c i n o g e n was u n a b l e t o i n t e r a c t w i t h t h e v i r a l genome and v i r a l r e p l i c a t i o n was not a f f e c t e d . However i f t h e p r e -c a r c i nogens were combined w i t h t h e 9S mouse l i v e r f r a c t i o n , enzymes p r e s e n t i n t h e l i v e r f r a c t i o n t r a n s f o r m e d t h e p r e c a r c i n o g e n s i n t o a c t i v e compounds c a p a b l e of i n t e r a c t i n g w i t h t h e v i r a l genome and i n t e r f e r i n g w i t h v i r a l r e p l i c a t i o n . The a s s u m p t i o n t h a t c h e m i c a l c a r c i n o g e n s i n t e r a c t e d w i t h t h e v i r a l DNA and t h a t DNA r e p a i r was n e c e s s a r y t o r e s t o r e v i r a l f u n c t i o n s i s s u p p o r t e d by t h e f o l l o w i n g o b s e r v a t i o n s : (a) The c a p a c i t y t o form IB seems t o depend on t h e r e p a i r c a p a c i t y of t h e c e l l s . F o r example, r e p a i r d e f i c i e n t XP c e l l ' s showed a r e d u c t i o n i n IB f o r m a t i o n w i t h c e r t a i n c h e m i c a l c a r c i n o g e n s such as 4NQ0 as compared t o c o n t r o l c e l l s and c e l l s of t h e h e t e r o z y g o u s XP p a r e n t s w hich have normal r e p a i r c a p a c i t y . • (b) N o n c a r c i n o g e n s such as 6N00 and n o n a c t i v a t e d p r e c a r c i n o g e n s w hich do not i n t e r a c t w i t h DNA had no e f f e c t s on IB f o r m a t i o n . 3. P o s s i b l e F a t e of C e l l s f o l l o w i n g an A b o r t i v e I n f e c t i o n Ad 12 i s c a p a b l e of i n f e c t i n g both human and r o d e n t c e l l s but t h e outcome i s c o m p l e t e l y d i f f e r e n t i n t h e two c a s e s . In human c e l l s ( i n c l u d i n g both normal and XP c e l l s ) , Ad 12 i n f e c t i o n i s p r o -d u c t i v e . V i r a l DNA i s t r a n s l a t e d and t r a n s c r i b e d and v i r a l r e p l i c a -t i o n r e s u l t s i n c e l l l y s i s . In r o d e n t c e l l s , Ad 12 i n f e c t i o n i s a b o r t i v e . N e i t h e r i n f e c t i o u s v i r u s nor v i r a l c o a t p r o t e i n s a r e s y n t h e s i z e d and no l y s i s o c c u r s . A few v i r a l genes a r e t r a n s l a t e d r e s u l t i n g i n t h e t r a n s f o r m a t i o n o f t h e r o d e n t c e l l s (Green e t a I . , 1970, 1971). The p r e s e n t r e s u l t s i n d i c a t e t h a t t h e p r o d u c t i v e i n f e c t i o n o f r e p a i r d e f i c i e n t XP c e l l s may be a l t e r e d by UV o r c e r t a i n c h e m i c a l c a r c i n o g e n s . When XP c u l t u r e s a r e i n f e c t e d w i t h UV-i m p a i r e d Ad 12 o r t r e a t e d s i m u l t a n e o u s l y w i t h a v i r u s and a c a r -c i n o g e n , v i r a l r e p l i c a t i o n and c e l l l y s i s a r e a b o l i s h e d . T h i s c l o s e l y r e s e m b l e s t h e a b o r t i v e i n f e c t i o n o f r o d e n t c e l l c u l t u r e s by Ad 12, and s u g g e s t s t h a t t r a n s f o r m a t i o n of r e p a i r d e f i c i e n t human c e l l s may be i n i t i a t e d by t h e combined t r e a t m e n t w i t h v i r u s and c a r c i n o g e n i c a g e n t s ( F i g u r e 19). T h i s i s c r u c i a l because t h e human p o p u l a t i o n i s o f t e n exposed s i m u l t a n e o u s l y t o v i r u s e s and p h y s i c a l and chemical' a g e n t s , w h i c h , a c c o r d i n g t o t h e p r e s e n t s t u d y , may i ' n i t i a t e ' n e o p l a s t i c t r a n s f o r m a t i o n i n r e p a i r d e f i c i e n t human g r o u p s . 42. F i g u r e 19:' Proposed model f o r t h e f a t e of Ad 12 i n normal and XP c e l l s t r e a t e d w i t h 4N00 o r MNNG: (A) R e p l i c a t i o n of Ad 12 i n u n t r e a t e d normal human c e l l s (B) T r a n s f o r m a t i o n of hamster c e l l s by Ad 12 (C) I n t e r a c t i o n o f 4NQ0 w i t h Ad 12 i n normal human c e l l s (D) I n t e r a c t i o n o f 4N00 w i t h Ad 12 i n X P c e l l s , (E) I n t e r a c t i o n o f MNNG w i t h Ad 12 i n XP c e l l s 43. i N F ^ r n i o N V I T I A L U N A I \ J I . i i i r v i A i . H U M A N I : L t u V I I 1 A L R i l l ' L I G A T I O N A m y INI--EC 1 I O N V I M A L D N A rsj u v i i I A I " f I C I ' L I C . A T I O N T I IA N f r I' I R (VIE C) i:u M O L Y S i l i r . I-I A I V I b . r i:-:11 i : i 7 1 . 1 A i n ; - 1 . I I S J F F C T K 1 N V I R A L O N A INJCH ItVIAL. H L I I V 1 A ^ J D C L L r i A i v i A i . . i .a t i H R A I I I I I.I V H ' I A I L Y ' , I : V I R A L D N A V I R A L I. I N A R L T M I C i A T II. I N A I J 1 S I C M I I r . T i o N V I R A L U N A O M Q O NO i" ir\iA i tr i-'Aii'i N L ) T H A N S f U H M f . L I V I R A L . C l : L I I'll I - ' L I f ' A T I O N _ N Q L Y S I S O A M A G F C J V I R A L D N A A D 1 2 M M F E C T I O N D N A M N N G A O 12 L Y S I S D A M A G E D REiPAIRfc 'O V I R A L V I R A L D N A V I R A L D N A R E P L I C A T I O N 44. 4. I d e n t i f i c a t i o n o f DNA R e p a i r D e f i c i e n t M u t a n t s i n t h e Human  P o p u I a t i o n The a p p l i c a t i o n o f u n s c h e d u l e d DNA r e p a i r s y n t h e s i s and s h i f t s i n s e d i m e n t a t i o n p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n i n a l k a l i n e s u c r o s e g r a d i e n t s has r e v e a l e d t h e r e p a i r d e f i c i e n c y of XP p a t i e n t s ( C l e a v e r , 1972). F u r t h e r i n v e s t i g a t i o n showed a Iack o f endonucI ease a c t i v i t y i n XP eel Is ( C l e a v e r , 1969). I t i s h i g h l y p r o b a b l e t h a t o t h e r r e p a i r d e f i c i e n t m u t a n t s , l a c k i n g e x o n u c l e a s e , I i g a s e o r po l y m e r a s e a c t i v i t y , may e x i s t i n t h e human . p o p u l a t i o n . As an example, i t was r e c e n t l y r e p o r t e d t h a t p a t i e n t s w i t h F a n c o n i ' s anaemia a r e a l s o d e f i c i e n t i n DNA r e p a i r p r o b a b l y due t o a l a c k o f e x o n u c l e a s e a c t i v i t y (Poon et_ aj_. , 1974). Methods such as u n s c h e d u l e d DNA r e p a i r and s e d i m e n t a t i o n i n s u c r o s e g r a d i e n t s a r e o n l y c a p a b l e o f r e v e a l i n g c e r t a i n d e f e c t s w i t h i n t h e DNA r e p a i r p r o c e s s . A h i g h l y s e n s i t i v e t e s t i s n e c e s s a r y f o r u n c o v e r i ng. v a r i ous DNA r e p a i r d e f i c i e n t mutants i n t h e human p o p u l a t i o n . The r e a c t i v a -t i o n of i m p a i r e d v i r u s may be used f o r such a pur p o s e . I t s e r v e s as a more s e n s i t i v e a s s a y f o r r e p a i r d e f i c i e n c y t h a n u n s c h e d u l e d DNA r e p a i r and DNA s e d i m e n t a t i o n s i n c e any d e f e c t w i t h i n t h e DNA r e - . p a i r p r o c e s s wi.I I r e s u l t . i n t h e i n a b i l i t y t o r e a c t i v a t e i m p a i r e d v i r u s e s . I t i s a l s o a f a s t e r and more ec o n o m i c a l method f o r d e t e c t i n g r e p a i r d e f i c i e n c i e s i n v a r i o u s human g r o u p s . However, t h e ma j o r d i s a d v a n t a g e s a r e t h a t (a) i t i s u n a b l e t o i d e n t i t y t h e n a t u r e o f t h e r e p a i r d e f i c i e n c y , and (b) i t i s u n a b l e t o d e t e c t h e t e r o z y g o t e s who have normal r e p a i r c a p a c i t y . T h e r e f o r e t h e r e a c t i v a t i o n o f i m p a i r e d v i r u s can o n l y be used as a f a s t but u n d e t a i l e d a s s a y f o r r e p a i r , d e f i c i e n t p a t i e n t s . < 5. I d e n t i f i c a t i o n of Chemical C a r c i n o g e n s and P r e c a r c i n o g e n s Today i t i s known t h a t man l i v e s i n a sea of c a r c i n o g e n s . The y e a r l y i n t r o d u c t i o n o f th o u s a n d s o f new c h e m i c a l s i n t o o u r e n v i r o n m e n t , of which a p o r t i o n a r e c a r c i n o g e n i c , has be-come a h a z a r d t o o u r h e a l t h . I t has become n e c e s s a r y t o d e v e l o p c a r c i n o g e n i c i t y t e s t s t o d e t e c t p o t e n t i a l c a r c i n o g e n s . In v i v o t e s t s such as s k i n p a i n t i n g and i n j e c t i o n o f e x p e r i m e n t a l a n i m a l s w i t h c h e m i c a l s a r e u s u a l l y l e n g t h y and e x p e n s i v e . In t h e p a s t 10 y e a r s , v a r i o u s i n v i t r o t e s t s have been d e s i g n e d f o r t e s t i n g c h e m i c a l s f o r t h e i r c a r c i n o g e n i c i t y as i n d i c a t e d by t h e i r a c t i o n on chromosome a b e r r a t i o n s , m u t a g e n e s i s , DNA r e p a i r s y n t h e s i s and i n v i t r o ceI I t r a n s f o r m a t i o n ( r e v i e w e d by Sto I t z e t a_l_. , I 974) The p r e s e n t r e a c t i v a t i o n method s e r v e s as a f a s t e r and more economical assay f o r c a r c i n o g e n i c a c t i v i t y t h a n o t h e r i n v i t r o t e s t s . By e x p o s i n g r e p a i r d e f i c i e n t XP c u l t u r e s t o c h e m i c a l s o r c h e m i c a l s combined w i t h an a c t i v a t i o n system f o l l o w i n g v i r a l i n -f e c t i o n , any damage t o t h e v i r a l DNA wiI I a b o l i s h v i r a l r e p l i c a -t i o n and IB f o r m a t i o n . The major c r i t i c i s m of t h e p r e s e n t method, however, as of o t h e r i n v i t r o methods, i s t h a t t h e c h e m i c a l con-c e n t r a t i o n employed i s o f t e n many f o l d h i g h e r t h a n t h a t found i n t h e e n v i r o n m e n t , Moreover r e s u l t s i n v i t r o may not be comparable t o r e s u l t s i n v i v o . However, f o r t h e mass s c r e e n i n g of th o u s a n d s of c h e m i c a l s , i n v i t r o t e c h n i q u e s seem t o be t h e o n l y s o l u t i o n . 46. 6. O u t l o o k The l i n k between DNA r e p a i r d e f i c i e n c y and c a r c i n o g e n e s i s i n p a t i e n t s w i t h Xeroderma pigmentosum has de m o n s t r a t e d t h e i m p o r t a n t r o l e o f DNA r e p a i r i n t h e maintenance of a n o r m a l l y f u n c t i o n i n g c e l l . However, t h e m o l e c u l a r mechanism o f DNA r e p a i r i s by no means s i m p l e . The p r e s e n t r e s u l t s have shown t h a t c e l l s of v a r i o u s XP p a t i e n t s (XP-E, XP-C, XP-K) have d i f f e r e n t c a p a c i t i e s t o r e a c t i v a t e U V - i m p a i r e d Ad 12. T h i s s u g g e s t s t h a t m u t a t i o n a t d i f f e r e n t l o c i may produce t h e r XP phenotype. In s u p p o r t o f t h i s v i e w i s t h e r e c e n t f i n d i n g o f f o u r XP c o m p l e m e n t a t i o n groups by c e l l h y b r i d i z a t i o n e x p e r i m e n t s (Robbins e_t a j _ . , 1974). Moreover c e r t a i n XP p a t i e n t s have a. normal l e v e l o f uns c h e d u l e d DNA r e p a i r s y n t h e s i s f o l l o w i n g UV t r e a t m e n t (Burk e t a I . , 1971; C l e a v e r , 1972). I t i s i n t e r e s t i n g t h a t t h e c e l l s o f t h e s e p a t i e n t s show a reduced c a p a c i t y t o r e a c t i v a t e U V - i m p a i r e d v i r u s (Day, 1974). O b v i o u s l y t h e s e XP p a t i e n t s a r e d e f i c i e n t i n c e r t a i n r e p a i r p r o c e s s e s o t h e r t h a n t h e e n d o n u c l e a s e s t e p . Thus i t i s i n c o r r e c t t o d e f i n e XP as a d i s e a s e which l a c k s e n d o n u c l e a s e a c t i v i t y . Perhaps DNA r e p a i r d e f i c i e n c y i s common among p a t i e n t s w i t h g e n e t i c d i s e a s e s . In s u p p o r t of t h i s i s t h e r e c e n t f i n d i n g t h a t p a t i e n t s w i t h F a n c o n i ' s anaemia appear t o l a c k e x o n u c l e a s e a c t i v i t y (Poon e_t_ a j _ . , 1974). The p r e s e n t r e a c t i v a t i o n method seems t o be a h i g h l y p r o m i s i n g t e c h n i q u e f o r de-t e c t i n g such r e p a i r d e f i c i e n t mutants i n t h e human p o p u l a t i o n . A d d i -t i o n a l r e s e a r c h s h o u l d be. d i r e c t e d a t u n c o v e r i n g t h e s e DNA r e p a i r d e f i c i e n t mutants s i n c e t h e y r e p r e s e n t a group which i s h i g h l y s u s -c e p t i b l e t o c a r c i n o g e n e s i s , and t h e y make t h e a s s i g n m e n t o f a " s a f e " l e v e l o f c a r c i n o g e n s i n o u r e n v i r o n m e n t a d i f f i c u l t i f not i m p o s s i b l e t a s k . 47. REFERENCES Aa r o n s o n , S.A. and L y t l e , C D . , N a t u r e 228, 359, 1970. Andrewes, C , " V i r u s e s and C a n c e r " , W e i d e n f e l d and N i c o l s o n , 1970. A r c o s , J.C. and A r g u s , M.F., Advan. Cancer Res. J J _ , 305, 1968. B a r d o s , T., D a t t a - G u p t a , N., Hebborn, R. and T r i g g l e , D.J., J . Med. Chem. 8_, 1.67, 1965. B o y l a n d , E., i n " P h y s i o - C h e m i c a l Mechanisms o f C a r c i n o g e n e s i s " ed. Bergman, E.D. and P u l l m a n , B., p. 25, I s r a e l Academy of S c i e n c e s and H u m a n i t i e s , J e r u s a l e m . B o y l a n d , E. and G r e e n , B., B r i t . J . C a n c e r ]_6, 507 , 1 9 6 2 . Burk, P.G., L u t z n e r , M. A.,- C l a r k e , D.D. and R o b b i n s , J.H., J . C l i n . Med. 77_, 759, I 971 . C a m p b e l l , A., Adv. G e n e t i c s J J _ , 101, 1962. C a s t o , B.C., C a n c e r Res. 3_3, 402, 1973. C a s t o , B.C., P i e c z y n s k i , W.J. and D i p a o l o , J.A., C a n c e r Res. 33, 819, 1973. C l e a v e r , J . E . , N a t u r e 218, 652, 1968. C l e a v e r , J . E . , P r o c . Nat. Acad. S c i . 63, 428, 1969. C l e a v e r , J . E . , Mut. Res. VZ, 453, 1971. C l e a v e r , J.E., John Hopkins Med. J . , S u p p l . J_, 195, 1972. '. . C l e a v e r , J . E., C a n c e r Res. 33, 362, 1973. 1 ; j C l e a v e r , J . E . , Advan. Rad. B i o l . 4_, I, 1974. C l e n d e n n i n g , W., "Xeroderma pigmentosum", i n Dermatology i n G e n e r a l M e d i c i n e " , ed. F i t z p a t r i c k , T.B. e t a_l_. , M c G r a w - H i l l Book I n c . , I 971, page 436. Day, R.S., Cancer Res.'3_4, 1965, 1974. i i . ' ! ; 48. E l - H e f n a w i , H., "Xeroderma pigmentosum", i n " C l i n i c a l Dermato-l o g y , V o l . 4", ed. Demis, D. e t a_l_., H a r p e r and Row P u b l i s h e r s I n c . , I 972, page I. F a z e k a s de S t : G r o t h , S., N a t u r e 162, 294, 1948. G a u d i n , D., Gregg, R.S. and Y i e l d i n g , K.L., Biochem. B i o p h y s . Res. Commun. 45, 630, 1971. G r e e n , M., P a r s o n s , J.T., P i n a , M., F u j i n a g a , K., C a f f i e r , H. and L a n d g r a f - L e u r s , I . , C o l d S p r i n g Harbour Symp. 35, 803, 1970. G r e e n , M., P a r s o n s , J.T., C a f f i e r , H., L a n d g r a f - L e u r s , M. and T s u e i , D., " T r a n s c r i p t i o n of A d e n o v i r u s Genes i n P r o d u c t i v e l y I n f e c t e d and i n T r a n s f o r m e d C e l l s " , i n "The B i o l o g y of Onco-g e n i c V i r u s e s " , ed. S i l v e s t r i , L.G., N o r t h - H o l l a n d P u b l i s h i n g Company, 1971, page 88. H e i d e l b e r g e r , C , Fed. P r o c . 32, 2154, 1973. H i e g e r , I . , " C a r c i n o g e n e s i s " , Academic P r e s s , 1961. H o w a r d - F l a n d e r s , P., Ann. Rev. Biochem. 37_, 175, 1968. I r v i n g , C C , Methods i n C a ncer Res. 8_, 189, 1973. K a l n i n s , V . I . , S t i c h , H.F., G r e g o r y , C. and Yohn, D.S. Cancer Res. Z7_, 1874, 1967. L a i s h e s , B.A., PhD T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1974. Lawley, P.O., P r o c . N u c l . A c i d Res. M o l . B i o l . 5_, 89, 1966. L y t l e , C P . , A a r o n s o n , S.A. and Harvey, E., I n t . J . Rad. B i o l . 22_, 159, 1972. 49. Mart i nez-Pa lomo, A., L e B u i s , J . and B e r n a r d , W., J . V i r o l . j _ , 817, 1967. M i l l e r , J.A., Cancer Res. 30_, 559, 1970. ^ M i l l e r , J.A. and M i l l e r , E.C., J e r u s a l e m Symp. on Quantum C h e m i s t r y and Biochem. J_, 237, 1969a. ' M i l l e r , J.A. and M i l l e r , E.C., P r o g . Exp. Tumour Res. _M_, 273, 1969b. M i l l e r , J.A. and M i l l e r , E.C., J . Nat. Cancer I n s t . 47_, 5, 1971. Montgomery, H., "DermatopathoIogy", H a r p e r and Row P u b l i s h e r s I n c . , . 1967 , page 123. N i c h o l s , W.W., P e l u s e , M., G o o d h e a r t , C.R., M c A l l i s t e r , R. and B r a d t , C. , V i r o l o g y 34_, 303, 1968. Oxman, M.N. and B l a c k , P.H., P r o c . Nat. Acad. S c i . - 5 5 , 1133, 1966. Oxman, M.N,, Rowe W.P. and B l a c k , P.H., P r o c , Nat. Acad. S c i . 57_, 941, 1967. P a i n t e r , R.B., C u r r . T o p i c s R a d i a t . Res. 45, 1971. P a u l , J.S. and Montgomery, P.O'B, and L o u i s , J.B.,.Cancer Res. 31, 413, 1971, P h i l i p s o n , L., J . V i r o l . J _ , 868, 1967. Poon, P.K., O ' B r i e n , R.L. and P a r k e r , J.W*, N a t u r e 250, 223, 1974. P o t t , P., r e p r i n t e d i n Nat. Cancer I n s t . Monograph J_0, 7, 1963. Reed, W., L a n d i n g , B., Sugarman, G., C l e a v e r , J.E. and Me l n y k , J.., J .A.M.A. 207, 2073, 1969. R o b b i n s , J.H., Kraemer, K.H., L u t z n e r , M.A., F e s t o f f , B.W. and Coon, . H.G., A n n a l s o f I n t e r n a l M e d i c i n e 80, 221, 1974. 50. Sarmay D.S.R., Raj a I a k s m i , S. and F a r b e r , E., i n " P r i n c i p l e s of L i v e r D i s e a s e " , ed. B e c k e r , F.F., M a r c e l Dekker, New Y o r k , 1974. S e t l o w , R.B. and Regan, D., Biochem. B i o p h y s . Res. Commun. 46, 1019, 1972. S e t l o w , R.B., Regan, J.D., German, J . and C a r r i e r , W.L., P r o c . Nat. Acad. S c i . 64, 1035, 1969. S t i c h , H.F., K a l n i n s , V . I . , McKinnon, E. and Yohn, D.S., J . U l t r a -s t r u c t . Res. J_9, 556, 1967. S t i c h , H.F., A v i l a , L.R. and Yohn, D..S., Expt. C e l l Res. 53_, "44, 1968. S t i c h , H.F. and San, R.H.C., Mut. Res. _I0_, 389, 1970. S t i c h , H.F. and San R.H.C, Mut. Res. _I3_, 279, 1971. S t i c h , H.F., Hammerberg, 0. and C a s t o , B., Can. J . Genet. C y t o l . JM_, 911, I 972a. S t i c h , H.F., San, R.H.C., M i l l e r , J.A. and M i l l e r , E.G., N a t u r e New B i o l . 238, 9, 1972b. S t i c h , H.F. and San, R.H.C., S o c i e t y Exp. B i o l and Med. 142, 155, 1973. S t i c h , H.F., San, R.H.C. and Kawazoe, Y., Mut. Res. \J_, 127, 1973. S t i c h , H.F., K i e s e r , D., L a i s h e s , B.A. and San, R.H.C.,-Prod. Cana-: i d i a n C a ncer C o n f e r e n c e 10, 1974a. ' I S t i c h , H.F., S t i c h , W. and Lam, P., Na t u r e 250, 599, 1974b. S t o l t z , D.R., P o i r i e r , L.A., I r v i n g , C . C , S t i c h , H.F., W e i s b u r g e r , J.H. and G r i c e , H.C., T o x i c o l o g y and A p p l i e d Pharm. 29_, 157, I 974. 51 . Sussenbach, J.S., V i r o l o g y 33_, 567, 1.967. Zur Hausen, H., J . V i r o l . 1174, 1967. Zur,Hausen, H., J . V i r o l . 2, 218, 1968. I 

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