Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Repair of DNA damage in mammalian cells after low doses of ionizing radiation Berger, Stuart Alan 1982

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1982_A6_7 B47.pdf [ 4.19MB ]
Metadata
JSON: 831-1.0095183.json
JSON-LD: 831-1.0095183-ld.json
RDF/XML (Pretty): 831-1.0095183-rdf.xml
RDF/JSON: 831-1.0095183-rdf.json
Turtle: 831-1.0095183-turtle.txt
N-Triples: 831-1.0095183-rdf-ntriples.txt
Original Record: 831-1.0095183-source.json
Full Text
831-1.0095183-fulltext.txt
Citation
831-1.0095183.ris

Full Text

R E P A I R OF DNA DAMAGE IN MAMMALIAN C E L L S AFTER LOW DOSES OF I O N I Z I N G RADIAT ION by STUART ALAN BERGER B . S c , M c G i l l U n i v e r s i t y , 1980 T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SC IENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f P h y s i c s ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A u g u s t 1982 S t u a r t A l a n B e r g e r , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Physics The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date August 19, 1982 DE-6 (3/81) A b s t r a c t : i s g e n e r a l l y a c c e p t e d t h a t the c r i t i c a l t a r g e t o f S ) 1 •J !' i o n i z i n g r a d i a t i o n i n c e l l u l a r r e p r o d u c t i v e d e a t h i s DNA. What i s l e s s known i s what t y p e s of l e s i o n s a r e i n v o l v e d , how t h ey are r e p a i r e d and how v a r i o u s a g e n t s enhance, p r o t e c t o r m o d i f y them. One o f t h e main r e a s o n s f o r t h e s e d i f f i c u l t i e s has been the l a c k o f a s s a y s f o r m o l e c u l a r damage s e n s i t i v e enough t o measure y i e l d s at doses where s i g n i f i c a n t numbers of c e l l s s u r v i v e . R e c e n t l y however, v a r i o u s t e c h n i q u e s have appeared t h a t e x h i b i t t h i s s e n s i t i v i t y . One o f t h e s e , Rydbergs* d o u b l e - l a b e l u n w i n d i n g method, can r o u t i n e l y d e t e c t damage i n the DNA o f mammalian c e l l s from doses as low as 0.1 Gy. We have used t h i s t e c h n i q u e t o measure i n i t i a l y i e l d s , r e p a i r k i n e t i c s and r e s i d u a l y i e l d s o f damage i n t h e dose range 1-6 Gy. We have f o u n d , i n s u b s t a n t i a l agreement w i t h measurements at h i g h e r d o s e s , the e x i s t e n c e o f at l e a s t two d i s t i n c t r e p a i r p r o c e s s e s w i t h h a l f - l i v e s o f 7 m i n u t e s and 250 m i n u t e s r e s p e c t i v e l y . We have a l s o found t h a t the d i s t r i b u t i o n o f b r e a k s between the f a s t and s l o w p r o c e s s e s can be a l t e r e d by v a r i o u s c o n d i t i o n s ; f o r example: whether o r not the i r r a d i a t i o n was done i n the p r e s e n c e o f oxygen or n i t r o g e n , p o s t - t r e a t m e n t i n h y p e r t o n i c s o l u t i o n , i n c u b a t i o n w i t h ARA-A, i i i Can i n h i b i t o r o f DNA s y n t h e s i s ) o r p o s t - t r e a t m e n t i n d i m e t h y l s u l p h o x i d e . We have a l s o found t h a t t h e y i e l d o f u n r e p a i r e d l e s i o n s Cas d e f i n e d by damage r e m a i n i n g a f t e r two hours i n c u b a t i o n a t 37° C ) seems t o i n c l u d e a q u a d r a t i c v a r i a t i o n w i t h i n i t i a l dose s u g g e s t i n g t h a t among the s l o w l y r e p a i r i n g b r e a k s , t h e r e e x i s t complex, c o m b i n a t i o n - t y p e l e s i o n s Cwhich may i n c l u d e DNA d o u b l e - s t r a n d b r e a k s ) . S u c h s t u d i e s , combined w i t h r e s u l t s from o t h e r a s s a y s s h o u l d h e l p i n u n d e r s t a n d i n g t h e n a t u r e o f the l e s i o n s i n DNA and t h e r e p a i r p r o c e s s e s a c t i n g on them and w i l l e v e n t u a l l y a i d In i d e n t i f y i n g the c r i t i c a l l e s i o n ( s ) r e s p o n s i b l e f o r c e l l d e a t h . i v A B S T R A C T M T A B L E OF CONTENTS . Iv L I S T OF T A B L E S v i L I S T OF F I G U R E S . v i i A B B R E V I A T I O N S i x ACKNOWLEDGEMENTS x 1 . INTRODUCTION 1 1 . 1 C e n t r a l c h a l l e n g e o f r a d i o b i o l o g y 3 1 . 2 M o l e c u l a r r a d i o b i o l o g y 6 1 . 3 R e p a i r k i n e t i c s 8 1 . 4 P o t e n t i a l l y l e t h a l d a m a g e 9 2 . M A T E R I A L S AND METHODS 13 2 . 1 C e l l g r o w t h a n d m a i n t e n a n c e 13 2 . 2 DNA l a b e l 1 i ng 14 2 . 3 C e l l t r e a t m e n t s ." 14 2 . 3 . 1 O x i c i r r a d i a t i o n s 14 2 . 3 . 2 A n o x i c i r r a d i a t i o n s 14 2 . 4 I n c u b a t i o n c o n d i t i o n s 15 2 . 4 . 1 H y p e r t o n i c p o s t - t r e a t m e n t 16 2 . 4 . 2 A R A - A . 17 . 2 . 4 . 3 DMSO 17 2 . 4 . 4 P l a t e a u - p h a s e c e l l s . 17 2 . 5 U n w i n d i n g a s s a y 19 2 . 6 U n w i n d i n g o f DNA i n a l k a l i 21 V 2 . 7 D o u b l e - l a b e l u n w i n d i n g m e t h o d 25 3 . R E S U L T S 27 3 . 1 E l u t i o n p r o f i l e s a n d u n w i n d i n g k i n e t i c s 27 3 . 2 D o s e r e s p o n s e - e x t e r n a l c o n t r o l a n d e v / b r e a k . 33 3 . 3 D o s e r e s p o n s e - i n t e r n a l c o n t r o l - a e r o b i c . . . . 36 3 . 4 R e p a i r k i n e t i c s - a e r o b i c i r r a d i a t i o n 36 3 . 5 R e m a i n i n g d a m a g e 41 3 . 6 D o s e r e s p o n s e - h y p o x i a 41 3 . 7 R e p a i r k i n e t i c s - h y p o x i c i r r a d i a t i o n 4 3 3 . 8 P l a t e a u - p h a s e c e l l s 46 3 . 9 H y p e r t o n i c p o s t - t r e a t m e n t 49 3 . 1 0 A R A - A . . 49 3 . 1 1 DMSO 52 4 . . . D I S C U S S I O N . 54 4 . 1 R e p a i r k i n e t i c s 54 4 . 1 . 1 S a t u r a t i o n 55 4 . 1 . 2 S u b p o p u 1 a t I o n w i t h d i f f e r e n t k i n e t i c s . . 59 4 . 1 . 3 I n a c c e s s i b l e d a m a g e 59 4 . 1 . 4 M u l t i p l e r e p a i r p a t h w a y s 60 4 . 1 . 5 DSB i n d u c t i o n 61 4 . 2 OER 6 8 4 . 3 P o s t - t r e a t m e n t s a n d PLD 70 4 . 4 R e l a t i n g m o l e c u l a r d a m a g e a n d c e l l d e a t h 73 5 . R E F E R E N C E S 8 0 V i L I S T OF T A B L E S : Tab%e 1 . Summary o f p a r a m e t e r s f i t t e d t o r e p a i r k i n e t i c s d a t a . V I I L I S T OF F I G U R E S : I'-' "? , 1 . G r o w t h c u r v e - C H 2 B 2 ' c e l l s i n P e t r i d i s h e s 18 2 . L e n g t h d i s t r i b u t i o n o f f r a g m e n t l e n g t h s f o r r a n d o m l y i n d u c e d s t r a n d b r e a k s 23 3 . E l u t i o n p r o f i l e s 28 4 . U n w i n d i n g k i n e t i c s 30 5 . D o s e r e s p o n s e - e x t e r n a l c o n t r o l 32 6\. M e a s u r e d b r e a k s v s . s a l t c o n c e n t -r a t i o n o f u n w i n d i n g s o l u t i o n 35 7 . D o s e r e s p o n s e - i n t e r n a l c o n t r o l 37 8 . R e p a i r k i n e t i c s 39 9 . B r e a k s r e m a i n i n g a f t e r 2 h o u r s - l o w d o s e s 42 1 0 . B r e a k s r e m a i n i n g a f t e r 2 h o u r s - h i g h d o s e s 42 1 1 . O x i c a n d a n o x i c d o s e r e s p o n s e -s t a n d a r d g a s s i n g p r o c e d u r e 44 1 2 . O x i c a n d a n o x i c d o s e r e s p o n s e -a l t e r n a t e g a s s i n g p r o c e d u r e 45 1 3 . R e p a i r k i n e t i c s - c e l l s i r r a d i a t e d i n 0 2 o r N 2 47 1 4 . R e p a i r k i n e t i c s f o r c e l l s g r o w n t o p l a t e a u - p h a s e a f t e r 3 , 9 a n d 15 d a y s o f g r o w t h 48 1 5 . R e p a i r k i n e t i c s a f t e r h y p e r t o n i c p o s t - t r e a t m e n t 50 v i i i LIST OF FIGURES (CONT'D) 16. R e p a i r k i n e t i c s o f c e l l s i n c u b a t e d i n medium c o n t a i n i n g ARA-A 51 17. E f f e c t o f DMSO on r e p a i r k i n e t i c s o f i r r a d i a t e d c e l l s 53 18. S i m u l a t i o n o f r e p a i r k i net i c s f o r s a t u r a t e d and u n s a t u r a t e d s i t u a t i o n 58 I X A B B R E V I A T I O N S : A R A - A A R A - C ASG B BUdR DMF DMSO DNA D 20 DSB F G y . LET NSG N a C l N2 OER °2 PBS PLD RBE SDS SSB u . v . a r a b i n o f u r a n o s y l a d e n i n e a r a b i n o f u r a n o s y l c y t o s i n e a l k a l i n e s u c r o s e g r a d i e n t s b r e a k s 5' - b r o m o d e o x y u r i d i n e d o s e - m o d i f y i n g f a c t o r ' ; r a t i o o f d o s e s r e q u i r e d t o a c h i e v e t h e same b i o l o g i c a l e f f e c t . d i m e t h y l s u l p h o x i d e d e o x y r i b o n u c l e i c a c i d d e u t e r i u m o x i d e ( h e a v y w a t e r ) d o u b l e - s t r a n d b r e a k i n DNA f r a c t i o n o f d o u b l e - s t r a n d e d DNA t o t o t a l DNA. G r a y ; u n i t o f a b s o r b e d d o s e ( 1 J o u l e / K g . ) L i n e a r E n e r g y T r a n s f e r ' n e u t r a l s u c r o s e g r a d i e n t s s o d i u m c h l o r i d e m o l e c u l a r n i t r o g e n o x y g e n e n h a n c e m e n t r a t i o ; r a t i o o f d o s e s r e q u i r e d o b t a i n t h e same e f f e c t i n h y p o x i a a s u n d e r o x i c c o n d i t i o n s . m o l e c u l a r o x y g e n p h o s p h a t e - b u f f e r e d s a l i n e p o t e n t i a l l y l e t h a l d a m a g e r e l a t i v e b i o l o g i c a l e f f e c t i v e n e s s s o d i u m l a u r y l s u l p h a t e s i n g l e - s t r a n d b r e a k i n DNA u l t r a v i o l e t X ACKNOWLEDGEMENTS: I w o u l d l i k e t o t h a n k a l l my c o l l e a g u e s a t t h e B . C . C a n c e r R e s e a r c h C e n t r e . I n p a r t i c u l a r , I w i s h t o t h a n k D r s . K . S k o v a n d M. K o r b e l i k , a n d J u l i e t B r o s i n g f o r v a l u a b l e d i s c u s s i o n s a n d s u g g e s t i o n s . I a l s o w i s h t o a c k n o w l e d g e t h e c a p a b l e t e c h n i c a l a s s i s t a n c e o f I s a b e l H a r r i s o n a n d H a n s A d o m a t a n d t h e f i n e t y p i n g a n d s e c r e t a r i a l s k i l l s o f B e v E r s o y who a l w a y s h a d a c h e e r f u l w o r d f o r e v e r y b o d y . I w o u l d 1 i ke t o e x p r e s s my g r a t i t u d e t o t h e B . C C a n c e r F o u n d a t i o n f o r a c c o m o d a t i o n , ; f i n a n c i a l s u p p o r t a n d t r a v e l s u p p o r t a n d t o t h e N a t i o n a l C a n c e r I n s t i t u t e o f C a n a d a f o r f i n a n c i a l s u p p o r t i n t h e f o r m o f a R e s e a r c h S t u d e n t s h i p . I w i s h t o e s p e c i a l l y t h a n k D r . B r a n k o P a l c i c a n d D r . L l o y d S k a r s g a r d , H e a d , M e d i c a l B i o p h y s i c s U n i t a n d my s u p e r v i s o r . T o g e t h e r , t h e y m a n a g e d t o c r e a t e a n e x c e l l e n t r e s e a r c h e n v i r o n m e n t by p r o v i d i n g v a l u a b l e d i r e c t i o n a n d s u p p o r t w h i l e s t i l l a l l o w i n g me t h e f r e e d o m t o p u r s u e my r e s e a r c h i n d i r e c t i o n s o f my c h o o s i n g . T h e i r k i n d n e s s a n d g o o d h u m o u r h a v e g u a r a n t e e d t h a t I w i l l a l w a y s l o o k b a c k on my s t a y h e r e a t t h e B . C . C a n c e r R e s e a r c h C e n t r e w i t h f o n d m e m o r i e s . F i n a l l y , I w i s h t o t h a n k my f a m i l y who g a v e me t h e l o v e a n d s u p p o r t t h a t I n e e d e d t o s u r v i v e t h e r a i n y s e a s o n s h e r e i n V a n c o u v e r . To a l l o f t h e p e o p l e a b o v e , I d e d i c a t e t h i s w o r k . X I I 'Nature i s not o n l y s t r a n g e r t h a n we imagine; i t s t r a n g e r t h a n we can p o s s i b l y i m agine.' J.B.S. Haldane 1 1 . I n t r o d u c t i o n : T h e g r o w i n g p r e v a l e n c e o f r a d i a t i o n In t h e e n v i r o n m e n t t o d a y u n d e r s c o r e s m o r e t h a n e v e r t h e n e e d f o r b e t t e r u n d e r s t a n d i n g i t s e f f e c t s i n b i o l o g i c a l m a t e r i a l s . I t h a s l o n g b e e n known t h a t r a d i a t i o n c a n c a u s e s e r i o u s d a m a g e i n b i o l o g i c a l macromo1 e c u 1es w h i c h c a n l e a d t o a v a r i e t y o f d e l e t e r i o u s e f f e c t s i n c l u d i n g m u t a t i o n s , c h r o m o s o m a l a b e r r a t i o n s a n d c e l l d e a t h . R a d i a t i o n s a r e u s u a l l y b r o a d l y d j v i d e d I n t o 2 c a t e g o r i e s ; n o n - i o n i z i n g a n d i o n i z i n g . The f i r s t , w h i c h i n c l u d e s e l e c t r o m a g n e t i c r a d i a t i o n s up t o a n d i n c l u d i n g t h e u l t r a v i o l e t ( w a v e l e n g t h s a b o u t 10 ^ m ) , m a n i f e s t s i t s e l f m a i n l y t h r o u g h e x c i t a t i o n s o f m o l e c u l e s a n d r e a r r a n g e m e n t o f c h e m i c a l b o n d s . I o n i z i n g r a d i a t i o n s i n c l u d e e l e c t r o m a g n e t i c r a d i a t i o n s w i t h — 8 w a v e l e n g t h s b e l o w a b o u t 10 m s u c h a s x - r a y s a n d y - r a y s a s w e l l a s a c c e l e r a t e d p a r t i c l e s b o t h f r o m n a t u r a l ( c o s m i c r a y s ) a n d m a n - m a d e ( s u c h a s f r o m p a r t i c l e a c c e l e r a t o r s ) s o u r c e s . T h e i r h i g h e r e n e r g y a l l o w s t h e m t o b r e a k b o n d s , i o n i z e a n d c r e a t e many l o n g - l i v e d r e a c t i v e r a d i c a l s . R a d i o b i o l o g y a s a s c i e n c e e m e r g e d o u t o f t h e g r o w i n g n e e d t o u n d e r s t a n d t h e e f f e c t s o f t h e s e r a d i a t i o n s i n . b i o 1 o g i c a 1 2 m a t e r i a l s . A s s u c h i t i s a m u l t i - f a c e t e d d i s c i p l i n e b r i n g i n g t o t h e p r o b l e m a p p r o a c h e s f r o m a l l t h e m a j o r n a t u r a l s c i e n c e s . I n r e c e n t y e a r s , t h e m a i n e f f o r t s o f r a d i o b i o l o g i s t s h a v e b e e n d i r e c t e d t o w a r d s s u g g e s t i n g w a y s o f i m p r o v i n g t h e u s e s o f r a d i a t i o n i n c a n c e r r a d i o t h e r a p y a n d i m p r o v i n g o u r u n d e r s t a n d i n g s o a s t o be a b l e t o r e l i a b l y e s t i m a t e r i s k a n d p r o v i d e p r o t e c t i o n f r o m i t s h a r m f u l e f f e c t s . A m a j o r e m p h a s i s h a s e m e r g e d i n r a d i o b i o l o g y r e c e n t l y o n t h e i m p o r t a n c e o f s t u d y i n g t h e e f f e c t s o f r a d i a t i o n a t l o w d o s e s . One r e a s o n f o r t h i s e m p h a s i s i s t h a t f o r a v a r i e t y o f r e a s o n s , r a d i o t h e r a p y h a s t u r n e d i n c r e a s i n g l y t o t h e u s e o f s m a l l d o s e f r a c t i o n s ( o n t h e o r d e r o f 2 Gy o r l e s s ) s p r e a d o v e r e x t e n s i v e p e r i o d s o f t i m e i n t h e t r e a t m e n t o f m a l i g n a n t d i s e a s e . I t h a s b e e n s u g g e s t e d ( T h o m l i n s o n & G r a y 1 9 5 5 ; P o w e r s S T o l m a c h 1 9 6 3 ) t h a t t h e r e may e x i s t a s u b p o p u 1 a t i o n o f t u m o r c e l l s t h a t a r e h y p o x i c w h i c h w o u l d make t h e m r a d i o r e s i s t a n t c o m p a r e d t o t h e i r m o r e o x y g e n a t e d n e i g h b o u r s . I t i s a l s o known ( E l k i n d & S u t t o n I 9 6 0 ; S i n c l a i r 1 9 6 8 ; G i l l e s p i e e t a l . 1 9 7 5 ; H a l l 1 9 7 8 , c h a p t e r 7 ) t h a t c e l l s i n c e r t a i n p a r t s o f t h e c e l l c y c l e a r e l e s s r a d i o s e n s i t i v e t h a n o t h e r s w h i c h w o u l d mean t h a t a f t e r r a d i a t i o n t r e a t m e n t , a f r a c t i o n o f t h e s e r a d i o r e s i s t a n t c e l l s c o u l d r e m a i n v i a b l e . The i d e a b e h i n d f r a c t i o n a t i o n i s t h a t a l l o w i n g t i m e t o e l a p s e b e t w e e n f r a c t i o n s w o u l d a l l o w t h e c e l l s t o r e - o x y g e n a t e ( F l e t c h e r 1 9 7 3 ) a n d r e - d i s t r i b u t e t h e m s e l v e s i n t o l e s s r a d i o r e s i s t a n t p a r t s o f t h e c e l l c y c l e ( W i t h e r s 1 9 7 5 a , b ) . T h e r e i s a l s o some 3 e v i d e n c e b e i n g a c c u m u l a t e d w h i c h s u g g e s t s t h a t t h e O x y g e n E n h a n c e m e n t R a t i o ( O E R ) may be l o w e r a t l o w e r d o s e s ( L i t t b r a n d S R e v e s z 1 9 6 9 ; Chapman e t a l . 1 9 7 5 b ; M c N a l l y e t a l . 1 9 7 5 ; P e t t e r s e n e t a l . 1 9 7 5 ; P a l c i c , B r o s i n g S S k a r s g a r d 1 9 8 2 ) , a J s i t u a t i o n w h i c h w o u l d a l s o be e x p l o i t e d by u s i n g f r a c t i o n a t e d r e g i m e s . W h a t e v e r t h e r a t i o n a l e o r i t s s o u r c e , t h e I n c r e a s i n g u s e o f s m a l l d o s e f r a c t i o n s In t h e t r e a t m e n t o f m a l i g n a n t d i s e a s e h a s l e d C h a p m a n & G i l l e s p i e ( 1 9 8 1 ) t o p r o p o s e t h a t m o r e e f f o r t be e x p e n d e d i n t h e s t u d y o f r a d i o b i o l o g y a t 200 r a d s ( 2 G y ) . U n d e r s t a n d i n g t h e e f f e c t s o f l o w d o s e s i s a l s o o f i m p o r t a n c e i n c o n s i d e r a t i o n s o f p r o t e c t i o n . T y p i c a l x - r a y e x a m i n a t i o n s c a n d e l i v e r a n y w h e r e b e t w e e n m i l l i G r a y s f o r s i m p l e c h e s t x - r a y s t o 0 . 1 Gy ( 1 0 r a d s ) f o r m o r e c o m p l i c a t e d p r o c e d u r e s . I t h a s u s u a l l y b e e n a s s u m e d t h a t r i s k e s t i m a t e s o b t a i n e d a t h i g h e r d o s e s a p p l y t o l o w e r l e v e l s b u t t h e l a c k o f t e c h n i q u e s a v a i l a b l e f o r s t u d y i n g t h e e f f e c t s o f r a d i a t i o n a t l o w d o s e s h a s p r e c l u d e d t e s t i n g o f t h e s e a s s u m p t i o n s . T h i s s i t u a t i o n i s now c h a n g i n g a s i m p r o v e d t e c h n i q u e s b o t h a t t h e c e l l u l a r a n d t h e m o l e c u l a r l e v e l a r e e m e r g i n g t h a t h a v e t h e c a p a b i l i t y t o s t u d y t h e e f f e c t s o f r a d i a t i o n a t ' r a d I o b i o 1 o g i c a 1 1 y r e l e v a n t ' d o s e s . 1 . 1 C e n t r a l C h a l l e n g e o f R a d i o b i o l o g y : The e f f e c t s o f r a d i a t i o n on w h o l e a n i m a l s a r e w e l l u n d e r s t o o d In t e r m s o f i t s e f f e c t s a t t h e t i s s u e a n d o r g a n l e v e l ( H a l l 1 9 7 8 , c h a p t e r s 10 & 1 1 , A l p e r 1 9 7 9 , c h a p t e r 1 ) . 4 S i m i l a r l y , t h e l a t e e f f e c t s s e e n a t t h e t i s s u e l e v e l c a n be t r a c e d t o t h e k i l l i n g o f s p e c i a l p r e c u r s o r c e l l s w h o s e n o r m a l f u n c t i o n i s t o p r o l i f e r a t e . The same c a n n o t be s a i d o f u n d e r s t a n d i n g c e l l u l a r e n d p o i n t s i n m o l e c u l a r t e r m s , a l t h o u g h i t i s f i r m l y e s t a b l i s h e d t h a t d a m a g e t o b i o l o g i c a l m a c r o m o 1 e c u 1 e s i s t h e m a i n r e s u l t o f r a d i a t i o n a t t h e m o l e c u l a r l e v e l . I t i s a l s o g e ' n e r a l l y a c c e p t e d t h a t DNA, t h e g e n e t i c m a t e r i a l o f t h e c e l l i s t h e c r i t i c a l t a r g e t f o r i o n i z i n g r a d i a t i o n ( a n d U . V . l i g h t ) i n r e p r o d u c t i v e d e a t h . H i s t o r i c a l l y , t w o e v e n t s h a v e h e l p e d t o p o i n t t o w a r d s t h e i m p o r t a n c e o f DNA a s t h e c r i t i c a l t a r g e t . The f i r s t h a s b e e n t h e l a r g e r o l e t h a t t h e ' c e n t r a l d o g m a ' ( W a t s o n 1 9 7 7 ) o f m o l e c u l a r b i o l o g y h a s p l a y e d . By i d e n t i f y i n g DNA a s t h e r e p o s i t o r y o f b i o l o g i c a l i n f o r m a t i o n , i t h a s s e r v e d t o e m p h a s i z e t h e i m p o r t a n c e o f m a i n t a i n i n g i t s I n t e g r i t y t o j u s t a b o u t e v e r y f u n c t i o n c a r r i e d o u t i n t h e c e l l . The s e c o n d h a s b e e n t h e d e v e l o p m e n t a n d s u b s e q u e n t i m p r o v e m e n t s o f c l o n i n g t e c h n i q u e s f o r m a m m a l i a n c e l l s ( P u c k , M a r c u s S C i e c i u r a 1 9 5 6 ; P u c k & M a r c u s 1 9 5 6 ) . T h i s l e d t o t h e p r o m i n e n t u s e o f c o l o n y - f o r m i n g a b i l i t y a s a s u i t a b l e e n d p o i n t f o r s t u d y i n g t h e e f f e c t s o f r a d i a t i o n o n m a m m a l i a n c e l l s . T h i s a b i l i t y i s a p r o c e s s i n w h i c h m a i n t a i n i n g t h e i n t e g r i t y o f DNA I s a l s o e s s e n t I a 1 . S p u r r e d by t h e s e t w o i n f l u e n c e s , v a r i o u s e x p e r i m e n t s h a v e b e e n p e r f o r m e d w h i c h I m p l i c a t e DNA a s a c r i t i c a l t a r g e t . T h e s e e x p e r i m e n t s i n c l u d e s e l e c t i v e i r r a d i a t i o n o f t h e n u c l e u s ( Z i r k l e 1 9 5 7 ; M u n r o 1 9 7 0 ; C o l e e t a l ' . 1 9 7 4 ; D a t t a e t a l ' . 5 1 9 7 6 ) , ' s u i c i d e ' e x p e r i m e n t s w i t h l a b e l l e d n u c l e o t i d e s ( H e r s h e y e t a l . 1 9 5 7 ; M a r i n & B e n d e r 1 9 6 3 ; B u r k i & O k a d a 1 9 6 8 ; B u r k i e t a l . 1 9 7 3 ) , t h e r e s u l t a n t i n c r e a s e i n r a d i o s e n s i t i v i t y f r o m t h e i n c o r p o r a t i o n o f BUdR ( K a p l a n , S m i t h g T o m l i n 1 9 6 3 ; S z y b a l s k i 1 9 6 7 ; Adams 1 9 6 7 ; Dewey e t a l . 1 9 7 1 ; Sawad -a^S O k a d a 1 9 7 2 ) , t h e c o r r e l a t i o n b e t w e e n DNA c o n t e n t a n d r a d i o s e n s I t i v i t y ( K a p l a n & M o s e s 1 9 6 4 ; S p a r r o w , U n d e r b r o o k S S p a r r o w 1 9 6 7 ) , v a r i o u s c o r r e l a t i o n s b e t w e e n DNA d a m a g e a n d c e l l s u r v i v a l i n c l u d i n g t h e c o r r e l a t i o n b e t w e e n m o l e c u l a r a n d s u r v i v a l d a t a o n t h e r e l a t i o n s h i p b e t w e e n RBE a n d LET ( R i t t e r , C l e a v e r & T o b i a s 1 9 7 7 ) , t h e c o r r e l a t i o n s o b s e r v e d o n t h e OER a t t h e m o l e c u l a r a n d c e l l u l a r l e v e l ( S k o v , P a l c i c & S k a r s g a r d 1 9 7 9 ) , t h e c o r r e l a t i o n s b e t w e e n c h r o m o s o m a l a b e r r a t i o n s a n d s u r v i v a l ( B e n d e r S W o l f f 1 9 6 1 ; S k a r s g a r d e t a l . 1 9 6 7 ; D e w e y , M i l l e r S L e e p e r 1 9 7 1 ; C a r r a n o 1 9 7 3 a , b ; D e w e y , S a p a r e t o S B e t t e n 1 9 7 8 ; N a k a t s u g a w a , I s h i z a k i & S u g a h a r a 1 9 7 8 ; R a a p h o r s t & Dewey 1 9 7 9 a ) a n d t h e d e c r e a s e s In s u r v i v a l s e e n i n m u t a n t s d e f i c i e n t i n t h e r e p a i r o f DNA d a m a g e ( C l e a v e r 1 9 6 8 ; P a t e r s o n e t a l . 1 9 7 6 ) . H o w e v e r , i n s p i t e o f t h e l a r g e a n d e v e r i n c r e a s i n g a m o u n t o f w o r k t h a t h a s b e e n d o n e , u n d e r s t a n d i n g o f t h e e x a c t r e l a t i o n s h i p b e t w e e n DNA d a m a g e a n d c e l l d e a t h r e m a i n s e l u s i v e . S t i l l l a c k i n g i s t h e k n o w l e d g e o f t h e n a t u r e o f t h e c r i t i c a l l e s i o n s , how t h e y a r e a f f e c t e d by t h e p h y s i c a l a n d c h e m i c a l e n v i r o n m e n t o f t h e c e l l a n d , p e r h a p s m o s t i n t e r e s t i n g , bow t h e b i o l o g i c a l a c t i v i t y o f t h e c e l l a c t s t o m o d i f y , e n h a n c e o r r e p a i r t h e m . T h e s e p r o b l e m s a r e a t t h e 6 f o r e f r o n t o f r a d i o b i o l o g y a s I n d e e d t h e y s h o u l d b e . F o r a b e t t e r u n d e r s t a n d i n g o f t h e e f f e c t s o f r a d i a t i o n i n m o l e c u l a r t e r m s w i l l s u g g e s t w a y s o f i m p r o v i n g r a d i o t h e r a p y . p r o t o c o l s . I t w i l l a l s o i m p r o v e o u r u n d e r s t a n d i n g o f i t s e f f e c t s a t t h e c e l l u l a r l e v e l ( e s p e c i a l l y i n t h e c r i t i c a l l o w d o s e r e g i o n ) a n d t h u s p r o v i d e a f i r m e r b a s i s f r o m w h i c h t o p r o v i d e r a t i o n a l p r o t e c t i o n p r o c e d u r e s . 1 . 2 M o l e c u l a r R a d i o b i o 1 o g y : M o l e c u l a r r a d i o b i o l o g i s t s t o d a y h a v e a w h o l e a r s e n a l o f t e c h n i q u e s w i t h w h i c h t o s t u d y t h e e f f e c t s o f r a d i a t i o n a t t h e m o l e c u l a r l e v e l . In p a r t i c u l a r , a n u m b e r o f t e c h n i q u e s a r e a v a i l a b l e w i t h w h i c h t o s t u d y DNA d a m a g e . A l k a l i n e s u c r o s e g r a d i e n t s ( A S G ) , a n d N e u t r a l s u c r o s e g r a d i e n t s ( N S G ) ( M c G r a t h S W i l l i a m s 1 9 6 6 ) , t w o m e t h o d s w h i c h w e r e d e v e l o p e d e a r l y o n , c a n m e a s u r e s i n g l e - s t r a n d a n d d o u b l e - s t r a n d b r e a k s r e s p e c t i v e l y . T h e i r d i s a d v a n t a g e i s t h a t t h e y a r e l i m i t e d i n t h e i r r e s o l u t i o n t o h i g h d o s e s . The a l k a l i n e e l u t i o n ( K o h n S E w i g 1 9 7 3 ; K o h n e t a l . 1 9 7 6 ) a n d n e u t r a l e l u t i o n m e t h o d s ( B r a d l e y S K o h n 1 9 7 9 ) a l s o s u p p o s e d l y m e a s u r e s i n g l e a n d d o u b l e - s t r a n d b r e a k s . T h e i r a d v a n t a g e i s t h a t t h e i r s e n s i t i v i t y i s much i m p r o v e d o v e r t h e g r a d i e n t m e t h o d s . T h e i r d i s a d v a n t a g e i s t h a t t h e n a t u r e o f t h e l e s i o n s b e i n g m e a s u r e d I s n o t w e l l u n d e r s t o o d . The M . l u t e u s a s s a y ( C a r r i e r & S e t l o w 1 9 7 0 ; P a t e r s o n & S e t l o w 1 9 7 2 ; P a t e r s o n 1 9 7 5 ) m e a s u r e s b a s e d a m a g e by e x p o s i n g c e l l s t o a n e x t r a c t p r e p a r e d f r o m M i c r o c o c c u s l u t e u s w h i c h , t h r o u g h i t s e n z y m a t i c a c t i v i t y 7 c o n v e r t s d a m a g e i n b a s e s t o b r e a k s t h a t c a n be m e a s u r e d on A S G . I t i s t h u s c o m p l i m e n t a r y t o t h e o t h e r m e t h o d s . The u n w i n d i n g m e t h o d ( A h n s t r o m S E d v a r d s s o n 1 9 7 4 ; R y d b e r g 1 9 7 5 ; R y d b e r g 1 9 8 0 ) i s t h e m o s t s e n s i t i v e o f a l l a n d h a s b e e n u s e d i n t h i s w o r k m a i n l y b e c a u s e o f t h i s p r o p e r t y . I t h a s l o n g b e e n k n o w n t h a t b o t h p r o k a r y o t i c a n d e u k a r y o t i c e e l 1s p o s e s s e f f i c i e n t a n d c o m p l e x m e c h a n i s m s f o r r e p a i r i n g d a m a g e i n DNA b u t , u n l i k e d a m a g e f r o m u . v . l i g h t w h i c h i s q u i t e s p e c i f i c a n d t h u s a m e n a b l e t o i n v e s t i g a t i o n , i r r a d i a t i o n w i t h i o n i z i n g r a d i a t i o n r e s u l t s i n a w i d e v a r i e t y o f b i o c h e m i c a l l e s i o n s i n DNA ( W a r d 1 9 7 5 ; Von S o n n t a g e t a l . 1 9 8 1 ) . N e v e r t h e l e s s , s t r a n d b r e a k a s s a y s w h i c h do n o t d i s t i n g u i s h b e t w e e n d i f f e r e n t t y p e s o f l e s i o n s h a v e p r o v e d t o be q u i t e f r u i t f u l i n p r o v i d i n g i m p o r t a n t i n f o r m a t i o n . One s u c h t y p e o f I n f o r m a t i o n h a s b e e n h o w , o v e r t h e c o u r s e o f t i m e , t h e c e l l r e p a i r s t h e d a m a g e i n i t s DNA, a s u b j e c t w h i c h h a p p e n s t o be t h e c e n t r a l t o p i c o f t h i s t h e s i s . A u s e f u l p e r s p e c t i v e o n t h i s b i o l o g i c a l a c t i v i t y c a n p e r h a p s be o b t a i n e d by c o m p a r i n g i t t e m p o r a l l y t o t h e o t h e r e v e n t s i n i t i a t e d by t h e r a d i a t i o n . The e f f e c t s o f r a d i a t i o n c a n be r o u g h l y d i v i d e d i n t o 3 b r o a d ' s t a g e s ' i n t i m e ( T o b i a s e t a l . 1 9 8 0 ) ; — 18 - 8 1 ) p h y s i c a l s t a g e b e t w e e n 10 s e c a n d 10 s e c : i n i t i a l e l e c t r o n i c e n e r g y t r a n s f e r a n d d e c a y i n t o v i b r a t i o n a l , r o t a t i o n a l s t a t e s a n d h e a t - 1 4 - 4 2 ) c h e m i c a l , s t a g e b e t w e e n 10 s e c a n d 10 s e c : 8 p r i m a r y I o n a n d f r e e r a d i c a l p r o d u c t i o n , r a d i c a 1 - r a d i c a 1 i n t e r a c t i o n , r a d I c a 1 - m o 1 e c u 1e i n t e r a c t i o n , l o n g - l i v e d m a c r o m o 1 e c u 1 a r l e s i o n s 3 ) b i o l o g i c a l s t a g e b e t w e e n m a c r o m o 1 e c u 1 a r r e l a x a t i o n r e p a i r , c e l l d i v i s i o n , i n t e g r a t i o n 10 s e c a n d 10 s e c : a n d r e c o g n i t i o n , e n z y m a t i c g e n e t i c r e c o m b i n a t i o n a n d The e x p e r i m e n t s t h a t w i l l be d e s c r i b e d i n t h e f o l l o w i n g p a g e s h a v e a l l b e e n p e r f o r m e d i n t h e l a t e r ' b i o l o g i c a l s t a g e ' . A l t h o u g h t h e 3 s t a g e s a r e s e p a r a t e d i n t i m e , t h e y a r e n o t i n d e p e n d e n t i n t h a t t h e p h y s i c a l c h a r a c t e r i s t i c s o f t h e r a d i a t i o n a n d t h e c h e m i c a l e n v i r o n m e n t o f t h e c e l l a t t h e t i m e o f i r r a d i a t i o n h a v e i m p o r t a n t c o n s e q u e n c e s i n t h e l a t e r b i o l o g i c a l s t a g e l o n g a f t e r t h e r a d i a t i o n h a s b e e n a b s o r b e d . 1 . 3 R e p a i r k i n e t i c s : E a r l y r e p o r t s on t h e k i n e t i c s o f r e p a i r o f t h e s e l e s i o n s i n m a m m a l i a n c e l l s , w h i c h w e r e m o s t l y p e r f o r m e d w i t h c e n t r i f u g a t 1 o n t e c h n i q u e s , w e r e l i m i t e d by t h e i r r e s o l u t i o n t o h i g h d o s e s . M o s t o b s e r v e d s i n g l e , f i r s t - o r d e r k i n e t i c s w i t h h a l f - t i m e s o n t h e o r d e r o f m i n u t e s ( L e t t e t a l . 1 9 6 7 ; H u m p h r e y , S t e w a r d S S e d i t a 1 9 6 8 ; F o x S F o x 1 9 7 3 ; D u g l e & G i l l e s p i e 1 9 7 4 ) f o r S S B s a n d g e n e r a l l y s l o w e r k i n e t i c s f o r D S B s ( C o r r y & C o l e 1 9 7 3 ; L a n g e 1 9 7 4 ) . W i t h s u b s e q u e n t i m p r o v e m e n t s i n t e c h n i q u e , t h e e x i s t e n c e o f a s l o w e r c o m p o n e n t 9 i n v o l v i n g between 10 and 25% o f the t o t a l amount of i n i t i a l damage has been r e c o g n i z e d ( R o o t s & Smith 1975; Dugle, G i l l e s p i e S Chapman 1976; C o l e et a l . 1979; Weibezahn S C o q u e r e l l e 1981; B r y a n t & B l o c h e r 1980; Ward, B l a k e l y & J o i n e r 1982; Skov et a l . 1982; W i e r o w s k i S Wheeler 1982). Because t h e s e measurements have, f o r the most pa rt;> * vbeen performed a t s u p r a l e t h a l d o s e s , i t has n e v e r been known whether o r not t h e s e r e s u l t s c o u l d be e x t r a p o l a t e d back t o the lower dose r e g i o n where a s i g n i f i c a n t p r o p o r t i o n o f t h e c e l l s w i l l s u r v i v e . R e c e n t l y however, v a r i o u s m o l e c u l a r t e c h n i q u e s have appeared (Kohn S Ewig 1973; Kohn et a l . 1976; Rydberg 1975, 1980) w h i c h have begun t o p r o v i d e measurements i n t h e low dose range. We have used one o f t h e s e t e c h n i q u e s (Rydbergs* d o u b l e - l a b e l u n w i n d i n g method) t o measure th e k i n e t i c s o f r e p a i r o f x - r a y induced l e s i o n s In mammalian c e l l s i n-v i t ro a f t e r r e l a t i v e l y low d o s e s . We have a l s o l o o k e d a t the damage r e m a i n i n g a f t e r r e p a i r so as t o b e t t e r u n d e r s t a n d the n a t u r e of the u n r e p a i r e d damage, i t s r e l a t i o n s h i p t o t h e i n i t i a l damage and e v e n t u a l l y , how i t i s r e l a t e d t o c e l l d e a t h . 1.4 P o t e n t i a l l y l e t h a l damage: C o r r e l a t i n g two e n d p o i n t s u s u a l l y i n v o l v e s m a n i p u l a t i n g c o n d i t i o n s under w h i c h both e n d p o i n t s are measured and n o t i n g whether o r not they respond i n the same f a s h i o n . F o l l o w i n g t h i s c o n c e p t , we have employed some t r e a t m e n t s n o r m a l l y used 10 t o r e v e a l t h e p h e n o m e n o n o f P o t e n t i a l l y l e t h a l Damage ( P L D ) , a c e l l u l a r e f f e c t , t o s e e how t h e y a f f e c t t h e . r e p a i r o f DNA d a m a g e . I t i s w e l l known t h a t t h e e f f i c a c y o f i o n i z i n g r a d i a t i o n i n i n a c t i v a t i n g p r o l i f e r a t i n g c e l l s c a n be a f f e c t e d by t h e e n v i r o n m e n t a n d / o r s t a t e o f t h e c e l l s a f t e r i r r a d i a t i o n . F o r i n s t a n c e , i f c e l l s a r e m a i n t a i n e d u n d e r s u b - o p t i m a l g r o w t h c o n d i t i o n s s o t h a t p r o g r e s s i o n t h r o u g h t h e c e l l - c y c l e i s i n h i b i t e d , s u r v i v a l w i l l be e n h a n c e d ( B e l l i & S h e l t o n 1 9 6 9 ; H a h n e t a l . 1 9 7 3 ; L i t t l e 1 9 7 3 ) . C e l l s g r o w n t o p l a t e a u - p h a s e m a y , d e p e n d i n g o n t h e c e l l l i n e , h a v e e n h a n c e d o r d e c r e a s e d s u r v i v a l ( H a h n & L i t t l e 1 9 7 2 ) . I f t h e c e l l s a r e e x p o s e d t o h i g h t e m p e r a t u r e s f o r a p e r i o d o f t i m e b e f o r e p l a t i n g , s u r v i v a l w i l l be l o w e r ( B e n - H u r , E l k i n d & B r o n k 1 9 7 4 ; D e w e y , S a p a r e t o £ B e t t e n 1 9 7 8 ) . O t h e r p o s t - t r e a t m e n t s h a v e a l s o b e e n r e p o r t e d t o d e c r e a s e s u r v i v a l . T h e s e i n c l u d e ; p o s t - i r r a d i a t i o n i n c u b a t i o n f o r some m i n u t e s i n a n i s o t o n i c s o l u t i o n s ( D e t t o r e t a l . 1 9 7 2 ; R a a p h o r s t S Dewey 1 9 7 9 a , b ; P o h l i t & H e y d e r 1 9 8 1 ) , i n c u b a t i o n i n m e d i u m c o n t a i n i n g A R A - A ( I l i a k i s 1 9 8 0 ) , i n c u b a t i o n i n m e d i u m c o n t a i n i n g D^O ( B e n - H u r , U t s u m i S E l k i n d 1 9 8 0 ) , I n c u b a t i o n i n m e d i u m c o n t a i n i n g d i m e t h y l s u l p h o x i d e (DMSO) ( R a a p h o r s t & A z z a m 1 9 8 1 ) a s w e l l a s o t h e r s . T h e s e p o s t - t r e a t m e n t s a l l t a k e p l a c e l o n g a f t e r t h e p h y s i c a l a n d c h e m i c a l e f f e c t s o f t h e r a d i a t i o n h a v e t a k e n p l a c e . F u r t h e r m o r e , t h e y h a v e l i t t l e o r no e f f e c t o n t h e p l a t i n g e f f i c i e n c y o f u n i r r a d i a t e d c e l l s . I t i s f o r t h e s e 11 r e a s o n s t h a t t h e r e s u l t s h a v e b e e n i n t e r p r e t e d a s b e i n g m a i n l y a n e f f e c t o n t h e r e p a i r o f r a d i a t i o n i n d u c e d d a m a g e t h a t i s p o t e n t i a l l y l e t h a l . P o t e n t i a l l y l e t h a l damage ( P L D ) i s a n o p e r a t i o n a l t e r m a t t h e c e l l u l a r l e v e l a n d i s d e f i n e d s o l e l y i n t e r m s o f t h e m e t h o d s u s e d i n u n c o v e r i n g i t s e f f e c t s . T h e m a i n r e a s o n f o r t h i s i s t h a t t h e c r i t i c a l l e s i o n ( s ) i n v o l v e d h a v e n o t a s y e t b e e n i d e n t i f i e d . Few p r o p o s a l s h a v e b e e n p u t f o r w a r d a s t o t h e c o n n e c t i o n b e t w e e n PLD m e a s u r e d a t t h e c e l l u l a r l e v e l a n d m o l e c u l a r d a m a g e . C h a d w i c k a n d L e e n h o u t s ( 1 9 7 3 ) f o r i n s t a n c e h a v e p r o p o s e d t h a t . PLD i s a DNA d o u b l e - s t r a n d b r e a k . B e n - H u r , U t s u m i S E l k i n d ( 1 9 8 0 ) , w h i l e r e c o g n i z i n g t h e i m p o r t a n c e o f DNA h a v e p r o p o s e d t h a t s i t e s w h e r e DNA i s a s s o c i a t e d w i t h t h e n u c l e a r e n v e l o p e a r e r e s p o n s i b l e ; h o w e v e r , c o n c l u s i v e p r o o f r e m a i n s t o be f o u n d . The e f f e c t s on DNA a n d i t s r e p a i r o f some a g e n t s known t o e x p r e s s PLD h a v e a l r e a d y b e e n s t u d i e d . Among t h e s e a r e : A R A - C ( H i s s S P r e s t o n 1 9 7 7 ; Dunn S R e g a n 1 9 7 9 ; W a r d , B l a k e l y S J o i n e r 1 9 8 2 ) , h y p e r t h e r m i a ( C o r r y S R o b i n s o n 1 9 7 7 ) , c a f f e i n e ( L e h m a n n 1 9 7 2 ) , A c t i n o m y c i n - D ( L e e , B e n n e t S B y f i e l d 1 9 7 2 ) , h y p e r t o n i c p o s t - t r e a t m e n t ( B e n - H u r , U t s u m i S E l k i n d 1 9 8 0 ) a n d o t h e r s ( K a p l a n 1 9 7 2 ; W a r d , B l a k e l y & J o i n e r 1 9 8 2 ) . In t h i s t h e s i s , we r e p o r t e x t e n s i o n s o f t h e s e s t u d i e s u s i n g o u r a s s a y t o i n c l u d e t h e e f f e c t s o f A R A - A , DMSO a n d h y p e r t o n i c p o s t - t r e a t m e n t a f t e r r e l a t i v e l y l o w d o s e s o f r a d i a t i o n ( 6 G y ) . We h a v e a l s o m e a s u r e d t h e r e p a i r k i n e t i c s o f c e l l s g r o w n t o 12 p l a t e a u - p h a s e i n o r d e r t o c o m p a r e t h e m w i t h n o r m a l l y c y c l i n g c e l l s . 13 2 . M a t e r i a l s a n d M e t h o d s : 2 . 1 C e l l g r o w t h a n d m a i n t e n a n c e : C h i n e s e h a m s t e r f i b r o b l a s t s o f t h e C H 2 B 2 l i n e , a c l o n a l d e r i v a t i v e o f t h e C H E F - 1 2 5 1 i n e ( P r e s c o t t S B e n d e r 1 9 6 3 ) w e r e u s e d t h r o u g h o u t . T h e s e c e l l s h a v e 22 c h r o m o s o m e s p e r d i p l o i d c o m p l e m e n t a n d a b o u t 7 p l c o g r a m s o f DNA p e r c e l l ( M c B r i d e S 1 2 P e t e r s e n 1 9 7 0 ) . T h i s t r a n s l a t e s t o a p p r o x i m a t e l y 4 . 2 x 10 d a l t o n s o f D N A / c e l l . T h e i r d o u b l i n g t i m e i n g r o w t h m e d i u m i s b e t w e e n 12 a n d 13 h o u r s . The c e l l s w e r e g r o w n a s m o n o l a y e r c u l t u r e s i n p l a s t i c 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 s ) w i t h m i n i m a l e s s e n t i a l m e d i u m F - 1 6 ( G r a n d I s l a n d B i o l o g i c a l C o . , G r a n d I s l a n d , N . Y . ) s u p p l e m e n t e d w i t h 10% f e t a l c a l f s e r u m ( G i b c o ) a n d s o d i u m b i c a r b o n a t e b u f f e r . G r o w t h w a s a t 37°C i n a 5% C O ^ , 95% a i r a t m o s p h e r e t o m a i n t a i n p r o p e r p H . R e s e e d l n g o f t h e c u l t u r e s w a s p e r f o r m e d e v e r y 3 - 4 d a y s by e x p o s u r e t o 0 . 1 % t r y p s i n ( D i f c o L a b o r a t o r i e s , D e t r o i t , M I . ) f o r 8 m i n u t e s a t r o o m t e m p e r a t u r e . The s u s p e n s i o n w a s c o l l e c t e d , s p u n down ( 6 0 0 r p m , 6 m i n ) a n d r e s u s p e n d e d i n g r o w t h m e d i u m . C e l l c o u n t i n g w a s d o n e In a C o u l t e r c o u n t e r ( C o u l t e r E l e c t r o n i c s , H i a l e a h , F l o r i d a ) . 14 2 . 2 DNA 1 a b e l l i n g : R a d i o a c t i v e t h y m i d i n e w a s i n c o r p o r a t e d i n t o t h e DNA o f t h e c e l l s by e x p o s i n g 1 - d a y o l d c u l t u r e s t o e i t h e r C^H] 14 t h y m i d i n e ( 4 7 C i / m m o l e , A m e r s h a m a t 0 . 1 u C i / m l ) o r [ C ] t h y m i d i n e ( 5 9 C i / m o l e , A m e r s h a m a t 0 . 0 2 u C I / m l ) s u p p l e m e n t e d w i t h 2 x 1 0 ~ ^ M u n l a b e l l e d t h y m i d i n e f o r 18 t o 24 h o u r s p r i o r t o t r y p s i n i z a t i o n . One h o u r b e f o r e h a r v e s t i n g , t h e c e l l p o o l w a s d e p l e t e d o f r a d i a o a c t i v e p r e c u r s o r s by i n c u b a t i n g c e l l s i n n o n - r a d i o a c t i v e m e d i u m . The r a t i o o f l a b e l l e d t o u n l a b e l l e d t h y m i d i n e w a s a p p r o x i m a t e l y 1 , 0 0 0 t o 1 t h u s a s s u r i n g a u n i f o r m d i s t r i b u t i o n o f t h e l a b e l l e d n u c l e o t i d e s t h r o u g h o u t t h e DNA. D e p l e t i o n o f r a d i o a c t i v i t y f r o m t h e m e d i u m o v e r t h e c o u r s e o f t h e l a b e l l i n g p e r i o d w a s , f o r b o t h l a b e l s b e t w e e n 30 a n d 4 0 % . 2 . 3 C e l l t r e a t m e n t s : 2 . 3 . 1 O x i c i r r a d i a t i o n s : C e l l s w e r e i r r a d i a t e d a s s i n g l e c e l l s u s p e n s i o n s i n 12 x 75 mm p l a s t i c t u b e s ( F a l c o n P l a s t i c s ) , 0 . 5 ml t o t a l v o l u m e p e r t u b e a t a c o n c e n t r a t i o n o f 1 x 10 / m l . The t u b e s w e r e s u p p o r t e d by a p l a s t i c h o l d e r i m m e r s e d i n i c e - w a t e r w h i c h c o u l d h o l d up t o 20 t u b e s a t a t i m e . I r r a d i a t i o n w a s p e r f o r m e d w i t h x - r a y s ( P i c k e r , 280 k V p , HVL 1.7mm C u , 1 . 7 3 G y / m i n ) . T h i s p r o c e d u r e w i l l be r e f e r r e d t o a s t h e ' s t a n d a r d a e r o b i c p r o c e d u r e ' . 2 . 3 . 2 A n o x i c i r r a d i a t i o n s : The s t a n d a r d p r o c e d u r e f o r i r r a d i a t i n g c e l l s i n t h e a b s e n c e o f o x y g e n w a s a s f o l l o w s . The c e l l s u s p e n s i o n w a s p l a c e d i n a s p e c i a l l y d e s i g n e d g l a s s 15 i r r a d i a t i o n v e s s e l ('type I I ' ) w i t h a f l a t bottom and a l o n g neck f o r a d d i n g o r removing samples. From a n o t h e r o p e n i n g , gas c o n t a i n i n g l e s s t h a n 5 ppm 0^ was a l l o w e d t o e n t e r and pass o v e r the s u s p e n s i o n w h i c h was s t i r r e d by a m a g n e t i c s t i r r i n g rod ( P a r k e r , S k a r s g a r d S Emmerson 1969) . The purpose of the g a s s i n g was t o purge any r e m a i n i n g 0^ and m a i n t a i n a p o s i t i v e p r e s s u r e t o p r e v e n t 0^ from e n t e r i n g . P u r g i n g i n t h i s manner was m a i n t a i n e d f o r a t l e a s t 45 m i n u t e s p r i o r t o i r r a d i a t i o n . Throughout t h i s p e r i o d and d u r i n g t h e a c t u a l i r r a d i a t i o n p e r i o d i t s e l f , the i r r a d i a t i o n v e s s e l was immersed i n an i c e - w a t e r m i x t u r e . I r r a d i a t i o n was w i t h the same x - r a y s o u r c e as above e x c e p t the dose r a t e was 1.61 Gy/min. There i s some q u e s t i o n as t o whether or not t h e p r o c e d u r e d e s c r i b e d above i s adequate f o r removing a l l the oxygen p r e s e n t i n t h e medium. T h e r e f o r e , the f o l l o w i n g p r o c e d u r e was a t t e m p t e d . Here, t h e medium minus the c e l l s t o be t r e a t e d was gassed as above f o r 45 m i n u t e s and then g i v e n a dose o f 16 Gy t o remove any r e s i d u a l 0^ • The c e l l s , w h i c h had been c o n c e n t r a t e d t o 1 x lO^/ml i n t u b e s were gassed w i t h f o r 10 m i n u t e s and a t 37°C so t h a t they c o u l d metabo1ica11y d e p l e t e the r e m a i n i n g 0^ i n the t u b e . These c e l l s were t h e n t r a n s f e r r e d t o the i r r a d i a t i o n v e s s e l s by a p a s t e u r p i p e t t e and were then s u b j e c t e d t o an a d d i t i o n a l 45 m i n u t e s o f p u r g i n g . I r r a d i a t i o n t h e n proceeded as above. 2.4 I n c u b a t i o n c o n d i t i o n s : The m a j o r i t y o f the measurements t h a t w i l l be d e s c r i b e d below were performed u s i n g an ' i n t e r n a l c o n t r o l ' . Here, a f t e r 1 D c e l l t r e a t m e n t , a n e q u a l n u m b e r o f c e l l s d i f f e r e n t l y l a b e l l e d 3 14 CC H ] t h y m i d i n e v s . [ C ] t h y m i d i n e ) w h i c h , e x c e p t f o r t h e i r r a d i a t i o n , h a d b e e n s u b j e c t e d t o i d e n t i c a l c o n d i t i o n s , w e r e p o o l e d w i t h t h e t r e a t e d c e l l s . An i d e n t i c a l e x p e r i m e n t w i t h t h e l a b e l s s w i t c h e d a r o u n d w a s a l w a y s p e r f o r m e d c o n c u r r e n t l y . I n c u b a t i o n f o r r e p a i r w a s i n i t i a t e d by d i l u t i n g t h e c e l l s u s p e n s i o n s a p p r o x i m a t e l y t e n t i m e s w i t h warm m e d i u m a t 3 7 ° C . T h e c e l l s w e r e k e p t i n s m a l l E r l e n m e y e r f l a s k s w i t h m a g n e t i c s p i n n e r r o d s i n a n i n c u b a t o r t o m a i n t a i n t h e t e m p e r a t u r e . S i n c e f o r t h e m o s t p a r t , i n c u b a t i o n w a s no m o r e t h a n 2 h o u r s , CO^ f l o w t o m a i n t a i n pH w a s n o t n e c e s s a r y . I n c u b a t i o n w a s t e r m i n a t e d by r e m o v i n g a l i q u o t s o f a p p r o x i m a t e l y 5 x 10^ c e l l s f r o m t h e f l a s k s a n d v i g o r o u s l y i n j e c t i n g t h e m i n t o e i t h e r i c e - c o l d m e d i u m o r i c e - c o l d PBS t o 4 t i m e s t h e i n i t i a l v o l u m e . T h e c e l l s w e r e t h e n k e p t o n I c e u n t i l r e a d y f o r a s s a y i n g . 2 . 4 . 1 H y p e r t o n i c p o s t - t r e a t m e n t : A f t e r t r y p s i n i z a t i o n , c e l l s w e r e r e s u s p e n d e d i n i c e - c o l d P B S . I r r a d i a t i o n w a s p e r f o r m e d a s p e r t h e s t a n d a r d a e r o b i c p r o c e d u r e i n c l u d i n g p o o l i n g w i t h u n i r r a d i a t e d c o n t r o l c e l l s . A f t e r r e m o v i n g a p o r t i o n o f t h e c e l l s f o r a s s a y i n g I n i t i a l d a m a g e , t h e s u s p e n s i o n w a s s p u n d o w n , t h e PBS w a s r e p l a c e d w i t h 1 ml o f h y p e r t o n i c s o l u t i o n ( 0 . 5 M N a C l i n P B S , pH 7 . 1 ) a n d t h e s a m p l e s w e r e i n c u b a t e d a t 3 7 ° C . A f t e r 20 m i n u t e s , 10 ml o f c o l d PBS w a s a d d e d t o r e t u r n t h e c e l l s t o i s o t o n i c i t y . C e l l s . w h i c h a r e n o t g i v e n t i m e t o r e a d j u s t t o t h e s a l t c o n c e n t r a t i o n c h a n g e s h a v e b e e n s h o w n t o h a v e l o w e r p l a t i n g e f f i c i e n c i e s ( K o r b e l i k , P a l c i c & S k a r s g a r d i n p r e p a r a t i o n ) . A f t e r a f i v e m i n u t e 17 ad j u s t m e n t p e r i o d , t he c e l l s were spun down and resuspended In c o l d growth medium at a c o n c e n t r a t i o n o f 1 x 10^/ml from w h i c h a n o t h e r sample was t a k e n f o r a s s a y i n g . I n c u b a t i o n f o r t h e r e m a i n i n g c e l l s was i n i t i a t e d by d i l u t i n g 10 t i m e s a t 37°C and t e r m i n a t e d by f u r t h e r d i l u t i n g 4 t i m e s i n e i t h e r i c e - c o l d medium o r i c e - c o l d PBS. 2.4.2 ARA-A: C e l l s were i r r a d i a t e d as per s t a n d a r d a e r o b i c p r o c e d u r e i n growth medium minus sodium b i c a r b o n a t e b u f f e r . P o o l i n g w i t h u n i r r a d i a t e d c e l l s was a l s o as above. A f t e r removing an a l i q u o t o f t h e c e l l s f o r a s s a y i n g i n i t i a l damage, the c e l l s were d i l u t e d t o a c o n c e n t r a t i o n o f 1 x 10^/ml i n warm medium at 37°C c o n t a i n i n g 0.5mM ARA-A. As above, i n c u b a t i o n was t e r m i n a t e d by d i l u t i n g t h e samples 4 tim e s w i t h i c e - c o l d PBS. 2.4.3 DMSO: C e l l s were i r r a d i a t e d as per s t a n d a r d a e r o b i c p r o c e d u r e i n medium minus sodium b i c a r b o n a t e b u f f e r . P o o l i n g w i t h u n i r r a d i a t e d c o n t r o l s and the removal o f an a l i q u o t f o r a s s a y i n g i n i t i a l damage was a l s o as above. 10.7% (.1.5M) DMSO was added t o t h e r e m a i n i n g c e l l s at 0°C. A f t e r 15 m i n u t e s , c o l d medium was added, t h e s u s p e n s i o n was spun down, resuspended i n growth medium, a n o t h e r sample was removed f o r a s s a y i n g and i n c u b a t i o n was i n i t i a t e d and t e r m i n a t e d as above. 2.4.4 P l a t e a u - p h a s e c e l l s : P l a t e a u - p h a s e c e l l s were p r e p a r e d by s e e d i n g 1 x 10^ c e l l s i n 60 x 15 mm p l a s t i c p e t r i d i s h e s ( F a l c o n P l a s t i c s ) w i t h 5 ml o f growth medium. A f t e r 1 day o f i n c u b a t i o n , t he medium was a s p i r a t e d o f f and r e p l a c e d 18 Incubation Time (Days) F i g u r e 1. G r o w t h c u r v e - CH2B2 c e l l s i n P e t r i d i s h e s . 1 x 10 c e l l s w e r e s e e d e d i n P e t r i d i s h e s , f e d w i t h l a b e l l e d m e d i u m o n t h e f i r s t t w o d a y s a n d u n l a b e l l e d m e d i u m o n s u b s e q u e n t d a y s . C e l l s w e r e s c r a p e d o f f t h e s u r f a c e o f t h e d i s h w i t h a r u b b e r p o l i c e m a n , d i l u t e d a n d c o u n t e d i n a C o u l t e r c o u n t e r . 19 w i t h t h e l a b e l l e d m e d i u m d e s c r i b e d a b o v e . On t h e f o l l o w i n g d a y , t h e l a b e l l e d m e d i u m w a s r e p l a c e d w i t h m o r e l a b e l l e d m e d i u m w h i l e o n s u b s e q u e n t d a y s , u n l a b e l l e d m e d i u m w a s u s e d . F r e s h m e d i u m w a s a p p l i e d d a i l y . The g r o w t h c u r v e i n f i g u r e 1 s h o w s t h e l e v e l l i n g o f f t h a t o c c u r s a f t e r 3 d a y s u n d e r t h e s e c o n d i t i o n s . T h e c e l l s t h u s p r e p a r e d w e r e i r r a d i a t e d ( d o s e r a t e 1 . 5 8 G y / m i n , 3 . 6 5 Gy t o t a l d o s e ) o n i c e w i t h 5 ml o f i c e - c o l d P B S . I n c u b a t i o n w a s i n i t i a t e d by a s p i r a t i n g o f f t h e c o l d PBS a n d r e p l a c i n g w i t h 5 ml o f w a r m g r o w t h m e d i u m . I n c u b a t i o n w a s t e r m i n a t e d by r e p l a c i n g t h e w a r m m e d i u m w i t h c o l d PBS a n d p l a c i n g t h e p e t r i d i s h on a b e d o f i c e . C e l l s w e r e h a r v e s t e d by s c r a p i n g t h e m o f f t h e s u r f a c e o f t h e d i s h w i t h a r u b b e r p o l i c e m a n . C o n t r o l c e l l s d i f f e r e n t l y l a b e l l e d b u t w h i c h w e r e a l s o m a i n t a i n e d i n p e t r i d i s h e s a n d s u b j e c t e d t o t h e same c o n d i t i o n s a n d p r o c e d u r e s e x c e p t f o r t h e i r r a d i a t i o n w e r e s i m i l a r l y c o l l e c t e d a n d p o o l e d w i t h t h e i r r a d i a t e d c e l l s . 2 . 5 U n w i n d i n g a s s a y : DNA d a m a g e w a s , f o r t h e m o s t p a r t , a s s a y e d , u s i n g t h e d o u b l e - l a b e l u n w i n d i n g m e t h o d o f R y d b e r g ( 1 9 8 0 ) . The p o o l e d I r r a d i a t e d a n d c o n t r o l c e l l s w e r e f i r s t r e s u s p e n d e d t o a c o n c e n t r a t i o n o f 1 x l O ^ / m l . 5 0 u 1 o f t h i s s u s p e n s i o n w a s i n j e c t e d i n t o 1 ml o f a m o d e r a t e a l k a l i n e , h i g h i o n i c s t r e n g t h u n w i n d i n g s o l u t i o n ( 0 . 0 3 M N a O H , 0 . 0 1 M N a 9 H P O a , 0 . 9 M N a C l , pH 20 1 2 . 0 5 , R y d b e r g s 1 s o l u t i o n ' B ' . R y d b e r g s ' s o l u t i o n ' A ' i s i d e n t i c a l e x c e p t i t d o e s n o t i n c l u d ' e t h e s a l t . ) a t 2 5 ° C , a l l o w e d t o u n w i n d f o r 20 m i n u t e s u n d e r m i n i m a l , n o n - f l u o r e s c e n t i l l u m i n a t i o n a n d t h e n n e u t r a l i z e d w i t h 1 ml o f 0 . 0 3 4 M H C 1 . The r e s u l t i n g s o l u t i o n w a s t h e n s o n i c a t e d ( B r a n s o n S o n i f i e r s e t t i n g 2 , 10 s e c . ) a n d 0 . 1 ml o f 8% S D S , a d e t e r g e n t , w a s a d d e d . The m i x t u r e w a s e i t h e r e l u t e d i m m e d i a t e l y o r s t o r e d u n d e r r e f r i g e r a t i o n f o r up t o 1 w e e k w i t h no e f f e c t o n t h e r e s u l t s . B e f o r e e l u t i n g , t h e c o l u m n s w e r e w a s h e d w i t h 1 . 5 ml e a c h o f f i n a l a n d s t a r t i n g b u f f e r . The s o n i c a t e w a s p l a c e d on c o l u m n s o f h y d r o x y 1 a p a t i t e ( B i o - R a d , R i c h m o n d C a l i f . , 2 . 5 g m / c o l u m n ) k e p t a t 60°C i n a w a t e r b a t h . 4 e l u t i o n s ( A = 0 . 0 1 M , B = 0 . 1 2 5 M , C = 0 . 2 5 M a n d D = 0 . 5 M K H 2 - K 2 H P 0 4 b u f f e r s , pH 6 . 8 5 ) w e r e c o l l e c t e d , t h e f i r s t t w o ( A + B ) c o n t a i n i n g s i n g l e - s t r a n d e d ( s s ) , t h e l a s t t w o (C + D) d o u b l e - s t r a n d e d ( d s ) DNA. 15 ml * o f s c i n t i l l a t i o n f l u i d 3 14 ( A C S I I , A m e r s h a m ) w a s a d d e d a n d t h e H a n d C a c t i v i t i e s i n e a c h v i a l w e r e d e t e r m i n e d by c o u n t i n g f o r 10 m i n u t e s / v i a l i n a B e c k m a n s c i n t i l l a t i o n c o u n t e r . The c o u n t i n g d a t a w a s c o l l e c t e d o n p a p e r t a p e a n d r u n t h r o u g h a c o m p u t e r w h e r e c o r r e c t i o n s w e r e made f o r b a c k g r o u n d ( 5 c p m i n t h e c h a n n e l 14 14 a n d 11 cpm i n t h e C c h a n n e l ) a n d t h e s p i l l o v e r o f C c o u n t s i n t o t h e c h a n n e l ( 2 0 . 4 % ) . The f r a c t i o n o f d o u b l e - s t r a n d e d DNA f o r b o t h c o n t r o l a n d t r e a t e d c e l l s w a s c a l c u l a t e d . The m a j o r i t y o f t h e m e a s u r e m e n t s p e r f o r m e d w e r e d o n e u s i n g t h e p r o c e d u r e d e s c r i b e d a b o v e . O t h e r e x p e r i m e n t s t h a t w e r e a l s o p e r f o r m e d i n c l u d e , u n w i n d i n g k i n e t i c s w h e r e t h e 21 e x t e n t o f u n w i n d i n g o f t h e DNA f r o m t r e a t e d c e l l s w a s f o l l o w e d o v e r t i m e a n d e x t e r n a l c o n t r o l e x p e r i m e n t s w h e r e i n s t e a d o f i n c l u d i n g c o n t r o l c e l l s i n t h e t u b e s , c o n t r o l s a m p l e s w e r e a s s a y e d s e p a r a t e l y . 2 . 6 U n w i n d i n g o f DNA i n a l k a l i : The t h e o r y b e h i n d t h e u n w i n d i n g o f DNA i n a l k a l i i s now d i s c u s s e d . The t r e a t m e n t h e r e i s e s s e n t i a l l y t h a t o f R y d b e r g CI 9 7 5 ) . C o n s i d e r a v e r y l o n g d u p l e x s t r a n d o f DNA In w h i c h s t r a n d b r e a k s h a v e b e e n i n d u c e d r a n d o m l y . H e r e , r a n d o m n e s s i m p l i e s t h a t e a c h b o n d h o l d i n g t w o n u c l e o t i d e s t o g e t h e r h a s a n e q u a l p r o b a b i l i t y o f b e i n g r u p t u r e d . S i n c e t h e i n d i v i d u a l p r o b a b i l i t y o f a r u p t u r e o c c u r i n g I s s m a l l , P o i s s o n s t a t i s t i c s c a n be a p p l i e d t o p r e d i c t t h e l e n g t h d i s t r i b u t i o n o f n u c l e o t i d e s b e t w e e n t w o a d j a c e n t s t r a n d b r e a k s C L e h m a n n S O r m e r o d 1 9 7 0 b ) . T h e r e s u l t I s , f C n ) = e " n / n 0 . C D W h e r e f C n ) = l e n g t h d i s t r i b u t i o n f u n c t i o n C u n n o r m a l i z e d ) n = n u m b e r o f n u c l e o t i d e s b e t w e e n t w o s t r a n d b r e a k s . n^ = n u m b e r a v e r a g e n u c l e o t i d e l e n g t h b e t w e e n s t r a n d s 22 rig = N/CB + 1 ) w h e r e N = t o t a l n u m b e r o f n u c 1 e o t i d e s B = n u m b e r o f b r e a k s i n d u c e d i n t h e s t r a n d . When t h e DNA i s u n w o u n d i n a l k a l i n e s o l u t i o n , i t i s a s s u m e d t h a t t h e \ r a t e - 1 i m i t i ng f a c t o r o f t h e p r o c e s s i s t h e f r i c t i o n o f t h e r o t a t i n g s i n g l e - s t r a n d e d p o r t i o n s ( L o n g u e t - H i g g i n s & Z imm 1 9 6 0 ; C r o t h e r s 1 9 6 4 ; D a v i s o n 1 9 6 6 ) . The d o u b l e - s t r a n d e d p o r t i o n s a r e s u p p o s e d n o t t o r o t a t e . I t h a s b e e n s h o w n ( L o n g u e t - H i g g i n s S Z imm I 9 6 0 ) t h a t o n e c a n e x p e c t t h e k i n e t i c s o f u n w i n d i n g t o f o l l o w a s i m p l e p o w e r l a w o f t h e f o r m , n t = K t 3 ( 2 ) w h e r e n = n u m b e r o f n u c l e o t i d e s u n w o u n d i n t i m e t . K = p r o p o r t i o n a l i t y c o n s t a n t r e l a t e d t o t h e i n i t i a l m o l e c u l a r w e i g h t o f t h e f r a g m e n t s . 6 = a c o n s t a n t ( o n t h e o r d e r o f 0 . 5 ) . S i n c e t h i s a p p l i e s t o e v e r y f r a g m e n t b e t w e e n t w o s t r a n d b r e a k s , a t t i m e t , e a c h f r a g m e n t o f l e n g t h n w i l l h a v e u n w o u n d e I t h e r , 1 ) c o m p l e t e l y I f n < n . 2 ) up t o n i f n > n , T h u s o n e c a l c u l a t e s t h a t t h e t o t a l a m o u n t o f d o u b l e - s t r a n d e d DNA r e m a i n i n g a f t e r t i m e t I s , 23 t w h i c h , u p o n c a l c u l a t i o n ( m a k i n g t h e a p p r o x i m a t i o n t h a t b e c o m e s , F i g u r e 2 . L e n g t h d i s t r i b u t i o n o f f r a g m e n t l e n g t h s f o r r a n d o m l y I n d u c e d s t r a n d b r e a k s . 24 n Q 2 e n t / n Q ( 4 ) 2 The t o t a l l e n g t h ( i . e . n o r m a l i z a t i o n c o n s t a n t ) i s n^ s o t h a t t h e f r a c t i o n o f d o u b l e - s t r a n d e d t o t o t a l DNA ( F ) i s , F. = e " n t / n 0 . ( 5 ) S u b s t i t u t i n g f o r n a n d t a k i n g n a t u r a l l o g a r i t h m s o f b o t h s i d e s g i v e s , - l n ( F ) = K t 3 ( B + 1 ) / N . ( 6 ) So t h a t a p l o t o f - l n ( F ) v s . t o n a l o g - l o g s c a l e w i l l g i v e 3 a s i t s s l o p e a n d l o g [ K ( B + 1 ) / N ] a s I t s i n t e r c e p t . I f no s t r a n d b r e a k s h a v e b e e n i n t r o d u c e d ( s u c h a s i n c o n t r o l s a m p l e s , ) t h e n , - l n ( F ) c o n t r o l =• K t 8 / N • C 7 ) a n d d i v i d i n g e q u a t i o n ( 6 ) by e q u a t i o n ( 7 ) g i v e s , > 1 n ^ ^ t r e a t e d - l n ( F ) . . c o n t ro1 = B + 1 ( 8 ) 25 By comparing the t r e a t e d F t o t h e c o n t r o l F i n t h i s way, one g e t s an e s t i m a t e o f t h e number o f b r e a k s induced i n t h e s t r a n d , and t h i s i s the essence o f the e x t e r n a l c o n t r o l method. I t s h o u l d be emphasized t h a t the c o n t r o l fragment l e n g t h s cannot be measured by t h i s t e c h n i q u e t h u s one cannot get an a b s o l u t e measure o f t h e number o f b r e a k s s a y, per e e l 1 . More on t h i s w i l l be d i s c u s s e d i n S e c t i o n 3.2. 2.7 D o u b l e - l a b e l u n w i n d i n g method: The e x t e r n a l c o n t r o l method o f e s t i m a t i n g the number o f b r e a k s i n t h e s t r a n d i s s u i t a b l e f o r measurements where h i g h s e n s i t i v i t y i s not n e c e s s a r y . Because s l i g h t v a r i a t i o n s from sample t o sample i n e x p e r i m e n t a l c o n d i t i o n s o c c u r , t h e i n t e r n a l c o n t r o l method or d o u b l e - l a b e l method has been d e v e l o p e d (Rydberg 1980). Here, one i n c l u d e s c e l l s w h i c h a r e l a b e l l e d d i f f e r e n t l y from the t r e a t e d c e l l s and wh i c h a c t as c o n t r o l s t o t a k e account o f the v a r i a t i o n s . By a v e r a g i n g w i t h an i d e n t i c a l e x p e r i m e n t w i t h t h e l a b e l s s w i t c h e d around, one g e t s an a p p r o x i m a t e l y f i v e t i m e s more s e n s i t i v e measure o f the 3 14 number o f b r e a k s . In our c a s e , we have used H and C so t h a t , B = 1/2 K [ B 3 H t r e a t e d C t r e a t e d H c o n t r o l - 14 c o n t r o l ( 9 ) ] . 26 Where each o f the B v a l u e s have been c a l c u l a t e d from t h e i r r e s p e c t i v e F's. A g a i n , t h e c o n s t a n t K, w h i c h i s r e l a t e d t o the i n i t i a l m o l e c u l a r w e i g h t o f • t h e c o n t r o l f r a g m e n t s cannot be a b s o l u t e l y d e t e r m i n e d by t h i s t e c h n i q u e , t h u s t h e break v a l u e s measured here a r e r e l a t i v e . For t h e most p a r t , the d a t a r e p o r t e d below have been e i t h e r n o r m a l i z e d t o l",bYeak/Gy or have been r e p o r t e d as a p e r c e n t a g e o f the i n i t i a l damage. 27 3. Resu1ts : 3.1 E l u t i o n p r o f i l e s and u n w i n d i n g k i n e t i c s : The a b i l i t y o f h y d r o x y 1 a p a t i t e t o d i f f e r e n t i a l l y b i n d s i n g l e - s t r a n d e d and d o u b l e - s t r a n d e d DNA so t h a t they can be washed out s e p a r a t e l y i s w e l l c h a r a c t e r i z e d ( M a r t i n s o n 1973; M a r t i n s o n S Wagenaar 1974). To d e m o n s t r a t e t h i s p r o p e r t y , e l u t i o n p r o f i l e s were measured f o r DNA unwound i n a l k a l i f o r v a r i o u s t i m e s a f t e r I r r a d i a t i o n and t o d i f f e r e n t doses ( f i g u r e s 3a,b and c ) . I t i s c l e a r from t h e s e graphs t h a t t h e two peaks are w e l l s e p a r a t e d and t h a t by u s i n g our b u f f e r s , t h e s i n g l e - s t r a n d e d and d o u b l e - s t r a n d e d DNA can be washed out s e p a r a t e l y . I t i s a l s o c l e a r t h a t w i t h l o n g e r u n w i n d i n g t i m e s , DNA i s b e i n g c o n v e r t e d from th e d o u b l e - s t r a n d e d ( s e c o n d peak) t o the s i n g l e - s t r a n d e d ( f i r s t peak) form. A l s o , w i t h i n c r e a s i n g r a d i a t i o n , the f i r s t peak grows at the expense of t h e second I n d i c a t i n g f a s t e r u n w i n d i n g r a t e s and t h u s more b r e a k s . When the n e g a t i v e l o g a r i t h m o f t h e f r a c t i o n o f d o u b l e - s t r a n d e d DNA t o t o t a l [ - l n ( F ) ] f o r t h i s and o t h e r d a t a i s c a l c u l a t e d and p l o t t e d v e r s u s t ime ( t ) i n a l o g - l o g p l o t , s t r a i g h t 1 i n e s a r e o b t a i n e d w i t h s l o p e s t h a t v a r y from day t o 28 F i g u r e s 3 a , b S c. E l u t i o n Prof i 1 e s . DNA from c e l l s which were i r r a d i a t e d ^and then unwound f o r v a r i o u s times were place d on columns of h y d r o x y 1 a p a t i t e and washed w i t h b u f f e r s of i n c r e a s i n g m o l a r i t y . The f r a c t i o n s were c o l l e c t e d , counted and p l o t t e d as a percentage of the t o t a l recovered counts. The f i r s t peak Is the e l u t i o n of s i n g l e - s t r a n d e d DNA, the second peak i s the e l u t i o n of double-stranded DNA. 0^ A O/P 0.2 B C Buffer Molarity 0!5 D 29 day between 0.45 and 0.65. The goodness o f f i t o f the d a t a however, i s r e l a t i v e l y i n s e n s i t i v e t o the s l o p e v a l u e s so t h a t f o r c a l c u l a t i n g break v a l u e s , an average v a l u e o f 0.6 was us u a l 1y assumed. P l o t t e d i n f i g u r e 4 a r e t h e u n w i n d i n g k i n e t i c s f o r 8 v a r i o u s d o s e s . Here, F i s p l o t t e d as a f u n c t i o n o f ( t / 1 0 ) (where t i s i n m i n u t e s ) on a s e m i - l o g s c a l e . The f i t i s good f o r most c a s e s and as can be seen, the s l o p e o f the l i n e d e c r e a s e s w i t h i n c r e a s i n g dose. On some o f the u n w i n d i n g k i n e t i c s f o r c o n t r o l DNA, a downward bending o f the c u r v e was o b s e r v e d a f t e r l o n g e r u n w i n d i n g t i m e s . The d a t a f o r t h i s c u r v a t u r e i s l i m i t e d but i f t h i s was a r e a l e f f e c t , t h e n two reas o n s may acco u n t f o r i t . F i r s t , i f when the DNA i s f i r s t b r o k e n down t o i t s c o n t r o l fragment s i z e , t h e d i s t r i b u t i o n i s not random but m o n o d i s p e r s e , t h e n , f ( n ) = 6(n - N), ( 1 0 ) the d e l t a f u n c t i o n and, l n ( F ) = l n ( l - n t / N ) . ( 1 1 ) expa n d i n g e q u a t i o n ( 1 1 ) , one g e t s ; l n C F ) = " n t / N " 1 / 2 C n t / N ) 2 " 1 / 3 C n t / N ) 3 ( 1 2 ) 30 0.4| I I L_ 1.0 2.0 3.0 F F i g u r e 4. Unwinding K i n e t i c s . C e l l s i r r a d i a t e d to v a r i o u s doses were unwound In s o l u t i o n 'B' f o r v a r i o u s times. Unwinding was termi n a t e d by n e u t r a l i z i n g w i t h HC1. F, the f r a c t i o n of doub1e-stranded-to t o t a l DNA i s p l o t t e d a g a i n s t ( t / 1 0 ) where t i s in minutes and the exponent used was 0.6. The s c a l e i s semi-1ogarIthmic. 31 w h i c h says t h a t t o a f i r s t a p p r o x i m a t i o n , the m o n o d i s p e r s e d i s t r i b u t i o n f o l l o w s t h a t o f a random one but a f t e r l o n g e r u n w i n d i n g t i m e s , t h e l i n e a r i t y b r e a k s down and the c u r v e s h o u l d bend downwards. I t s h o u l d be added t h a t such c u r v a t u r e would not be e x p e c t e d from i r r a d i a t e d samples because i t has been shown ( C h a r l e s b y 1954) t h a t r e g a r d l e s s o f t h e i n i t i a l d i s t r i b u t i o n , a f t e r one i n t r o d u c e s a- m i n i m a l number of b r e a k s i n t o the s t r a n d , the l e n g t h d i s t r i b u t i o n r e t u r n s t o t h e random one. I t was assumed f o r the p urposes of c a l c u l a t i n g F ( e q u a t i o n ( 4 ) ) t h a t t h e l e n g t h o f the c o n t r o l f r a g m e n t s was i n f i n i t e l y l o n g . I f t h i s a s s u m p t i o n i s not made, one can a g a i n show t h a t t h e e f f e c t w i l l be t o i n t r o d u c e a s l i g h t downward c u r v a t u r e o f t h e u n w i n d i n g c u r v e . Thus the c o m b i n a t i o n o f t h e s e two e f f e c t s might e x p l a i n t h e downward c u r v a t u r e o f the c o n t r o l f r a g m e n t s . A l s o , t h e second e f f e c t becomes a p p a r e n t o n l y a f t e r l o n g e r u n w i n d i n g t i m e s ( a b o u t 60 m i n u t e s o r where F f a l l s t o about 30%) so i t would not a f f e c t the 20 m i n u t e e n d p o i n t used i n the d o u b l e - l a b e l e x p e r i m e n t s . The n o n - l i n e a r i t y at low v a l u e s o f F does however put an upper l i m i t on t h e doses one c o u l d use w i t h s o l u t i o n 'B 1. Thus f o r a l l p r a c t i c a l p u r p o s e s , o n l y doses lower than 7 o r 8 Gy c o u l d be used. S o l u t i o n 'A' ( w i t h o u t s a l t ) can be used f o r h i g h e r doses but t h i s w i l l be d i s c u s s e d below. 32 Dose (Gy) ' F i g u r e 5. Dose response - e x t e r n a l c o n t r o l C e l l s were i r r a d i a t e d , unwound In s o l u t i o n 'B' f o r 20 minutes and the r a t i o of the l o g a r i t h m s are p l o t t e d compared to the u n i r r a d i a t e d ( c o n t r o l ) sample. The slope of t h i s l i n e Is 0.81 breaks/Gy. 33 3.2 Dose response - e x t e r n a l c o n t r o l and e v / b r e a k : P l o t t e d i n f i g u r e 5 i s the r a t i o o f l o g a r i t h m s as per e q u a t i o n ( 8 ) as a f u n c t i o n o f dose. The r e l a t i o n s h i p i s l i n e a r w i t h an i n t e r c e p t o f 1 as i s p r e d i c t e d . On any g i v e n day, t h e v a r i a t i o n i n c o n t r o l v a l u e s f o r F from p o i n t t o p o i n t was r a r e l y g r e a t e r t h a n 5%. Average F c o n t r o l v a l u e s v a r i e d from day t o day between 75 and 90% a f t e r 20 m i n u t e s o f u n w i n d i n g . T h i s v a r i a t i o n was most l i k e l y due t o s m a l l changes i n t h e pH o f t h e u n w i n d i n g s o l u t i o n , changes i n t e m p e r a t u r e o f the w a t e r bath o r v a r i a t i o n s i n t h e s t a t e o f t h e c e l l s a l t h o u g h c a r e was t a k e n t o c o n t r o l t h e s e and o t h e r f a c t o r s as much as p o s s i b l e . As mentioned above, the break v a l u e s measured here a r e r e l a t i v e . For t h e purpose o f c o m p a r i s o n however, i t i s s t i l l o f i n t e r e s t t o c a l c u l a t e how much energy must be absorbed on 4 average t o produce a break In t h e DNA. The s l o p e o f t h i s 1 i n e - 8 i s 0.81 breaks/Gy w h i c h t r a n s l a t e s t o about 5 x 10 e v / b r e a k / d a l t o n . There i s much e v i d e n c e ( L e t t , K 1 u c i s S Sun 1970; E l k i n d S Kamper 1970; P a l c i c S S k a r s g a r d 1972) t h a t s u g g e s t s t h a t under a l k a l i n e c o n d i t i o n s , chromosomal DNA 8 d i s s o c i a t e s I n t o s u b u n i t s o f between 5 - 10 x 10 d a l t o n s . I f we assume such a range f o r our a s s a y , then our breakage e f f i c i e n c y number i s 25 - 50 e v / b r e a k . T h i s range i s not out of l i n e w i t h o t h e r measurements ( P a l c i c S S k a r s g a r d 1972; Kohn et a l . 1976) however two d i f f e r e n c e s e x i s t . The f i r s t i s t h a t f o r the most p a r t , the degree o f a l k a l i n e t r e a t m e n t f o r a l k a l i n e s u c r o s e g r a d i e n t s (ASG) i s much more e x t e n s i v e 34 l a s t i n g o f t e n on the o r d e r o f s e v e r a l h o u r s . Under such t r e a t m e n t , i t would be e x p e c t e d t h a t more d e g r a d a t i o n would t a k e p l a c e . The second d i f f e r e n c e i s t h a t our u n w i n d i n g s o l u t i o n i n c l u d e s s a l t and we have some e v i d e n c e t h a t s a l t not o n l y c a u s e s b r e a k s on i t s own but a l s o u n c o v e r s b r e a k s t h a t a r e not o t h e r w i s e u n c o v e r e d w i t h j u s t a l k a l i . F i g u r e 6 Is a p l o t o f the number o f b r e a k s measured a f t e r 6 Gy and 20 m i n u t e s u n w i n d i n g p l o t t e d a g a i n s t the s a l t c o n c e n t r a t i o n o f t h e u n w i n d i n g s o l u t i o n . W h i l e the f r a c t i o n o f d o u b l e - s t r a n d e d DNA i n the c o n t r o l samples went from about 87% t o 83% o v e r t h e s a l t range, the c o r r e s p o n d i n g I r r a d i a t e d samples went from 82% down t o 51%. T h i s i n d i c a t e s t h a t more damage r e s u l t i n g from t h e r a d i a t i o n i s b e i n g e x p r e s s e d when s a l t i s i n c l u d e d t h a n when i t i s n o t . I t i s w e l l known t h a t some forms o f damage become ' l a b i l e ' o n l y under a l k a l i n e c o n d i t i o n s ( L e n n a r t z , C o q u e r e l l e S Hagen 1973). That i s , the a l k a l i n e t r e a t m e n t c o n v e r t s damage t h a t under normal pH c o n d i t i o n s would not be c o n s i d e r e d a s t r a n d break i n t o a l e s i o n t h a t would be c o n s i d e r e d one under a l k a l i c o n d i t i o n s . In f a c t , some e s t i m a t e t h a t a l k a l i l a b i l e l e s i o n s may c o n t r i b u t e up t o 30% o f t h e damage measured u s i n g ASG ( L e n n a r t z , C o q u e r e l l e S Hagen 1973). The e v i d e n c e here t h a t damage i s b e i n g uncovered when s a l t i s i n c l u d e d i n the u n w i n d i n g s o l u t i o n may be e v i d e n c e f o r ' s a l t l a b i l e ' l e s i o n s . T h i s would a l s o i n d i c a t e t h a t t h e sp e c t r u m o f damage b e i n g uncovered by t h i s t e c h n i q u e i s p r o b a b l y d i f f e r e n t t h a n when 35 3.2 2.4 1.6 0.8 6 Gy irradiation 20 minutes unwinding JL J. 20 40 60 80 %maximum salt concentration 'B' F i g u r e 6. Measured breaks vs. s a l t c o n c e n t r a t i o n of unwinding s o l u t I on. C e l l s were i r r a d i a t e d w i t h 6 Gy. and allowed to unwind f o r 20 minutes i n unwinding s o l u t i o n s of v a r y i n g s a l t c o n c e n t r a t i o n s . The s o l u t i o n w i t h no s a l t Is s o l u t i o n 'A', the s a l t c o n c e n t r a t i o n of s o l u t i o n 'B* was 0. 9M NaCl. To c a l c u l a t e the number of breaks, each sample was compared t o an u n i r r a d i a t e d sample which was a l s o unwound In the same s o l u t i o n as per equation ( 8 ) . The common I n t e r c e p t of 1 was s u b t r a c t e d o f f . 36 t h e damage uncovered by ASG. T h i s would have i m p l i c a t i o n s t r y i n g t o compare d a t a from the two methods o r i n u n d e r s t a n d i n g t h e k i n d s o f l e s i o n s b e i n g measured w i t h t h e two t e c h n i q u e s . 3.3 Dose response - i n t e r n a l c o n t r o l - a e r o b i c : The purpose o f i n c l u d i n g i n t e r n a l c o n t r o l s was t o improve s e n s i t i v i t y by t a k i n g a c c o u n t o f the 5% v a r i a t i o n t h a t o c c u r s from sample t o sample w h i c h , as mentioned above, may be due t o any number o f f a c t o r s t h a t a re d i f f i c u l t t o c o n t r o l . F i g u r e 7 i s a p l o t o f B as c a l c u l a t e d by e q u a t i o n ( 9 ) as a f u n c t i o n o f dose. I t Is e v i d e n t t h a t o v e r t h i s dose range, the response Is l i n e a r w i t h dose and, because c o n t r o l break v a l u e s a re s u b t r a c t e d o f f , t h e l i n e goes t h r o u g h t h e o r i g i n . We have found t h a t under i d e a l c o n d i t i o n s , t h e f r a c t i o n o f d o u b l e - s t r a n d e d DNA can be d e t e r m i n e d t o w i t h i n 1.0% w h i c h , when combined w i t h the use o f i n t e r n a l c o n t r o l s p r o v i d e s us w i t h a degree o f s e n s i t i v i t y such t h a t damage from doses as low as 0.1 Gy can be d e t e c t e d r o u t i n e l y . In c o n t r a s t , t h e s e n s i t i v i t y o f the method when no i n t e r n a l c o n t r o l i s used i s a p p r o x i m a t e l y 0.5 Gy. The m a j o r i t y o f the e x p e r i m e n t s t o be d e s c r i b e d below were performed w i t h t h i s t e c h n i q u e . 3.4 R e p a i r k i n e t i c s - a e r o b i c i r r a d i a t i o n : The s e n s i t i v i t y o f t h e method a l l o w s us t o p e r f o r m a v a r i e t y o f m o l e c u l a r measurements at doses where s i g n i f i c a n t numbers o f c e l l s s t i l l s u r v i v e . In p a r t i c u l a r , we have done a 37 Initial Breaks v.s. Dose 1.0 2.0 3.0 4.0 Gy F i g u r e 7. Dose response - I n t e r n a l c o n t r o l . The number of breaks was c a l c u l a t e d as per equation (9). The e r r o r s are standard e r r o r s taken from unnormalized data over s e v e r a l days and thus represent the day to day v a r i a t i o n . The e r r o r s on any one day would be approximately the s i z e of the p o i n t s themselves. 38 s y s t e m a t i c s t u d y o f the r e p a i r k i n e t i c s f o r a v a r i e t y of doses between 1 and 6 Gy. Some o f t h e s e r e s u l t s a r e shown i n f i g u r e 8. I n s p e c t i o n o f t h e s e c u r v e s r e v e a l s t h a t the k i n e t i c s seem t o be composed o f the sum o f at l e a s t two s e p a r a t e e x p o n e n t i a l p r o c e s s e s , a f a s t p r o c e s s w i t h a h a l f - l i f e o f about 7 m i n u t e s i and a s l o w e r p r o c e s s w i t h a h a l f - l i f e o f about 250 m i n u t e s . F u r t h e r , t h e r e p a i r i s t o such an e x t e n t t h a t u s u a l l y o v e r 90% of t h e i n i t i a l damage i s r e p a i r e d a f t e r 2 h o u r s . The d a t a can u s u a l l y be a d e q u a t e l y f i t t e d t o a s i m p l e sum of two e x p o n e n t i a l s o f t h e form, B ( t ) = B Q [ ( l - f ) e ~ t / T l + f e " t / T 2 ] ( 1 3 ) where B Q = i n i t i a l number o f b r e a k s ( n o r m a l i z e d t o 1 break/Gy) f = f r a c t i o n o f b r e a k s r e p a i r e d v i a the slow r e p a i r p r o c e s s T^.= f a s t p r o c e s s time c o n s t a n t T 0 = slow p r o c e s s t i m e c o n s t a n t . T a b l e 1 i s a summary o f some o f the pa r a m e t e r s found f o r a v a r i e t y o f i n i t i a l doses u s i n g a n o n - l i n e a r l e a s t s q u a r e s f i t t i n g p r o c e d u r e ( B e v i n g t o n 1969). 1.0 Hrs. 2.0 1.0 Hrs. 2.0 F i g u r e 8 . Repair k i n e t i c s . R epair k i n e t i c s f o r v a r i o u s i n i t i a l doses. The data Is p l o t t e d on a semi-1ogarithmic s c a l e t o emphasize the dual nature of the k i n e t i c s . The l i n e s drawn through the p o i n t s represent the l e a s t - s q u a r e s f i t to a sum of two e x p o n e n t i a l s whose parameters are g i v e n In Table 1. I n i t i a l damage Is normalized t o 1 break/Gy. and so the p o i n t s are i n Gy-equiva1ent u n i t s . 40 B 0 (%) f + . T l min. + . T 2 min. + 2 X /v 1 . 0 17.0 0 . 8 8.8 2.6 364 7 0.33 1.5 9.3 0 . 1 12.3 1 . 2 361 2 1.59 2 . 5 4.4 0.3 . r - i 5 1 . 2 359 16 0 . 24 3.0 7.3 0 .4 8 . 8 0.1 360 1 3.53 3.25 - 12.0 0.8 10.2 0.4 363 17 0.86 3.75 15.5 0.3 9.9 1.5 365 8 0 .83 4.25 8.7 0 . 1 10.8 1. 0 360 9 0.45 4.5 14.7 0.3 10.9 0 . 1 368 28 0 . 68 5 . 0 10.8 0 . 1 8.6 0 . 3 361 2 1 . 2 6 .07 6.8 0 .1 7.2 0.4 360 13 0 .25 4 9. 6avg • 362avg • T a b l e 1. Summary o f p a r a m e t e r s found u s i n g a n o n - l i n e a r l e a s t - s q u a r e s f i t t i n g p r o c e d u r e . The e r r o r s a r e e s t i m a t e s o f the s t a n d a r d d e v i a t i o n c a l c u l a t e d from the i n v e r s e o f t h e c u r v a t u r e o f t h e reduced c h i - s q u a r e ( B e v i n g t o n 1969). The c o r r e s p o n d i n g time c o n s t a n t s ( f r o m T^ = T l n ( 2 ) ) a r e 6.7 mins. and 251 mins. r e s p e c t i v e l y . The reduced c h i - s q u a r e s t a t i s t i c ( f i n a l column) was c a l c u l a t e d u s i n g an e x p e c t e d d i s t r i b u t i o n o f ±10%. The f i t t i n g p r o c e d u r e Is r e l a t i v e l y i n s e n s i t i v e t o t h e v a l u e o f the s l o w time c o n s t a n t and, a l t h o u g h they r e p r e s e n t t h e l e a s t - s q u a r e s e s t i m a t e , l a r g e d i f f e r e n c e s w i l l l e a d t o o n l y s m a l l i n c r e a s e s i n the reduced c h i - s q u a r e . 41 3.5 Remaining damage: I t i s e x p e c t e d t h a t under most c o n d i t i o n s , i t w i l l be t h o s e l e s i o n s t h a t a re s l o w l y r e p a i r e d o r l e f t u n r e p a i r e d t h a t w i l l u l t i m a t e l y d e t e r m i n e the s u r v i v a b i l i t y o f the c e l l . Most a s s a y s , e s p e c i a l l y t h o s e used f o r m e a s u r i n g low l e v e l s o f damage, are r a r e l y c a p a b l e o f m e a s u r i n g damage s i g n i f i c a n t l y above c o n t r o l l e v e l s a f t e r moderate p e r i o d s o f r e p a i r . Thus t h e r e i s some q u e s t i o n as t o whether o r not a f t e r doses where a s i g n i f i c a n t p r o p o r t i o n o f the c e l l s w i l l go on t o p r o l i f e r a t e , any damage i s l e f t u n r e p a i r e d . However, i f t h e s l o w e r component o f r e p a i r i s p u r e l y e x p o n e n t i a l , once t h e f a s t e r r e p a i r p r o c e s s i s c o m p l e t e , the r e m a i n i n g damage s h o u l d be p r o p o r t i o n a l t o the f i n a l damage. From the r e p a i r c u r v e s , i t i s c l e a r t h a t o n l y t h e s l o w component i s p r e s e n t at 2 h o u r s . Thus i n f i g u r e 9, we have p l o t t e d t h e damage r e m a i n i n g a f t e r 2 hour s as a f u n c t i o n o f the i n i t i a l dose. L e a s t -s q u a r e s a n a l y s i s r e v e a l s a s i g n i f i c a n t q u a d r a t i c component i n c o n t r a s t t o the i n d u c t i o n o f i n i t i a l damage wh i c h i s p u r e l y l i n e a r . At h i g h e r d o s e s , t h e q u a d r a t i c component becomes more ap p a r e n t as seen i n f i g u r e 10 where o n l y damage r e m a i n i n g a f t e r 2 hours was a s s a y e d . The q u a d r a t i c component i n t h e r e m a i n i n g damage i n d i c a t e s t h a t the r e m a i n i n g damage i s not p r o p o r t i o n a l t o the i n i t i a l damage. 3.6 The Dose response - h y p o x i a : r a d i o s e n s i t i z i n g p r o p e r t i e s o f oxygen may have 42 F i g u r e 9. Breaks remaining a f t e r 2 hours - low doses Number of breaks remaining a f t e r 2 hours taken from the r e p a i r curves p l o t t e d as a f u n c t i o n of i n i t i a l dose. The l e a s t - s q u a r e s f i t t o these po i nts i s : Bks. 2 Hrs 5.8 x 10' 2D + 1.3 x which i s s l i g h t l y more s i g n i f i c a n t than a l i n e a r f i t u s i n g the reduced c h i - s q u a r e t e s t . The e r r o r s are e s t i m a t e s of the day to day reproducabI1 I t y . io-V to CO CD c 0.41 0.3 « 0.2| E CO CC 0.1 F i g u r e 10. Breaks remaining a f t e r 2 hours - high doses. 4 Q Number of breaks remaining a f t e r two hours. Here, o n l y remaining damage was assayed. The l e a s t - s q u a r e s f i t to these p o i n t s I s : Bks. ._. = 2 Hrs. 4 x 10~ 2D + 4 x lO'V. The e r r o r s are of the order of the s i z e of the p o i n t s . 09 co CD a a c « E CD 0C 3.0 2.0 1.0L 43 Important consequences e s p e c i a l l y i n r a d i o t h e r a p y . The OER can a l s o p l a y an i m p o r t a n t r o l e as a r a d i o b i o l o g i c a l parameter w h i c h may show c o r r e l a t i o n s between d i f f e r e n t e n d p o i n t s . For t h e s e r e a s o n s , the f o l l o w i n g e x p e r i m e n t s were performed i n the absence o f 0^. The f i r s t a t t e m p t s t o measure, the y i e l d o f b r e a k s under h y p o x i a a t low doses were performed as per t h e s t a n d a r d p r o c e d u r e d e s c r i b e d i n ' M a t e r i a l s and Methods'. The r e s u l t s a r e shown i n f i g u r e 11. A l t h o u g h the o x i c c a l i b r a t i o n c u r v e i s l i n e a r as e x p e c t e d , the a n o x i c c u r v e bends w i t h a d e c r e a s i n g s l o p e (and t h u s an i n c r e a s i n g OER) as the dose i n c r e a s e s . In f a c t , the OER v a r i e s from about 1.7 a t 1 Gy t o 2.9 at h i g h e r d o s e s . One p o s s i b l e e x p l a n a t i o n f o r t h i s r e s u l t i s t h a t at t h e lower d o s e s , some 0^ i s p r e s e n t i n the system w h i l e a t h i g h e r d o s e s , t h e 0^ g e t s d e p l e t e d , perhaps r a d 1 o l o g I c a l 1 y . To t e s t t h i s h y p o t h e s i s , the e x t r a s t e p s d e s c r i b e d i n S e c t i o n 2.3.2 were t a k e n . The r e s u l t i s shown i n f i g u r e 12. In c o n t r a s t t o t h e p r e v i o u s r e s u l t , the a n o x i c response i s a l s o l i n e a r down t o 1 Gy g i v i n g a c o n s t a n t OER o f 2.91. A c o n s t a n t OER i s In agreement w i t h m o l e c u l a r measurements at h i g h e r doses ( P a l c i c S S k a r s g a r d 1972). 3.7 R e p a i r k i n e t i c s . - h y p o x i c i r r a d i a t i o n : v I t has been shown u s i n g o t h e r methods ( R o o t s S Smith 1975; Skov et a l . 1982) t h a t t h e k i n e t i c s o f r e p a i r o f l e s i o n s i n DNA i r r a d i a t e d under h y p o x i a ( b u t a l l o w e d t o r e p a i r under a e r o b i c c o n d i t i o n s ) are d i f f e r e n t from r e p a i r a f t e r a e r o b i c Dose (Gy) F i g u r e 11. 0x5c and anoxic dose response -standard g a s s i n g procedure. The curve l a b e l l e d 0_ r e p r e s e n t s the response f o r c e l l s i r r a d i a t e d In a i r . The curve l a b e l l e d N„ r e p r e s e n t s the response f o r c e l l s made nypoxlc by the standard g a s s i n g procedure. The OER Increases from 1.7 (1 Gy In N«) to 2.9 at the hi g h e s t dose. 45 Dose (Gy) F i g u r e 12. Oxlc and anoxic response -a l t e r n a t e g a s s i n g procedure. The curve l a b e l l e d 0„ r e p r e s e n t s the response of c e l l s i r r a d i a t e d in a i r . The curve l a b e l l e d r e p r e s e n t s the response of c e l l s made anoxic by the more Involved procedure. The OER i s constant Cat 2.91) down to the lowest dose measured (1 Gy. in N 2 ) . 46 i r r a d i a t i o n i n t h a t l e s s damage i s r e p a i r e d v i a t h e f a s t p r o c e s s t h a n v i a the s l o w one. T h i s was v e r i f i e d u s i n g our assay a t low doses as seen In f i g u r e 13. Here, the r e p a i r k i n e t i c s o f c e l l s t r e a t e d by the s t a n d a r d h y p o x i c p r o c e d u r e were measured and a r e compared t o the k i n e t i c s o f c e l l s a e r o b i c a l l y i r r a d i a t e d t o - a p p r o x i m a t e l y t h e same l e v e l o f damage (6 Gy i n 0^ v s . 14 Gy i n and measured c o n c u r r e n t l y . W h i l e t h e f a s t and s l o w time c o n s t a n t s a r e a p p r o x i m a t e l y the same, the a e r o b i c a l l y i r r a d i a t e d c e l l s have 90% o f t h e i r b r e a k s r e p a i r e d v i a t h e f a s t p r o c e s s compared t o t h e a n o x i c a l l y i r r a d i a t e d c e l l s w h i c h have o n l y 80% o f t h e i r s r e p a i r e d v i a t h e same p r o c e s s . 3.8 P l a t e a u - p h a s e c e l l s : P l a t e a u - p h a s e c e l l s a re n o n - c y c l i n g , d e n s i t y i n h i b i t e d . c e l l s . As s u c h , t h e i r d i s t r i b u t i o n t h r o u g h o u t the c e l l c y c l e i s d r a s t i c a l l y d i f f e r e n t from n o r m a l l y c y c l i n g c e l l s i n t h a t t h e m a j o r i t y ( a b o u t 85%) (Hahn £ L i t t l e 1972) o f t h e c e l l s a r e i n the G l phase o f t h e c y c l e . T h i s p r o p e r t y t h u s p r o v i d e s a good t e s t t o see what e f f e c t s the c e l l c y c l e has on the r e p a i r k i n e t i c s . F i g u r e s 14a, b and c show the r e s u l t s o f e x p e r i m e n t s where the k i n e t i c s o f r e p a i r o f c e l l s grown t o p l a t e a u - p h a s e were measured. The k i n e t i c s on a l l t h r e e days a r e e s s e n t i a l l y i d e n t i c a l t o e x p o n e n t i a l c e l l s i r r a d i a t e d t o t h e same dose i n t h a t both t h e t i m e c o n s t a n t s and the amount o f l e s i o n s b e i n g r e p a i r e d v i a t h e two d i f f e r e n t p r o c e s s e s a re the same. 47 100 1.0 Time (Hrs.) F i g u r e 1 3 . R e p a i r k i n e t i c s - c e l l s I r r a d i a t e d In N „ o r I n 0 „ , C o m p a r i s o n o f r e p a i r k i n e t i c s f o r i r r a d i a t e d i n a i r C C ^ ) o r i n h y p o x i a C e l l s w e r e g i v e n a p p r o x i m a t e l y t h e a m o u n t o f i n i t i a l damge ( 6 Gy i n 0^, i n N_) a n d a l l o w e d t o r e p a i r i d e n t i c a l c o n d i t i o n s . A g r e a t e r p r o p o r t i o n ( 2 0 % v s . 1 0 % ) o f d a m a g e In N 2 i s r e p a i r e d v i a t h e s l o w r e p a i r p r o c e s s t h a n i n 0 „ . c e l l s C N 2 ) . s a m e 14 Gy u n d e r 48 Time (Hrs.) F i g u r e 14 a,b S c . R e p a i r k i n e t i c s f o r c e l l s grown t o p l a t e a u - p h a s e a f t e r 3, 9 and 15 d a y s o f g r o w t h . The t i m e c o n s t a n t s and f d i f f e r l i t t l e f r o m t h o s e f o r , c e l l s a s s a y e d w h i l e i n l o g p h a s e . 1.0 2.0 Time (Hrs.) 49 3.9 H y p e r t o n i c p o s t - t r e a t m e n t : F i g u r e 15 shows t h e r e p a i r k i n e t i c s o b t a i n e d a f t e r p o s t - t r e a t m e n t i n h y p e r t o n i c s o l u t i o n . The 20 minute i n c u b a t i o n r e s u l t s i n a d e c r e a s e i n the number o f b r e a k s a l t h o u g h the d e c r e a s e i s much l e s s t h a n would be e x p e c t e d w i t h o u t t h e t r e a t m e n t . *-.''A-fter the c e l l s were r e t u r n e d t o a n i s o t o n i c i t y and f u l l growth c o n d i t i o n s , d u a l k i n e t i c s r e t u r n e d w i t h s i m i l a r h a l f - t i m e s t o t h o s e measured under s t a n d a r d c o n d i t i o n s . The main d i f f e r e n c e was t h a t l e s s amounts o f damage were r e p a i r e d v i a t h e f a s t component t h a n w i t h the s t a n d a r d c o n d i t i o n s . Thus t h e f i n a l r e p a i r e v e n t u a l l y became p a r a l l e l t o t h e normal k i n e t i c s and never got down t o t h e same l e v e l s as t h e s t a n d a r d one.' Whereas one would e x p e c t t h e f r a c t i o n b e i n g r e p a i r e d v i a t h e sl o w k i n e t i c s ( f ) t o be about 10%, h e r e , f was 50% compared t o the i n i t i a l damage p o i n t and 20% compared t o damage p o i n t a f t e r 20 m i n u t e s p o s t - t r e a t m e n t . The main e f f e c t o f t h i s t r e a t m e n t then i s the t r a n s f e r o f damage w h i c h would n o r m a l l y be q u i c k l y r e p a i r e d t o damage whi c h i s e i t h e r h a r d e r t o r e p a i r o r c o m p l e t e l y unrepa i rab1e. 3.10 ARA-A: The k i n e t i c s o f r e p a i r f o r c e l l s i n c u b a t e d i n medium c o n t a i n i n g ARA-A a l s o show a s i m i l a r d i f f e r e n c e . In f i g u r e 16 are p l o t t e d t h e r e s u l t s o f measurements o f such an e x p e r i m e n t . The lower c u r v e i s t h e k i n e t i c s o f c e l l s i n c u b a t e d c o n c u r r e n t l y i n normal medium. A g a i n , the p r i n c i p a l 50 1.0 \to Time (Hrs.) F i g u r e 15. Repair k i n e t i c s a f t e r h y p e r t o n i c p o s t - t r e a t m e n t . A f t e r I r r a d i a t i o n (6 Gy.), c e l l s were incubated i n 0. 5M NaCl s o l u t i o n at 37°C f o r 20. minutes (upper c u r v e ) . When the s a l t s o l u t i o n was re p l a c e d w i t h normal growth medium, the dual k i n e t i c s returned although the p r o p o r t i o n of damage r e p a i r e d v i a the f a s t process decreased. For comparison, normal r e j o i n i n g k i n e t i c s ( l o wer c u rve) u s i n g the same c e l l s Is shown. 51 cr i i i 1.0 2.0 Time (Hrs.) F i g u r e 1 6 . R e p a i r k i n e t i c s o f c e l l s i n c u b a t e d i n m e d i u m c o n t a i n i n g A R A - A . A f t e r i r r a d i a t i o n C6 G y . ) , c e l l s w e r e i n c u b a t e d i n m e d i u m c o n t a i n i n g 0 . 5 m M A R A - A ( u p p e r c u r v e ) , a n a n a l o g u e o f a d e n o s i n e w h i c h I s t h o u g h t t o s p e c i f i c a l l y i n h i b i t DNA p o l y m e r a s e . L e s s d a m a g e I s b e i n g r e p a i r e d by t h e f a s t c o m p o n e n t t h a n f o r c e l l s i n c u b a t e d i n n o r m a l m e d i u m ( l o w e r c u r v e ) . i 52 d i f f e r e n c e i s t h a t w h i l e a dual k i n e t i c s o c c u r s , the amount o f damage b e i n g r e p a i r e d v i a t h e f a s t component i s reduced from 90% down t o 80%. 3.11 DMSO: The e f f e c t s o f DMSO are shown i n f i g u r e 17. The s i t u a t i o n here i s more c o m p l i c a t e d because DMSO caused b r e a k s ( o r at l e a s t what was measured as b r e a k s ) i n t h e c o n t r o l DNA. The i n t e r e s t i n g p o i n t i s t h a t t h e b r e a k s caused by DMSO and the r a d i a t i o n were l e s s t h a n a d d i t i v e . The damage measured In the u n i r r a d i a t e d c e l l s goes up t o 86% o f th e i n i t i a l r a d i a t i o n damage (6 Gy) and then s l o w l y d e c r e a s e s t o about 50%. The i r r a d i a t e d c e l l s however I n c r e a s e d t o o n l y 136 % o f t h e i n i t i a l damage. The damage measured here a l s o s l o w l y d e c r e a s e d t o about 50% o f t h e i n i t i a l damage. T h i s l e s s t h e n a d d i t i v e e f f e c t may be the r e s u l t o f t h e n o n - l i n e a r i t i e s i n the u n w i n d i n g k i n e t i c s at h i g h l e v e l s o f damage. F a f t e r t h e DMSO t r e a t m e n t was about 27% wh i c h i s below t he l i n e a r u n w i n d i n g r e g i o n . 5 3 500 CO CO CD k-CO J> 100 CO E CD CC 10 • • x-rays +DMSO • • DMSO only : / • * § — i i • i • i i i • a 1.0 Time (Hrs.) 2.0 F i g u r e 1 7 . E f f e c t o f DMSO o n t h e r e p a i r k i n e t i c s o f I r r a d i a t e d c e l l s . A f t e r I r r a d i a t i o n ( 6 G y . ) , I r r a d i a t e d c e l l s ( s q u a r e s ) a n d u n i r r a d i a t e d c e l l s ( c i r c l e s ) w e r e i n c u b a t e d i n m e d i u m c o n t a i n i n g 1 . 5 M DMSO f o r 15 m i n u t e s a t 0°C a f t e r w h i c h t h e c e l l s w e r e w a s h e d a n d r e - i n c u b a t e d i n n o r m a l m e d i u m . DMSO c a u s e d a n i n c r e a s e In t h e n u m b e r o f b r e a k s a n d i n c r e a s e d t h e p r o p o r t i o n o f b r e a k s b e i n g r e p a i r e d b y t h e s l o w p r o c e s s . DMSO a l s o i n d u c e d b r e a k s o f i t s own In u n i r r a d i a t e d c e l l s w h i c h w e r e a l s o s l o w l y r e j o i n e d . 54 4 . D i s c u s s i o n : 4 . 1 R e p a i r k i n e t i c s : R e p a i r w i t h d u a l k i n e t i c s h a s b e e n o b s e r v e d by v a r i o u s i n v e s t i g a t o r s u s i n g a v a r i e t y o f t e c h n i q u e s a n d many p r o p o s a l s h a v e b e e n p u t f o r w a r d i n t h e l i t e r a t u r e t o e x p l a i n t h e m . F o r i n s t a n c e C o l e e t a l . ( 1 9 7 9 ) h a v e s u g g e s t e d t h a t t h e f a s t c o m p o n e n t c o r r e s p o n d s t o r e p a i r o f r e l a t i v e l y s i m p l e l e s i o n s ( p r o b a b l y u t i l i z i n g DNA l i g a s e s i n some c a s e s ) w h i l e i n c r e a s i n g l y c o m p l e x l e s i o n s , w h i c h w o u l d r e q u i r e m o r e i n v o l v e d e n z y m a t i c a c t i v i t y ( s u c h a s e x c i s i o n r e p a i r ) w o u l d be s l o w l y r e p a i r e d . R e c e n t l y , u s i n g t h e e x t e r n a l c o n t r o l u n w i n d i n g t e c h n i q u e , B r y a n t a n d B l o c h e r ( 1 9 8 0 ) f o u n d v a r i o u s c o r r e l a t i o n s t h a t s u g g e s t e d t h a t t h e f a s t a n d s l o w p r o c e s s e s r e p r e s e n t e d r e p a i r o f S S B s a n d D S B s r e s p e c t i v e l y . W i e r o w s k i a n d W h e e l e r ( 1 9 8 2 ) o n t h e o t h e r h a n d h a v e s u g g e s t e d t h a t t h e s l o w e r c o m p o n e n t o f r e p a i r r e p r e s e n t s r e j o i n i n g o f b r e a k s t h a t a r e i n i t i a l l y i n a c c e s s i b l e t o t h e r e p a i r e n z y m e c o m p l e x e s . I t i s a l s o p o s s i b l e t h a t t h e s l o w e r k i n e t i c s i s d u e t o r e p r e s s e d o r d a m a g e d r e p a i r a c t i v i t y o f a s u b p o p u 1 a t i o n o f t h e t o t a l p o p u l a t i o n o f c e l l s . I f t h i s w e r e t h e c a s e , o n e c o u l d e n v i s i o n a s i m p l e m o d e l o f c y c l i n g c e l l s w h i c h , o n c e p a s t t h e c r i t i c a l p a r t o f t h e c y c l e c o u l d no l o n g e r r e p a i r t h e i r DNA. S u c h a m o d e l i s s i m i l a r t o t h e ' f i x a t i o n ' t y p e m o d e l s t h a t 55 have been proposed ( E l k i n d S Whitmore 1 9 6 7 ; G a r r e t t S Payne 1 9 7 8 ; F r a n k e n b e r g £ Frankenberg-Schwager 1 9 8 1 ) . Ward, B l a k e l y S J o i n e r ( 1 9 8 2 ) , m e a s u r i n g r e p a i r a t 25°C, found k i n e t i c s w i t h h a l f t i m e s , i n t e r m e d i a t e t o our f a s t and s l o w p r o c e s s e s . I t i s p o s s i b l e t h a t the two components t h a t we measure may a c t u a l l y be a r e s u l t i n g summation o f many d i f f e r e n t p r o c e s s e s g o i n g on and a l l w i t h s i m i l a r h a l f t i m e s but whose d i f f e r e n c e s a r e brought out at t h e lower t e m p e r a t u r e s where e n z y m a t i c a c t i v i t y i s s l o w e r . I t has a l s o been s u g g e s t e d (Haynes 1 9 6 6 ; C a l k i n s 1 9 7 1 ; P o h l i t & Heyder 1 9 8 1 ) t h a t s a t u r a t i o n o f r e p a i r enzymes may be i m p o r t a n t at the m o l e c u l a r l e v e l . S a t u r a t i o n t h e n i s a p o s s i b l e e x p l a n a t i o n f o r t h e dual k i n e t i c s . These p o s s i b i l i t i e s may a l l be c a t e g o r i z e d as f o l l o w s ; 1 ) S a t u r a t i o n o f r e p a i r enzymes 2) Subpopu1 a t i o n w i t h d i f f e r e n t k i n e t i c s 3) I n a c c e s s i b l e damage 4 ) M u l t i p l e r e p a i r pathways 4 . 1 . 1 S a t u r a t i o n : The enzyme s u b s t r a t e r e l a t i o n s h i p i s c h a r a c t e r i z e d by t h e number o f enzymes, the amount o f s u b s t r a t e ( o r number o f l e s i o n s ) and the p a r a m e t e r s d e s c r i b i n g the t u r n o v e r and d i f f u s i o n p r o c e s s e s . I f the number o f l e s i o n s i s s m a l l , t h e n the r a t e - 1 i m i t I n g f a c t o r w i l l be t h e t i m e t a k e n by t h e enzyme t o r e c o g n i z e t h e l e s i o n . In t h i s c a s e , the v e l o c i t y w i l l s imp1y be, 56 dn/dt = - k n ( t ) ( 1 4 ) ) where, dn/dt i s t h e r a t e o f d i s a p p e a r a n c e o f t h e l e s i o n , n ( t ) i s the number o f l e s i o n s a t t i m e t and k i s a r a t e c o n s t a n t . I f t h e number o f l e s i o n s i s l a r g e , t h e n t h e r a t e - 1 i m i t i n g f a c t o r w i l l be the amount o f time i t t a k e s f o r the enzyme t o co m p l e t e i t s t a s k i n r e p a i r i n g the l e s i o n . In t h i s c a s e , the v e l o c i t y i s , ' dn/dt = - k n Q ( 1 5 ) where n Q i s a c o n s t a n t r e l a t e d t o the i n i t i a l number o f enzymes. Thus, depending on t h e number o f l e s i o n s , the k i n e t i c s can be e x p o n e n t i a l o r l i n e a r . The same s i t u a t i o n Is d e s c r i b e d by t h e s i m p l e MIchae1 is - M e n t e n model o f enzyme s u b s t r a t e i n t e r a c t i o n ( C a l k i n s 1971; S t r y e r 1981, c h a p t e r 6 ) . Here, t h e c h e m i c a l r e a c t i o n i s supposed t o be, E + S + E S + E + P (16) 57 The k's' a r e the r a t e c o n s t a n t s , E i s the enzyme c o n c e n t r a t i o n and S i s the s u b s t r a t e c o n c e n t r a t i o n . T h i s s i m p l e model p r e d i c t s t h a t , V = V . m a X S + K C l 7 ) y : where ^ m a x = [ E T ] , ( C E T 3 i s t h e t o t a l enzyme c o n c e n t r a t i o n ) and K = (k„ + k_ ) / k . . Here t o o , when S >> K , t h e v e l o c i t y m 2 3 1 m reduces t o the c o n s t a n t V w h i l e when S << K , V becomes max nrr p r o p o r t i o n a l t o S and t h u s becomes e x p o n e n t i a l . The r e l e v a n c e t o our s i t u a t i o n can be seen by c o n s i d e r i n g a c a s e where s a t u r a t i o n has o c c u r e d . In t h i s c a s e , the k i n e t i c s w i l l s t a r t out w i t h the v e l o c i t y b e i n g c o n s t a n t so t h a t t h e t i m e c o u r s e o f t h e d i s a p p e a r a n c e o f l e s i o n s would be l i n e a r . As t h e number o f l e s i o n s was r e d u c e d , t h e k i n e t i c s would change from t h e l i n e a r s i t u a t i o n t o the e x p o n e n t i a l s i t u a t i o n . . T h i s Is i l l u s t r a t e d i n f i g u r e 18 where the k i n e t i c s o f such a s i t u a t i o n i s s i m u l a t e d . C l e a r l y t h e s e c u r v e s a r e not c h a r a c t e r i s t i c o f the r e p a i r k i n e t i c s t h a t we have measured. Our d a t a , i f a n y t h i n g , show a s l o w i n g down o f the r a t e o f removal o f t h e l e s i o n s whereas t h e l o s s o f s a t u r a t i o n i s c h a r a c t e r i z e d by an a c c e l e r a t i o n o f re m o v a l . We can t h u s c o n c l u d e t h a t enzyme s a t u r a t i o n can not e x p l a i n our dual k i net I c s . 58 ln (S/So) S-So+kln(S/So)--Vmaxt time F i g u r e 18. S i m u l a t i o n o f r e p a i r k i n e t i c s f o r s a t u r a t e d and u n s a t u r a t e d s i t u a t i on. o f P l o t and i n t e g r a t i ng u n s a t u r a t e d a s t r a i g h t l n ( S / S Q ) u n s a t u r a t e d v s . t i m e f o r s a t u r a t e d c a s e s c a l c u l a t e d by e q u a t i o n ( 1 7 ) . The s i t u a t i o n i s c h a r a c t e r i z e d by l i n e (and i s t h u s e x p o n e n t i a l ) wh I 1 e the c h a r a c t e r i zed s a t u r a t e d by a downward s i t u a t I on i s bendi n g c u r v e . 59 4.1.2 Subpopu1 a t i o n w i t h d i f f e r e n t k i n e t i c s : The s i m p l e s t e x p l a n a t o n o f t h i s s o r t would be t o p o s t u l a t e t h a t c e l l s i n some p a r t o f the c e l l c y c l e would be e i t h e r o n l y s l o w l y a b l e o r u n a b l e t o r e p a i r damage i n t h e i r DNA. L a t e G l c e l l s might be a p o s s i b l e c h o i c e . For i n s t a n c e i t i s known, t h a t the s h o u l d e r o f the s u r v i v a l c u r v e f o r t h e s e c e l l s Is much d e c r e a s e d ( S i n c l a i r 1968; G i l l e s p i e et a l . 1975). Some s u r v i v a l models i n t e r p r e t a r e d u c t i o n i n t h e s h o u l d e r t o mean reduced r e p a i r ( C a l k i n s 1971; Green S B u r k i 1972; T o b i a s et a l . 1980). Two p i e c e s o f e v i d e n c e however, te n d t o c o n f l i c t w i t h t h i s i n t e r p r e t a t i o n . F i r s t , p l a t e a u - p h a s e c e l l s w h i c h a r e not r e p l i c a t i n g and have t h e m a j o r i t y o f t h e i r c e l l s i n G l showed alm o s t I d e n t i c a l r e p a i r k i n e t i c s t o lo g - p h a s e c e l l s ( S e c t i o n 3.8). Second, the p r o p o r t i o n o f l e s i o n s b e i n g r e p a i r e d v i a the slow p r o c e s s i n c r e a s e d w i t h i n c r e a s i n g dose ( S e c t i o n 3.5). T h i s would not be i n a c c o r d w i t h a subpopu1 a t i o n h y p o t h e s i s . 4.1.3 I n a c c e s s i b l e damage: I t might be e x p e c t e d some damage might i n i t i a l l y be i n a c c e s s i b l e t o t h e r e p a i r enzymes, e s p e c i a l l y when c o n s i d e r i n g t h a t c h r o m a t i n c o n t a i n s not o n l y DNA but h i s t o n e s , n o n - h i s t o n e s and o t h e r p r o t e i n s a r r a n g e d i n many l e v e l s o f h i g h e r o r d e r s t r u c t u r e . Hanawalt et a l . (1979) In t h e i r r e v i e w o f DNA r e p a i r , c i t e d s t u d i e s where t h e s e n s i t i v i t y o f DNA t o S t a p h y l o c o c c a l n u c l e a s e d e g r a d a t i o n was used as a measure o f a c c e s s i b i l i t y t o enzymes. T h i s s t u d y d i d not 60 r e v e a l i n a c c e s s i b i l i t y i n i r r a d i a t e d human l y m p h o c y t e s . Normal r e p a i r a c t i v i t y was a l s o r e p o r t e d t o be seen i n h i g h l y condensed chromosomes of m i t o t i c HeLa e e l 1s s u b j e c t e d t o u.v. i r r a d i a t i o n . They i n t e r p r e t e d t h e s e r e s u l t s t o s u ggest t h a t c o n d e n s a t i o n per se does not seem t o p r e v e n t e n z y m a t i c systems from m o n i t o r i n g t h e DNA f o r damage.• "They t h u s c o n c l u d e d t h a t h i g h e r o r d e r o r g a n i z a t i o n does not appear t o p a r t i t i o n the DNA i n t o a c c e s s i b l e and i n a c c e s s i b l e r e g i o n s at t h e chromosome l e v e l . They d i d not however d i s c o u n t t h e p o s s i b i l i t y t h a t i n a c c e s s i b i l i t y might r e s u l t f r om th e b i n d i n g o f h i s t o n e s t o DNA. In any c a s e , our d a t a does not s u p p o r t t h i s h y p o t h e s i s . E s p e c i a l l y t h e f a c t t h a t t h e r e m a i n i n g damage i s not p r o p o r t i o n a l t o the i n i t i a l damage s i n c e the h i d d e n damage h y p o t h e s i s Is based on t h e a s s u m p t i o n t h a t a c o n s t a n t f r a c t i o n o f the DNA Is i n a c c e s s i b l e . I t s h o u l d be mentioned t h a t W i e r o w s k i and W h e e l e r s ' e x p e r i m e n t s were p e r f o r m e d In v i vo In r a t s . F u r t h e r , t h e y a l s o saw a dose dependence o f the s l o w component time c o n s t a n t at h i g h e r doses w h i c h we do not see. The d i f f e r e n c e s between t h e i r r e s u l t s and o u r s c o u l d be a consequence o f the d i f f e r e n t systems used. 4.1.4 M u l t i p l e r e p a i r pathways: T h i s i s perhaps the most a t t r a c t i v e o f a l l p o s s i b i l i t i e s because i t i s a l r e a d y known t h a t d i f f e r e n t , r e p a i r pathways are 61 p r e s e n t i n b o t h e u k a r y o t i c a n d p r o k a r y o t i c c e l l s . T h e r e i s a m p l e d o c u m e n t a t i o n ( P a i n t e r 1 9 7 0 ; R e g a n S S e t l o w 1 9 7 4 ; G r o s s m a n 1 9 7 4 ; G r o s s m a n e t a l . 1 9 7 5 ; H a n a w a l t e t a l . 1 9 7 9 ; M e n e g h i n i , M e n c k S S c h u m a c h e r 1 9 8 1 ) t h a t c e l l s p o s s e s s a t l e a s t t w o e x c i s i o n r e p a i r s y s t e m s f o r e x c i s i n g b a s e d a m a g e . T h i s e v i d e n c e c o m e s m o s t l y f r o m u . v . d a m a g e s t u d i e s w h e r e t h e t h y m i n e d i m e r w h i c h i s t h e p r i n c i p a l l e s i o n r e s u l t i n g f r o m t h i s t y p e o f i n s u l t , c a n b e e a s i l y f o l l o w e d . The p r o p o s a l t h a t t h e f a s t a n d s l o w c o m p o n e n t s r e p r e s e n t r e p a i r o f S S B s a n d D S B s r e s p e c t i v e l y i s a t t r a c t i v e b u t c o n f l i c t i n g a n d c o n f u s i n g d a t a a r e f o u n d i n t h e l i t e r a t u r e . I n p a r t i c u l a r , W i e b e z a h n , S e x a u e r & C o q u e r e l l e ( 1 9 8 0 ) h a v e m e a s u r e d t h e k i n e t i c s o f DSB r e p a i r a n d h a v e f o u n d t h a t i t t o o h a s a f a s t a n d s l o w c o m p o n e n t . F r a n k e n b e r g - S c h w a g e r e t a l . ( 1 9 8 0 ) , u s i n g a DSB a s s a y i n y e a s t c e l l s f o u n d a n a l m o s t p u r e l y q u a d r a t i c v a r i a t i o n w i t h d o s e f o r r e m a i n i n g d a m a g e w h e r e a s we f i n d a l a r g e l i n e a r c o m p o n e n t i n t h e r e m a i n i n g d a m a g e . In t h e l i g h t o f t h e s e d a t a , i t m i g h t be u s e f u l t o c a l c u l a t e how many D S B s o n e m i g h t e x p e c t t o f i n d a n d , i f p o s s i b l e , c o m p a r e t o t h e m e a s u r e m e n t s t h a t h a v e b e e n m a d e . 4 . 1 . 5 DSB I n d u c t i o n : In g e n e r a l , t h e r e a r e t w o w a y s i n w h i c h a DSB may be I n d u c e d i n d u p l e x DNA. The f i r s t i s 1 d i r e c t 1 p r o d u c t i o n w h e r e t h e l e s i o n i s c a u s e d by o n e e v e n t . T h e s e c o n d i s by a ' c o m b i n a t i o n ' p r o d u c t i o n w h e r e t h e l e s i o n h a s b e e n c a u s e d by 2 62 o r m o r e e v e n t s o n b o t h s t r a n d s . The y i e l d o f d i r e c t D S B s c a n be c o n s i d e r e d t o be p r o p o r t i o n a l t o t h e y i e l d o f S S B s i n d u c e d i n t h e DNA. The i d e a i s t h a t a p r o p o r t i o n o f S S B s t u r n o u t t o p r o d u c e D S B s . T h e r e f o r e , ttDSB = k A D ( 1 8 ) d i r e c t w h e r e , k = f r a c t i o n o f S S B s t h a t a r e D S B s A = a v e r a g e n u m b e r o f S S B s / c e l 1 / u n i t d o s e D = a b s o r b e d d o s e . The c a l c u l a t i o n f o r t h e n u m b e r o f c o m b i n a t i o n D S B s i n d u c e d i n DNA c a n be d o n e i n a n u m b e r o f w a y s . P e r h a p s o n e o f t h e b e s t i s t h a t o f F r e i f e l d e r ( 1 9 6 9 ) . H i s a p p r o a c h w a s t o a s k , g i v e n t h e n u m b e r o f S S B s i n b o t h s t r a n d s o f t h e d u p l e x , how t h e y d i s t r i b u t e t h e m s e l v e s s o a s t o c o m b i n e t o make D S B s . To a n s w e r t h i s q u e s t i o n , he made u s e o f t h e c o n c e p t o f a ' f o r b i d d e n r e g i o n ' on o n e s t r a n d a s s o c i a t e d w i t h a SSB on t h e o p p o s i t e s t r a n d . . I f t h e r e a r e n^ S S B s o n s t r a n d 1 , t h e n t h e r e w i l l be R ( h ) p o s i t i o n s on t h e 2 n d s t r a n d w h e r e , i f a SSB i s p r e s e n t w i l l r e s u l t i n a D S B . . R ( h ) = n ( 2 h + 1 ) ( 1 9 ) w h e r e , h = ' i n t e r a c t i o n d i s t a n c e ' ( i n n u c l e o t i d e s ) b e t w e e n t w o S S B s t o make a DSB n ^ . = n u m b e r o f S S B s o n t h e f i r s t s t r a n d The r e s u l t i s t h a t , 63 ttDSB comb i n a t I on 2 ( 2 0 ) where N 0 = T o t a l number o f bases i n t h e DNA. Combining th e two, ( 2 1 ) w h i c h i s a l i n e a r - q u a d r a t i c dose dependence. The numbers f o r t h e s e p a r a m e t e r s can be e s t i m a t e d . For i n s t a n c e , based on a v a l u e o f 30 ev/break f o r SSBs ( P a l c i c S S k a r s g a r d 1972) and a t o t a l DNA m a s s / c e l l o f 4.2 x 1 0 1 2 d a l t o n s ( M c B r i d e S P e t e r s e n 1970), a p p r o x i m a t e l y 1470 S S B / c e l l w i l l be induced f o r e v e r y Gray o f absorbed dose. N n, t h e number of bases per c e l l ( b a sed on an average o f 615 d a l t o n s /base p a i r ) i s 1.37 x 1 0 1 0 . The r a t i o o f DSBs induced per SSB has been e s t i m a t e d ( E l k i n d 1979) t o be a p p r o x i m a t e l y 1 DSB f o r e v e r y 25 SSBs. Thus k = 1/25 = 0.04. The i n t e r a c t i o n d i s t a n c e h i s more d i f f i c u l t t o e s t i m a t e . On p u r e l y c h e m i c a l g r ounds, F r e i f e l d e r (1969) has r e p o r t e d t h a t h can v a r y between 2.61 and 15.8 n u c l e o t i d e s depending on the i o n i c s t r e n g t h . Van der Schans (1978) r e p o r t e d i n a s i m i l a r .study t h a t 'temporal l o c a l d e n a t u r a t i o n 1 c o u l d i n c r e a s e t h e i n t e r a c t i o n d i s t a n c e t o between 16 and 34 n u c l e o t i d e s . The range of h v a l u e s can t h u s v a r y between 1 as a minimum v a l u e and about 30 as a maximum v a l u e . P u t t i n g t h e s e numbers t o g e t h e r g i v e s , 64 #DSB = 59 D + (3 - 61) x 7.9 x 10 5 D 2. ( 2 2 ) Thus the c o n t r i b u t i o n t o DSB i n d u c t i o n by t h e two terms w i l l 4 be a p p r o x i m a t e l y e q u i v a l e n t a t doses between 1.2 x 10 and 2.5 x 10^ Gy. One t h e r e f o r e would e x p e c t the i n d u c t i o n o f DSBs t o be e s s e n t i a l l y l i n e a r o v e r a v e r y wide dose range. In f a c t , t h i s i s what has been o b s e r v e d (Lehmann S Ormerod 1970a; Ho 1975; H u t c h i n s o n 1975). I t Is i n t e r e s t i n g t o compare t h e s e numbers w i t h t h e numbers measured f o r the r e m a i n i n g damage. U s i n g the same 30 e v / b r e a k v a l u e , one c a l c u l a t e s t h a t t h e n o r m a l i z a t i o n c o n s t a n t must be 1470 b r e a k / G r a y / c e l 1 . S i n c e t h e measured v a l u e s were n o r m a l i z e d t o 1 b r e a k / G r a y , the numbers must be so c o r r e c t e d . The b e s t f i t t o t h e r e m a i n i n g damage d a t a was, B k s . 0 . = 4 x 1 0 ~ 2 D + 4 x 1 0 " 4 D 2 ( 2 3 ) 2 h r s . M u l t i p l y i n g by 1470 g i v e s , Bks.. , = 59 D + 0.59 D 2. ( 2 4 ) 2 h r s . . I t i s i n t e r e s t i n g t o note t h a t t h i s p r e d i c t s t h a t about 60 ' l e s i o n s 1 remain i n t h e c e l l a f t e r 1 Gy and 2 hours r e p a i r . Upon comparing t h e two equat i o n s , •. -perhaps t h e most n o t i c e a b l e d i f f e r e n c e between t h e two i s t h e l a r g e d i s p a r i t y between t h e r e l a t i v e c o n t r i b u t i o n s from the q u a d r a t i c t e rms. 65 From th e measured d a t a , the two terms c o n t r i b u t e e q u a l l y when th e dose i s 100 Gy whereas the minimum above was 1.2 x 10 Gy. The numbers used i n the c a l c u l a t i o n a r e p r o b a b l y good e s t i m a t e s o f the p a r a m e t e r s . C e r t a i n l y t h e r e a r e no o r d e r o f magnitude d i f f e r e n c e s i n v o l v e d . Thus i n r e c o n c i l i n g t h e s e d i f f e r e n c e s , the f o l l o w i n g p o s s i b i l i t i e s e x i s t : 1) the r e m a i n i n g b r e a k s d a t a i n c l u d e s l i n e a r l y i n d u c ed damage such as SSBs w h i c h a r e s l o w l y repa i r e d ; 2) the i n t e r a c t i o n d i s t a n c e has been m i s i n t e r p r e t e d so t h a t i t c o n s i d e r s o n l y t h e chem i ca1 e n v i r o n m e n t of t h e c e l l and i g n o r e s the b i o1og i ca1 ( i . e . e n z y m a t i c ) e n vironment o f the c e l l ; 3) c o m b i n a t i o n o f 1) and 2 ) . The t h i r d o p t i o n i s f a v o u r e d f o r the f o l l o w i n g r e a s o n s . F i r s t , i t i s w e l l documented t h a t r e p a i r o f DSBs t a k e s p l a c e . Though t h e d a t a i s not c o m p l e t e l y i n a c c o r d , most r e p o r t a t l e a s t a one o r d e r o f magnitude r e d u c t i o n In the number o f DSBs a f t e r a p e r i o d o f two h o u r s . I f such a r e p a i r d i d t a k e p l a c e , t h e n t h e c a l c u l a t e d numbers would have t o be reduced by an o r d e r o f magnitude t o be p r o p e r l y compared t o the measured v a l u e s . The l i n e a r component o f the measured v a l u e would then be an o r d e r o f magnitude l a r g e r t h a n t h e c a l c u l a t e d v a l u e and would t h u s s u p p o r t the f i r s t p o s s i b i l i t y . Second, th e l a r g e o b s e r v e d q u a d r a t i c component o n l y becomes ap p a r e n t a f t e r i n c u b a t i o n i s a l l o w e d t o p r o c e e d . Even at h i g h d o s e s , no q u a d r a t i c component^ i s seen when i n i t i a l ( 66 d a m a g e i s a s s a y e d . I t i s t h u s r e a s o n a b l e t o a s s u m e t h a t t h e b i o l o g i c a l a c t i v i t y o f t h e c e l l i s n e c e s s a r y f o r t h e q u a d a r a t i c c o m p o n e n t t o a p p e a r . T h i s w o u l d s u g g e s t t h a t t h e r e i s a c e r t a i n a c t i v i t y t h a t a l l o w s l e s i o n s t h a t a r e h u n d r e d s o f n u c l e o t i d e s a p a r t t o i n t e r a c t . S u c h an a c t i v i t y d o e s e x i s t a n d t h a t i s t h e e x c i s i o n o f l e s i o n s i n DNA. I t i s known f r o m u . v . s t u d i e s t h a t t h e r e i s o n e r e p a i r p a t h w a y t h a t r e q u i r e s t h e e x c i s i o n o f v e r y l o n g p a t c h e s o f n u c l e o t i d e s ( o n t h e o r d e r o f 100 n u c l e o t i d e s i n m a m m a l i a n c e l l s ) s u r r o u n d i n g t h e l e s i o n f o r i t t o be p r o p e r l y r e p a i r e d . H a n a w a l t e t a l . ( 1 9 7 9 ) h a v e s u g g e s t e d t h a t t h i s may be b e c a u s e d i s t o r t i o n s o f t h e DNA h e l i x i n t e r f e r e w i t h t h e w o r k i n g s o f t h e e n z y m e . I n a n y e v e n t , o n e c o u l d t h u s e n v i s i o n t h a t i n t h e p r o c e s s o f e x c i s i n g a p a r t i c u l a r l y d i f f i c u l t l e s i o n o n o n e s t r a n d , t h e e n z y m e c o u l d m e e t up w i t h a n o t h e r l e s i o n o n t h e o p p o s i t e s t r a n d a n d t h u s c r e a t e a DSB t h a t w o u l d m o s t p r o b a b l y be d i f f i c u l t t o r e p a i r . S u c h a p r o p o s a l h a s o f t e n a p p e a r e d i n t h e l i t e r a t u r e a n d some m o d e l s a r e b a s e d o n s u c h a n a c t i v i t y ( H a n a w a l t 1 9 6 6 ; H a r m 1 9 6 8 ; M i l l e r 1 9 7 0 ; D a l r y m p l e e t a l . 1 9 7 0 ; B e n d e r , G r i g g s S B e d f o r d 1 9 7 4 ; B o n u r a , S m i t h S K a p l a n 1 9 7 5 ; M i l l e r e t a l . 1 9 7 5 ; T o m u r a £ V a n L a n c k e r 1 9 7 5 ) . I f we a s s u m e t h a t 1 ) l i n e a r l y i n d u c e d D S B s a r e r e p a i r e d down t o 10% ( W e i b e z a h n S C o q u e r e l l e 1 9 8 1 ) o f t h e i r i n i t i a l l e v e l s , 2 ) t h e n u c l e o t i d e e x c i s i o n p a t c h l e n g t h i s 100 n u c l e o t i d e s l o n g a n d 3 ) t h e l i n e a r c o m p o n e n t o f t h e m e a s u r e d 67 r e m a i n i n g damage i n c l u d e s s l o w l y r e p a i r i n g SSBs w h i c h a r e induced i n t h e r a t i o o f 25:1, t h e n t h e numbers become, ttDSBs = 5.9 D + 1.6 x 1 0 " 2 D 2 ( 2 5 ) B k s . 0 . = 2.4 D + 5.9 x 1 0 " 1 D 2. ( 2 6 ) l h r s . The numbers i n e q u a t i o n s ( 2 5 ) and ( 2 6 ) are c l o s e r but a l a r g e d i s p a r i t y s t i l l e x i s t s . There i s a l s o a p o s s i b i l i t y t h a t c e r t a i n k i n d s o f base damage w h i c h would not n o r m a l l y be measured as a s t r a n d break when a s s a y i n g f o r i n i t i a l damage may be r e p a i r e d v i a the same e x c i s i o n r e p a i r and can t h u s c o n t r i b u t e t o t h e q u a d r a t i c component o f t h e r e m a i n i n g damage. T h i s t y p e o f m o d i f i c a t i o n would mean t h a t t h e i n d u c t i o n o f SSBs f a c t o r A would be l a r g e r f o r t he q u a d r a t i c term t h a n f o r t h e l i n e a r term. I t has been f o u n d , u s i n g the M. l u t e u s assay ( S k o v , P a l c i c S S k a r s g a r d 1979) t h a t f o r e v e r y SSB i n d u c e d , 1.25 MLS s i t e s a r e a l s o i n d u c e d . T h i s then m o d i f i e s A by a f a c t o r o f 2.25 (assuming 2 t h a t a l l MLS s i t e s a r e r e p a i r e d In t h i s f a s h i o n ) . S i n c e A appears i n the q u a d r a t i c term, t h i s becomes a f a c t o r o f 5.1 and the e x p r e s s i o n becomes, ttDSBs = 5.9 D + 8.2 x 1 0 - 2 D 2. ( 2 7 ) There s t i l l r emains a d i f f e r e n c e but i t i s l e s s t h a n one o r d e r o f magnitude. 68 The purpose o f i n c l u d i n g t h i s n u m e r i c a l e x e r c i s e was not so much t o see how w e l l t h e numbers c o u l d be made t o f i t but r a t h e r t o h i g h l i g h t the number o f d i f f e r e n t f a c t o r s i n v o l v e d i n comparing t h e o r e t i c a l p r e d i c t i o n s w i t h e x p e r i m e n t . C l e a r l y more d a t a a r e needed on the i n d u c t i o n o f SSBs and DSBs, the i m p o r t a n c e o f b i o l o g i c a l a c t i v i t y i n c r e a t i n g DSBs and i e s p e c i a l l y on the i n d u c t i o n and subsequent c o n t r i b u t i o n t o DSBs of base damage. T h i s i n f o r m a t i o n i s c e r t a i n l y not out o f r e a c h o f t h e technique's a v a i l a b l e t o d a y and once a v a i l a b l e , c o u l d c l e a r up many u n c e r t a i n t i e s c o n c e r n i n g DNA damage and r e p a i r . 4.2 OER: The OER f o r i n i t i a l damage measured w i t h t h i s assay was 2.91 and, i n agreement w i t h h i g h e r dose d a t a , was 1 i n e a r down t o 1 Gy ( N 2 ) . ASG d a t a however s u g g e s t s an OER o f about 3.6 ( S k o v , P a l c i c S S k a r s g a r d 1979). The d i f f e r e n c e i n t h e s e two numbers i s a n o t h e r i n d i c a t i o n t h a t the two t e c h n i q u e s are not m e a s u r i n g t h e same s p e c t r u m o f damage f o r i f they were, t h e OER would be e x p e c t e d t o be the same. Once a g a i n , i t i s s u g g e s t e d t h a t t h i s s h o u l d be t a k e n i n t o a c c o u n t when c o m p a r i s o n s of d a t a t a k e n w i t h t h e d i f f e r e n t methods are done. Only one e x p e r i m e n t was performed u s i n g the e x t r a measures f o r o b t a i n i n g h y p o x i a . However, i f f u t u r e s t u d i e s c o n f i r m t h e r e s u l t , t h e n i t w i l l be s i g n i f i c a n t t h a t i t took e x t r a o r d i n a r y measures t o r e g a i n the l i n e a r dose dependence. The s t a n d a r d p r o c e d u r e used t o o b t a i n h y p o x i a i s one t h a t i s 69 i n u s e i n many l a b s f o r o b t a i n i n g s u r v i v a l d a t a . In t h e p a s t , m o s t o f t h e s e m e a s u r e m e n t s w e r e d o n e a t h i g h e r d o s e s w h e r e t h e p r e s e n c e o f s m a l l a m o u n t s o f o x y g e n i n t h e s y s t e m w o u l d h a v e h a d l i t t l e e f f e c t o n t h e r e s u l t s d u e t o t h e r a d i o c h e m i c a l d e p l e t i o n e f f e c t ( K o c h 1 9 7 5 ; W h i l l a n s S R a u t h 1 9 8 0 ) . A t t h e p r e s e n t t i m e h o w e v e r , m o r e m e a s u r e m e n t s a r e b e i n g p e r f o r m e d a t l o w e r d o s e s . The p r e s e n c e o f s m a l l a m o u n t s o f o x y g e n i n t h e s y s t e m w h e n h y p o x i a i s d e s i r e d may t h e n a l t e r t h e r e s u l t s a n d l e a d t o f a l s e c o n c l u s i o n s a b o u t t h e e f f e c t o f o x y g e n a t l o w d o s e s . T h e r e i s a p o s s i b i l i t y t h a t t h e J_n - v ? v o s i t u a t i o n may be s i m i l a r i n t h a t l o w e r l e v e l s o f o x y g e n may be p r e s e n t i n t h e h y p o x i c a r e a s o f t h e t u m o r ( G r a y e t a l . 1 9 5 3 ) . I f t h i s i s t h e c a s e , t h e n s m a l l d o s e f r a c t i o n s w h i c h do n o t c o m p l e t e l y d e p l e t e t h e o x y g e n t h a t i s p r e s e n t w o u l d t a k e a d v a n t a g e o f t h i s s i t u a t i o n . In a n y e v e n t , w h a t i s n e e d e d i s a c c u r a t e m e a s u r e m e n t o f t h e c o n c e n t r a t i o n o f o x y g e n c o i n c i d e n t w i t h r a d i o b i o l o g i c a l m e a s u r e m e n t s . A s i m i l a r m e a s u r e m e n t i n t h e _i_n - v i v o s i t u a t i o n w o u l d t h e n a l s o be d e s i r a b l e . The o b s e r v a t i o n t h a t t h e r e p a i r k i n e t i c s a r e d i f f e r e n t f o r c e l l s i r r a d i a t e d i n h y p o x i a t h a n f o r c e l l s i r r a d i a t e d u n d e r a e r o b i c c o n d i t i o n s p r e s e n t s a n i n t e r e s t i n g c o m p l i c a t i o n . I f t h e i d e a t h a t t h e t w o c o m p o n e n t s o f r e p a i r r e p r e s e n t d i f f e r e n t k i n d s o f l e s i o n s i s a c c e p t e d , t h e n t h e d i f f e r e n c e i n t h e r e p a i r k i n e t i c s s u g g e s t s t h a t a l t h o u g h f e w e r l e s i o n s a r e b e i n g p r o d u c e d i n h y p o x i a , t h e y a r e m o r e d i f f i c u l t t o r e p a i r a n d t h u s m o s t p r o b a b l y o f a d i f f e r e n t n a t u r e t h a n t h e l e s i o n s 70 produced i n a e r o b i c c o n d i t i o n s . I t a l s o s u g g e s t s t h a t p r o p e r c o m p a r i s o n s between s u r v i v a l and m o l e c u l a r d a t a on the e f f e c t o f oxygen w i l l e v e n t u a l l y have t o t a k e r e p a i r i n t o a c c o u n t . T h i s remains t o be done. 4.3 P o s t - t r e a t m e n t s and PLD: The p r i n c i p a l e f f e c t o f t h e s e p o s t - t r e a t m e n t s on t h e r e p a i r k i n e t i c s was the c o n v e r s i o n o f l e s i o n s w h i c h are n o r m a l l y q u i c k l y r e p a i r e d t o l e s i o n s w h i c h a r e e i t h e r s l o w l y r e p a i r e d o r not r e p a i r e d a t a l l . T h i s t h e n i s p r e l i m i n a r y e v i d e n c e w h i c h I d e n t i f i e s the f a s t component o f r e p a i r w i t h PLD measured at t h e c e l l u l a r l e v e l . We cannot however I d e n t i f y the a c t u a l l e s i o n i n v o l v e d e x c e p t t o say t h a t i t i s a component of t h e l e s i o n s t h a t under most c a s e s can be e a s i l y r e p a i r e d . Exposure t o h y p e r t o n i c s o l u t i o n s , among o t h e r t h i n g s , r e s u l t s i n a c o n d e n s a t i o n o f t h e n u c l e a r c h r o m a t i n ( D e t t o r et a l . 1972), a r e d u c t i o n i n DNA s y n t h e s i s ( R o b b i n s , Pederson £ K l e i n 1970), r e l a x a t i o n o f nucleosomes CSahasrabuddhe £ Saunders 1977) and r e v e r s i b l e I n h i b i t i o n o f p r o t e i n s y n t h e s i s ( R o b e r t s o n £ Bermek 1977). I t i s c o n c e i v a b l e t h a t the s t r u c t u r a l changes of the DNA r e s u l t i n g from t h e s e f a c t o r s can i n t e r f e r e w i t h t h e w o r k i n g s o f enzymes t h a t a r e s t r u c t u r e s p e c i f i c . I t i s a l s o p o s s i b l e t h a t t h e i n t r o d u c t i o n o f s a l t may cause l o c a l d e n a t u r a t l o n at s t r a n d b r e a k s and t h u s c o n v e r t r e l a t i v e l y s i m p l e l e s i o n s i n t o more d i f f i c u l t ones. 71 Ben-Hur, Utsumi S E l k i n d (1980) used ASG t o measure the e f f e c t o f s a l t on t h e r e p a i r - k i n e t i c s and found t h a t a l t h o u g h d u r i n g t r e a t m e n t , r e p a i r was i n h i b i t e d , when the c e l l s were r e t u r n e d t o normal i n c u b a t i o n c o n d i t i o n s , t h e damage e v e n t u a l l y r e t u r n e d t o the l e v e l s e x p e c t e d f o r c e l l s not t r e a t e d . I t i s n,otv c l e a r whether the d i f f e r e n c e between our r e s u l t s and t h o s e o f Ben-Hur et a l . i s due t o t h e d i f f e r e n c e s i n t he t e c h n i q u e o r i n the l e v e l o f the doses used (6 Gy v s . 130Gy). I t would a l s o be i n t e r e s t i n g t o see how t h e s a l t t r e a t m e n t a f f e c t s r e p a i r o f DSBs and base damage. ARA-A Is r e p o r t e d t o be a s p e c i f i c i n h i b i t o r o f DNA polymerase ( I l i a k i s 1980). I f t h i s Is t r u e , t h e n t h e f a c t t h a t ARA-A a f f e c t e d t h e r e p a i r k i n e t i c s s u g g e s t s t h a t DNA polymerase i s an i m p o r t a n t p a r t o f the r e p a i r s y s t e m f o r a t l e a s t a segment o f x- r a y induced damage. Of c o u r s e i f a p o l y m e r i z a t i o n s t e p i s i n v o l v e d , t h e n most l i k e l y an e x c i s i o n s t e p preceeded i t . T h i s s u p p o r t s the p r o p o s a l d i s c u s s e d above t h a t e x c i s i o n r e p a i r p l a y s an Important r o l e In x - r a y induced damage. • The l e v e l s o f p o s t - t r e a t m e n t used were chosen because they have an a p p r o x i m a t e l y equal e f f e c t a t the s u r v i v a l l e v e l ( r e d u c t i o n In s u r v i v a l o f between 1 and 2 l o g s ) ( K o r b e l i k , P a l c i c 8 S k a r s g a r d i n p r e p a r a t i o n ) . I t i s i n t e r e s t i n g t o note t h a t b o t h t r e a t m e n t s had a p p r o x i m a t e l y t h e same n u m e r i c a l e f f e c t on t h e f r a c t i o n r e p a i r e d v i a the sl o w component. T h i s s u g g e s t s a p o t e n t i a l a r e a t o l o o k f o r f u r t h e r c o r r e l a t i o n s between t h e two d i f f e r e n t e n d p o i n t s . I t a l s o s u g g e s t s t h a t 72 t a k i n g a l o o k a t t h e e f f e c t o f c o m b i n a t i o n s o f t r e a t m e n t s may i n d i c a t e whether o r not the same s e c t o r o f damage i s b e i n g a f f e c t e d by the d i f f e r e n t t r e a t m e n t s . DMSO, i n c o n t r a s t t o h y p e r t o n i c p o s t - t r e a t m e n t i s not an o s m o t i c a g e n t . I t has been r e p o r t e d t o have e f f e c t s on c e l l membranes (Lyman S P r e i s l e r 1976), c o n f o r m a t i o n o f DNA ( L a p e y r e £ Bekhor 1974), i t can induce d i f f e r e n t i a t i o n i n some c e l l s ( S c h e r £ F r i e n d 1978; Marks £ R i f k i n d 1978) and caus e s r e d u c t i o n s i n t h e m o l e c u l a r w e i g h t o f t h e DNA ( D a r z y n k i e w i c z et a l . 1976) w h i c h s u g g e s t s the i n d u c t i o n o f some k i n d o f s t r a n d b r e a k s . The i n c r e a s e d u n w i n d i n g seen i n t h e c o n t r o l samples i s i n agreement w i t h t h e s u g g e s t i o n t h a t DMSO causes b r e a k s . That t h e s e b r e a k s by t h e m s e l v e s do not a f f e c t s u r v i v a l s u g g e s t s t h a t they may be an example o f ' n o n - l e t h a l ' damage - damage t h a t under normal c i r c u m s t a n c e s can be e a s i l y d e a l t w i t h by t h e c e l l . The f a c t t h a t DMSO ca u s e s b r e a k s a l s o s u g g e s t s a mechanism o f a c t i o n f o r i t s PLD e f f e c t . I t i s p o s s i b l e t h a t t h e i n d u c t i o n o f a DMSO break o p p o s i t e an a l r e a d y induced SSB may r e s u l t In a DSB whic h would- be d i f f i c u l t t o r e p a i r . I f DSBs a r e c r i t i c a l l e s i o n s , t h e n such a t r e a t m e n t would be e x p e c t e d t o reduce s u r v i v a l . The -.experIments d e s c r i b e d above a re o n l y p r e l i m i n a r y i n d i c a t i o n s o f the c o n n e c t i o n between PLD at the m o l e c u l a r and c e l l u l a r l e v e l s . Much more work needs t o be done, e s p e c i a l l y u s i n g o t h e r a s s a y s w h i c h p r o v i d e d i f f e r e n t i n f o r m a t i o n , u s i n g c o m b i n a t i o n s o f t r e a t m e n t s and p e r f o r m i n g m o l e c u l a r and 73 c e l l u l a r e x p e r i m e n t s c o n c u r r e n t l y . R e g a r d l e s s o f t h e s t a t e o f t h e s e s t u d i e s , some t r e n d s may be seen i n t h e r e s u l t s so f a r . F i r s t , t h e r e i s a s t r o n g s u g g e s t i o n t h a t a t l e a s t a component o f t h e f a s t r e p a i r i n g damage i s damage t h a t can be d e s c r i b e d as p o t e n t i a l l y l e t h a l . I t remains t o be seen whether o r not a l l o f i t can be so d e s c r i b e d . Second, s l o w l y r e p a i r i n g damage o r , most l i k e l y , damage t h a t i s not r e p a i r e d a t a l l i s what i s i m p o r t a n t i n r e l a t i n g DNA damage w i t h c e l l d e a t h . T h i r d , i n a l l t h e s e t r e a t m e n t s , t h e r e i s a s u g g e s t i o n t h a t complex damage r e s u l t i n g from a l t e r a t i o n s o f t h e o r i g i n a l l e s i o n i s more l e t h a l than t h e u n a l t e r e d l e s i o n i t s e l f . W i th h y p e r t o n i c p o s t - t r e a t m e n t , a l t e r a t i o n s i n the s t r u c t u r e o f DNA around t h e l e s i o n may r e s u l t i n i t s e x p r e s s i o n as PLD, With ARA-A, t h e r e i s a s u g g e s t i o n t h a t e x c i s e d damage w h i c h i s not r e s t o r e d i s i m p l i c a t e d and w i t h DMSO, the c o m b i n a t i o n o f an x-r a y induced l e s i o n w i t h a DMSO induced break may then r e s u l t i n a more c r i t i c a l t y p e o f damage than j u s t the x - r a y l e s i o n a l o n e . I f t h e s e t r e n d s s t a n d t h e t e s t o f i n v e s t i g a t i o n , t h e n they w i l l u n d o u b t e d l y h e l p i n i d e n t i f y i n g j u s t what the e e l 1 can o r cannot cope w i t h . . 4.4 R e l a t i n g m o l e c u l a r damage w i t h c e l l d e a t h : A l a r g e number o f models w h i c h a t t e m p t t o d e s c r i b e the s u r v i v a l r e s ponse o f c e l l s t o r a d i a t i o n have been proposed o v e r t h e y e a r s . E a r l i e r models d e s c r i b e d the a c t i o n o f r a d i a t i o n as a s e r i e s o f d i s c r e t e ' h i t s ' i n one or more 74 ' t a r g e t s ' ( L e a , H a i n e s S C o u l s o n 1936; Lea 1955; E l k i n d S S u t t o n 1960; Powers 1962; K i e f e r 1971; Gunther & S c h u l t z 1972; Ahnstrom & E h r e n b e r g 1977). More o f t e n t h a n n o t , the t a r g e t o f the r a d i a t i o n was not i d e n t i f i e d a l t h o u g h some s p e c u l a t e d t h a t DNA was i n v o l v e d . W i t h t h e r e c o g n i t i o n t h a t r e p a i r o r r e c o v e r y was an i m p o r t a n t c h a r a c t e r i s t i c o f the r a d i a t i o n response ( E l k i n d 8 S u t t o n I 9 6 0 ) , models w i t h t i m e dependent p a r a m e t e r s were d e v e l o p e d w h i c h c o u l d a c c o u n t f o r s u b - l e t h a l damage, l i q u i d - h o l d i n g r e c o v e r y e x p e r i m e n t s , f r a c t i o n a t i o n s t u d i e s and dose r a t e e f f e c t s ( D i e n e s 1966; Haynes 1966; Payne & G a r r e t t 1975; N i e d e r e r S Cunningham 1976; G a r r e t t & Payne 1978; Braby S Roesch 1978; P o h l i t S Heyder 1981). T r i p l e - s t a t e models w h i c h proposed t h a t c e l l s might go t h r o u g h an i n t e r m e d i a t e s t a t e b e f o r e c o m m i t t i n g t h e m s e l v e s t o s u r v i v e o r d i e (Kappos & P o h l i t 1972; T o b i a s e t a l . 1980) have a l s o been s u g g e s t e d . Many t h e o r i e s r e s u l t i n g i n l i n e a r - q u a d r a t i c dose d e p e n d e n c i e s have a l s o been s u g g e s t e d ( J a c o b s e n 1957; Neary 1965; S i n c l a i r 1966; Chadwick S Leenhouts 1973, 1981; K e l l e r e r S R o s s i 1972, 1978; Douglas & F o w l e r 1976; Leenhouts and Chadwick 1978,). The adequacy o f a model cannot be d e t e r m i n e d by c u r v e f i t t i n g a l o n e a l t h o u g h such s t a t i s t i c a l t e s t i n g can be i n d i s p e n s i b l e f o r e l i m i n a t i n g m i s l e a d i n g ones. The v a l i d i t y o f a model must be based on an a b i l i t y t o p r e d i c t v a r i o u s r a d i o b i o l o g i c a l q u a n t i t i e s ( s u c h as OERs, DMFs, RBEs e t c . ) , on i t s a b i l i t y t o d e s c r i b e t h e r e s p o n s e o f c e l l s t o e x p e r i m e n t s i n v o l v i n g t ime ( s u c h as s p l i t - d o s e e x p e r i m e n t s and dose r a t e 75 e f f e c t s ) , i t must be u s e f u l f o r t h e r a d i o l o g i s t who i s t r y i n g t o d e s i g n b e t t e r t r e a t m e n t p r o t o c o l s and, o f i n t e r e s t h e r e , i t s p a r a m e t e r s must be a c c e s s i b l e t o e x p e r i m e n t a l v e r i f i c a t i o n . Too many models i n c o r p o r a t e t a r g e t s t h a t a r e l o o s e l y d e f i n e d o r not i d e n t i f i e d at a l l , s u g g e s t r e c o v e r y p r o c e s s e s w h i c h a r e not based on e x p e r i m e n t or propose s t a t e s o f the c e l l t h a t would be most, d i f f i c u 1 t , i f not i m p o s s i b l e t o i d e n t i f y . The Chadwick and Leenhouts model, w h i c h p r o p o s e s t h a t the c r i t i c a l l e s i o n i s a DSB, though i t perhaps i s an o v e r s i m p l i f i c a t i o n o f an u n d o u b t e d l y more complex s i t u a t i o n , Is a u s e f u l model i n w h i c h t o frame h y p o t h e s e s because 1) t h e mathematics i s s i m p l e and a c c e s s i b l e , 2) the number o f p a r a m e t e r s i s l i m i t e d and 3) because they have come out and i d e n t i f i e d the c r i t i c a l l e s i o n , they have made t h e i r t h e o r y amenable t o e x p e r i m e n t a l v e r i f i c a t i o n a t both the c e l l u l a r and m o l e c u l a r 1 e v e l s . I t i s f o r t h e s e r e a s o n s t h a t t h e f o l l o w i n g d i s c u s s i o n w i l l be w i t h t h i s model In mind. Chadwick and Leenhouts have proposed t h a t the DSB i s the c r i t i c a l l e s i o n i n c e l l d e a t h . They have c a l c u l a t e d t h a t DSBs must be induced w i t h both a l i n e a r and q u a d r a t i c dose dependence so t h a t , #DSBs = aD + BD 2. ( 2 8 ) 76 The c o - e f f i c i e n t s i n c l u d e both p h y s i c a l and tempor a l f a c t o r s by t a k i n g a c c o u n t o f t h e f a c t t h a t DSBs can be r e p a i r e d . S i n c e t h e i n d u c t i o n o f DSBs i s random, one can use P o i s s o n s t a t i s t i c s t o d e s c r i b e the number o f c e l l s t h a t would have no DSBs r e m a i n i n g . By i n c l u d i n g a ' f a c t o r ( p ) r e l a t i n g DSBs w i t h c e l l d e a t h ' , the r e s u l t i s , I t has been found ( S i n c l a i r 1966; G i l l e s p i e et a l . 1975) t h a t t h i s dose dependence a d e q u a t e l y d e s c r i b e s the s u r v i v a l r esponse o f many mammalian c e l l s t o i o n i z i n g r a d i a t i o n . For p l a t i n g , t h e l i n e a r c o - e f f i c i e n t i s a p p r o x i m a t e l y 2.4 x 10 Gy * w h i l e t h e q u a d r a t i c c o - e f f i c i e n t i s a p p r o x i m a t e l y 2.7 x -2 -2 10 Gy . The c o n t r i b u t i o n s o f t h e s e two terms i s thus equal at about 9 Gy. From the d i s c u s s i o n above on the i n t e r p r e t a t i o n o f t h e n a t u r e o f the r e m a i n i n g damage, i t was c o n c l u d e d t h a t t h e r e m a i n i n g damage measured was a c o m b i n a t i o n o f both s l o w l y r e p a i r i n g SSBs and DSBs. I t was s u g g e s t e d t h a t i f a c o r r e c t i o n i n the l i n e a r term was made t o a c c o u n t f o r t h e SSBs, t h e n t h e number o f DSBs measured a f t e r 2 hours was, (2 9 ) c e l l s , under a e r o b i c c o n d i t i o n s and w i t h immediate #DSBs 2. h r s . = 2.4 D + 0.59 D . (26) 77 Here, t h e c o n t r i b u t i o n o f the two terms i s equal at about H-Gy, o n l y a f a c t o r o f about 2 d i f f e r e n t from t h a t measured w i t h s u r v i v a l . Chadwick and Leenhouts proposed t h a t t h e c e l l t r e a t s l i n e a r l y induced DSBs and quad r a t i c a 1 1 y induced DSBs e q u a l l y . I t i s c l e a r however ( s e e above) t h a t f o r t h e q u a d r a t i c term t o be i m p o r t a n t at r e l e v a n t d o s e s , t h i s cannot be so. One o f t h e key s t u m b l i n g b l o c k s i n t h e i r t h e o r y has always been how two l e s i o n s c o u l d I n t e r a c t o v e r the lon g d i s t a n c e s p r e d i c t e d by 2 the magnitude o f t h e D dependence. : The p o s s i b i l i t y t h a t t h e b i o l o g i c a l a c t i v i t y o f the c e l l i s i m p o r t a n t i n t h i s r e s p e c t may be one p o s s i b l e e x p l a n a t i o n . I t i s c o n s i s t e n t w i t h t h e f a c t t h a t many t r e a t m e n t s can a f f e c t t h e l i n e a r and q u a d r a t i c components d i f f e r e n t l y (Chapman e t a l . 1975a; F r a n k e n b e r g £ Frankenberg-Schwager 1981; I l i a k i s 1980). ' I t i s a l s o c o n s i s t e n t w i t h the f a c t t h a t a l l measurements show t h a t DSBs are induced l i n e a r l y . I t i s an a r e a t h a t needs e x p l o r i n g . In any e v e n t , i t does not t a k e away from t h e Chadwick and Leenhouts ? dea t h a t the DSB i s the c r i t i c a l l e s i o n . I t i s not known whether o r not the 2 hour e n d p o i n t used i s a good measure o f the t r u e amount o f damage t h a t w i l l e v e n t u a l l y be l e f t i n t h e c e l l . I t i s c o n c e i v a b l e t h a t though t h e r e a r e s l o w l y r e p a i r i n g SSBs as w e l l as s l o w l y r e p a i r i n g DSBs, t h e r e might be more u n r e p a i r a b l e DSBs th a n SSBs. T e s t i n g t h i s by a s s a y i n g f o r damage a f t e r l o n g e r i n c u b a t i o n t i m e s w o u l d , by n e c e s s i t y i n v o l v e u s i n g much h i g h e r doses ( a p p r o x i m a t e l y d o u b l i n g t h e dose f o r e v e r y 4 hours e x t r a 78 I n c u b a t i o n ) a n d i t i s n o t c l e a r w h e t h e r t h e l i n e a r - q u a d r a t i c d o s e d e p e n d e n c e w o u l d h o l d a t t h e h i g h e r d o s e l e v e l s . I t i s a l s o c o n c e i v a b l e t h a t a t h i g h d o s e s , c e l l i n a c t i v a t i o n may be by a d i f f e r e n t m e c h a n i s m . F o r i n s t a n c e , i t i s known t h a t a f t e r d o s e s w h i c h r e d u c e s u r v i v a l down i n t o t h e s e c o n d a n d t h i r d l o g , c e l l s may n o t e v e n go t h r o u g h o n e c e l l d i v i s i o n . A t t h e l o w e r d o s e e n d , i t i s uncommon t o f i n d c e l l s t h a t h a v e n o t g o n e t h r o u g h a t l e a s t a c o u p l e o f d i v i s i o n s b e f o r e c e a s i n g t o d i v i d e ( H u r w i t z & T o l m a c h 1 9 6 9 ; T h o m p s o n & S u i t 1 9 6 9 ; J u n g 1 9 8 2 ) . T h i s i s a n o t h e r ' g o o d r e a s o n f o r p e r f o r m i n g m o l e c u l a r m e a s u r e m e n t s a t l o w e r d o s e s . N e v e r t h e l e s s , t h e r e a s o n a b l y c l o s e c o r r e s p o n d e n c e b e t w e e n t h e t w o r a t i o s s t i l l s u g g e s t s t h a t t h e C h a d w i c k a n d L e e n h o u t s m o d e l may h a v e v a l i d i t y . I t s h o u l d a l s o be m e n t i o n e d t h a t t h e t h e o r y a s i t s t a n d s i s o n l y v a l i d f o r s y n c h r o n i z e d c e l l s , F o r a s y n c h r o n o u s p o p u l a t i o n s ( s u c h a s t h o s e u s e d i n t h i s s t u d y ) , t h e l i n e a r - q u a d r a t i c d o s e d e p e n d e n c e may s t i l l h o l d b u t a s G i l l e s p i e e t a l . ( 1 9 7 5 ) h a v e s h o w n , w h e n c o m p a r i n g m o l e c u l a r a n d c e l l u l a r d a t a f o r t h e s e c e l l s , o n e m u s t c o r r e c t f o r t h e f a c t t h a t a s i g n i f i c a n t p r o p o r t i o n o f t h e p o p u l a t i o n h a s m o r e t h a n o n e g e n o m e . T h i s w o u l d t e n d t o u n d e r e s t i m a t e t h e l i n e a r t e r m f o r s u r v i v a l w h e n c o m p a r e d t o t h e m o l e c u l a r d a t a . A s i m p l e c a l c u l a t i o n s h o w s t h a t t h i s f a c t o r c a n be a s much a s 2 . 5 . In a n y e v e n t , w h a t i s b e c o m i n g c l e a r i s t h a t f r o m a m o l e c u l a r p o i n t o f v i e w , t h e C h a d w i c k a n d L e e n h o u t s i d e a i s s t i l l a f r u i t f u l h y p o t h e s i s w i t h w h i c h o n e c a n w o r k . M o r e 79 i n f o r m a t i o n i s needed but i t s h o u l d be emphasized t h a t the answers a r e w i t h i n r e a c h o f t h e t e c h n i q u e s a v a i l a b l e t o d a y . Once t h e more s e n s i t i v e t e c h n i q u e s a r e w e l l c h a r a c t e r i z e d , low dose d a t a w i l l be a b l e t o answer some o f the q u e s t i o n s r a i s e d above. The e l u c i d a t i o n of t h e r e l a t i o n s h i p between the v a r i o u s e n d p o i n t s ( m o l e c u l a r , c e l l u l a r , chromosomal a b e r r a t i o n s ) w i l l p r o v i d e t h e f i r m b a s i s needed f o r t h e b e t t e r use and c o n t r o l o f I o n i z i n g r a d i a t i o n . 80 5 . R e f e r e n c e s : A d a m s , G . E . ( 1 9 6 7 ) The g e n e r a l a p p l i c a t i o n o f p u l s e r a d i o l y s i s t o c u r r e n t p r o b l e m s i n r a d i o b i o 1 o g y . C u r r . T o p . Rad i a t . R e s . Q . , 3_, 3 5 . A h n s t r o m , G . & E d v a r d s s o n , K . A . ( 1 9 7 4 ) R a d i a t i o n - i n d u c e d s i n g l e - s t r a n d b r e a k s In DNA d e t e r m i n e d by r a t e o f a l k a l i n e s t r a n d s e p a r a t i o n a n d h y d r o x y 1 a p a t I t e c h r o m a t o g r a p h y : An a l t e r n a t i v e t o v e l o c i t y s e d i m e n t a t i o n . I n t . J . R a d i a t . B i o l . . 2 6 , 4 9 3 . A h n s t r o m , G . S E h r e n b e r g , L . ( 1 9 7 7 ) The n a t u r e o f t h e t a r g e t In t h e b i o l o g i c a l a c t i o n o f i o n i z i n g r a d i a t i o n s . A d v . B i o l . M e d . P h y s . , 1_7, 1 2 9 . A l p e r , T . ( 1 9 7 9 ) C e l l u l a r R a d I o b 1 o 1 o g y , C a m b r i d g e U n i v e r s i t y P r e s s , New Y o r k . B e l l i , J . A . & S h e l t o n , M. ( 1 9 6 9 ) P o t e n t i a l l y l e t h a l r a d i a t i o n d a m a g e : r e p a i r by m a m m a l i a n c e 1 1 s i n c u 1 t u r e . S c I e n c e , 1 6 5 , 4 9 0 . B e n d e r , M . A . S W o l f f , S . ( 1 9 6 1 ) X - r a y i n d u c e d c h r o m o s o m e a b e r r a t i o n s a n d r e p r o d u c t i v e d e a t h i n m a m m a l i a n c e l l s . ' N Am. N a t u r a l i s t , 9 5 , 39 . ' B e n d e r , M . A , & G r i g g s , H . G . ( 1 9 7 4 ) M e c h a n i s m s o f c h r o m o s o m a l a b e r r a t i o n p r o d u c t i o n : I I I C h e m i c a l s a n d i o n i z i n g r a d i a t i o n . M u t a t . R e s . , 2 3 , 1 9 7 . B e n - H u r , E . , E l k i n d , M . M . S B r o n k , B . V . ( 1 9 7 4 ) T h e r m a l l y e n h a n c e d r a d i o r e s p o n s e o f c u l t u r e d C h i n e s e h a m s t e r c e l l s : I n h i b i t i o n o f r e p a i r o f s u b l e t h a l d a m a g e a n d e n h a n c e m e n t o f l e t h a l d a m a g e . Rad i a t . R e s . , 58 , 3 8 . 81 B e n - H u r , E . , U t s u m i , H . , S E l k i n d , M . M . ( 1 9 8 0 ) P o t e n t i a l l y l e t h a l a n d DNA r a d i a t i o n d a m a g e : S i m i l a r i t i e s i n i n h i b i t i o n o f r e p a i r by m e d i u m c o n t a i n i n g D_0 a n d by h y p e r t o n i c b u f f e r . R a d i a t . R e s . , 8 4 , 2 5 . B e v i n g t o n , P . R . ( 1 9 6 9 ) D a t a - R e d u c t i o n a n d E r r o r A n a l y s i s f o r t h e P h y s i c a l  S c i e n c e s , M c G r a w H i l l B o o k C o . , T o r o n t o . B o n u r a , T . , S m i t h , K . C . & K a p l a n , H . S . ( 1 9 7 5 ) E n z y m a t i c i n d u c t i o n o f DNA d o u b l e - s t r a n d b r e a k s i n x - i r r a d i a t e d E . c o l i K - l 2 . P r o c . N a t l . A c a d . S c i . U S A , 7 2 , 4 2 6 5 . B r a b y , L . A . S R o e s c h , W . C . ( 1 9 7 8 ) T e s t i n g o f d o s e - r a t e m o d e l s w i t h C h 1 a m y d o m o n a s r e i n h a r d i . R a d i a t . R e s . , 76 , 259 . B r a d l e y , M . O . S K o h n , K . W . ( 1 9 7 9 ) X - r a y I n d u c e d DNA d o u b l e - s t r a n d b r e a k p r o d u c t i o n a n d r e p a i r i n m a m m a l i a n c e l l s a s m e a s u r e d by n e u t r a l f i l t e r e l u t i o n . Nuc 1 . A c i d s R e s . , 7_, 79 3 . B r y a n t , P . E . & B l o c h e r , D. ( 1 9 8 0 ) M e a s u r e m e n t o f t h e k i n e t i c s o f DNA D S B ' s r e p a i r In E h r l i c h A s c i t e s t u m o r c e l l s u s i n g t h e u n w i n d i n g m e t h o d . I n t . J . R a d i a t . B i o l . , 3 8 , 3 3 5 . B u r k i , H . J . S O k a d a , S . ( 1 9 6 8 ) A c o m p a r i s o n o f t h e k i l l i n g o f c u l t u r e d m a m m a l i a n c e l l s i n d u c e d by d e c a y o f i n c o r p o r a t e d t r i t i a t e d m o l e c u l e s a t - 1 9 6 d e g r e e s C . B i o p h y s . J . , 8_, 4 4 5 . B u r k i , H . J . , R o o t s , R . , F e i n e n d e g e n , L . E . & B o n d , V . P . ( 1 9 7 3 ) I n a c t i y a t i o n o f m a m m a l i a n c e l l s a f t e r d i s i n t e g r a t i o n o f 5 H o r 5 I i n c e l l DNA a t - 1 9 6 ° C . I n t . J . R a d 1 a t . B i o l . , 2 4 , 3 6 3 . C a l k i n s , J . ( 1 9 7 1 ) A m e t h o d o f a n a l y s i s o f r a d i a t i o n r e s p o n s e b a s e d o n e n z y m e k i n e t i c s . R a d i a t . R e s . , 4_5, 5 0 . C a r r a n o , A . V . ( 1 9 7 3 a ) C h r o m o s o m e a b e r r a t i o n s a n d r a d i a t I o n - i n d u c e d c e l l d e a t h . I. T r a n s m i s s i o n a n d s u r v i v a l p a r a m e t e r s o f a b e r r a t i o n s . M u t a t . R e s . , 1 7 , 3 5 5 . 82 C a r r a n o , A . V . ( 1 9 7 3 b ) C h r o m o s o m e a b e r r a t i o n s a n d r a d i a t i o n - i n d u c e d c e l l d e a t h . I I . P r e d i c t e d a n d o b s e r v e d , e e l 1 s u r v i v a l . M u t a t . -Res . , 17 , 366 . ' C a r r i e r , W . F . S S e t l o w , R . B . ( 1 9 7 0 ) E n d o n u c l e a s e f r o m M i c r o c o c c u s l u t e u s w h i c h h a s a c t i v i t y t o w a r d s u l t r a v i o l e t i r r a d i a t e d DNA: P u r i f i c a t i o n a n d p r o p e r t i e s . J . B a c t e r i o l . , 102 , 1 7 8 . C h a d w i c k , K . H . S L e e n h o u t s , H . P . ( 1 9 7 3 ) A m o l e c u l a r t h e o r y o f c e l l s u r v i v a l . P h y s . M e d . B i o l . , 1 8 , 7 8 . C h a d w i c k , K . H . S L e e n h o u t s , H . P . ( 1 9 8 1 ) T h e M o l e c u l a r T h e o r y o f R a d i a t i o n B i o l o g y , S p r i n g e r V e r l a g , New Y o r k . C h a p m a n , J . D . , G i l l e s p i e , C . J . , R e u v e r s , A . P . S D u g l e , D . L . ( 1 9 7 5 a ) The i n a c t i v a t i o n o f C h i n e s e h a m s t e r c e l l s by. x - r a y s ; The e f f e c t s o f c h e m i c a l m o d i f i e r s on s i n g l e - a n d d o u b l e -e v e n t s . R a d i a t . R e s . , 6 4 , 36 5 . C h a p m a n , J . D . , G i l l e s p i e , C . J . , R e u v e r s , A . P . S D u g l ' e , D . L . ( 1 9 7 5 b ) R a d I o p r o t e c t o r s , r a d i o s e n s i t i z e r s , a n d t h e s h a p e o f t h e mammal i a n e e l 1 s u r v i v a l c u r v e . I n : C e l l S u r v i v a l a f t e r Low D o s e s o f R a d i a t i o n :  T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , ( T . A l p e r e d . ) , J o h n W i l e y S S o n s , L o n d o n , 1 3 5 . C h a p m a n , J . D . S G i l l e s p i e , C . J . ( 1 9 8 1 ) R a d I a t I o n - i n d u c e d e v e n t s a n d t h e i r t i m e s c a l e i n mamma 1 i a n c e 1 1 s . A d v . R a d i a t . B i o l . , 9., 1 4 3 . C h a r l e s b y , A . ( 1 9 5 4 ) \ M o l e c u l a r w e i g h t c h a n g e s i n t h e d e g r a d a t i o n o f l o n g - c h a i n p o l y m e r s . P r o c . R o y . S o c . A , 2 2 4 , 12 0 . C l e a v e r , J . E . ( 1 9 6 8 ) D e f e c t i v e r e p a i r r e p l i c a t i o n o f DNA i n x e r o d e r m a ' p i g m e n t o s u m . N a t u r e ( l o n d o n ) , 2 1 8 , 65 2 . C o l e , A . , C o o p e r , W . G . , S h o n k a , F . , C o r r y , P . M . , H u m p h r e y , R . M . & A n s e v i n , A . T . ( 1 9 7 4 ) DNA s c i s s i o n i n H a m s t e r c e l l s a n d i s o l a t e d n u c l e i s t u d i e d by l o w - v o l t a g e e l e c t r o n beam i r r a d i a t i o n . R a d I a t . R e s . , 6 0 , 1 . 83 C o l e , A . , M e y n , R . E . , C h e n , R . , C o r r y , P . M . g H i t t e l m a n , W. C 1 9 7 9 ) M e c h a n i s m s o f c e l l i n j u r y . I n : R a d i a t i o n B i o l o g y i n C a n c e r R e s e a r c h , ( R . E . M e y n g H . R . W i t h e r s e d s . ) , R a v e n P r e s s , New Y o r k , 3 3 . C o r r y , P . M . g. C o l e , A . ( 1 9 7 3 ) D o u b l e - s t r a n d r e j o i n i n g i n m a m m a l i a n DNA. N a t u r e (New B i o l . ) , 2 4 5 , 1 0 0 . C o r r y , P . M . , R o b i n s o n , S . g G e t z , S . ( 1 9 / 7 ) J ' 1 H y p e r t h e r m i c e f f e c t s on DNA r e p a i r m e c h a n i s m s . R a d i o l o g y , 12 3 , 47 5 . C r o t h e r s , D . M . ( 1 9 6 4 ) T h e k i n e t i c s o f DNA d e n a t u r a t i o n . J . M o l . B i o l . . 9., 7 1 2 . D a l r y m p l e , G . V . , B a k e r , M . L . , S a n d e r s , J . L . , M o s s J r . , A . J . , N a s h , J . C . g W i l k i n s o n , K . P . ( 1 9 7 0 ) Do m a m m a l i a n c e l l s r e p a i r r a d i a t i o n i n j u r y by t h e c u t - a n d - p a t c h m e c h a n i s m ? I I . An e x t e n s i o n o f t h e o r i g i n a l mode 1 . J . T h e o r . B i o l . , 2 8 , 1 2 1 . D a r z y n k i e w i c z , Z . , T r a g a n o s , F . , S h a r p l e s s , T . , F r i e n d , C . g - M e l a m e d , M . R . ( 1 9 7 6 ) N u c l e a r c h r o m a t i n c h a n g e s d u r i n g e r y t h r o i d d i f f e r e n t i a t i o n o f F r i e n d v i r u s i n d u c e d l e u k e m i c c e l l s . E x p . C e l l R e s . , 9 9 , 3 0 1 . D a t t a , R . , C o l e , A . g R o b i n s o n , S . ( 1 9 7 6 ) 2 i f l U s e o f t r a c k - e n d a l p h a p a r t i c l e s f r o m Am t o s t u d y r a d i o s e n s i t i v e s i t e s In CHO c e l l s . R a d i a t . R e s . , 6 5 , 1 3 9 . D a v i s o n , P . F . ( 1 9 6 6 ) The r a t e o f s t r a n d s e p a r a t i o n i n a 1ka1 i - t r e a t e d DNA. J . M o l . B i o l . , 22 , 97 . D e t t o r , C M . , D e w e y , W . C . , W i n a n s , L . F . g N o e l , J . S . ( 1 9 7 2 ) E n h a n c e m e n t o f x - r a y d a m a g e i n s y n c h r o n o u s C h i n e s e h a m s t e r c e l l s by h y p e r t o n i c t r e a t m e n t s . Rad i a t . R e s . , 52 , 35 2 . D e w e y , W . C . , M i l l e r , H . H g L e e p e r , D . B . ( 1 9 7 1 ) C h r o m o s o m a l a b e r r a t i o n s a n d m o r t a l i t y o f x - i r r a d i a t e d m a m m a l i a n c e l l s : E m p h a s i s o n r e p a i r . P r o c . N a t l . A c a d . S c i . U S A , 6 8 , 66 7 . 84 D e w e y , W . C . , S t o n e , L . E . , M i l l e r , H . H . g G i b l a k , R . E . ( 1 9 7 1 ) R a d i o s e n s i t i z a t i o n w i t h 5 - b r o m o d e o x y u r i d i n e o f C h i n e s e h a m s t e r c e l l s x - i r r a d i a t e d d u r i n g d i f f e r e n t p h a s e s o f t h e c e l l e y e 1e . R a d i a t . R e s . , 4 7 , 6 7 2 . D e w e y , W . C . , S a p a r e t o , S . A . g B e t t e n , D . A . ( 1 9 7 8 ) H y p e r t h e r m i c r a d i o s e n s i t i z a t i o n o f s y n c h r o n o u s C h i n e s e m a s t e r c e l l s : R e l a t i o n s h i p b e t w e e n l e t h a l i t y a n d c h r o m o s o m a l a b e r r a t i o n s . Rad1 a t . R e s . , 7 6 , 4 8 . D i e n e s , G . J . ( 1 9 6 6 ) A k i n e t i c m o d e l o f b i o l o g i c a l r a d i a t i o n r e s p o n s e . Rad i a t . R e s . . 28 , 183 . D o u g l a s , B . G . g F o w l e r , J . F . ( 1 9 7 6 ) The e f f e c t o f m u l t i p l e s m a l l d o s e s Of x - r a y s o n s k i n r e a c t i o n s i n t h e m o u s e a n d a b a s i c i n t e r p r e t a t i o n . Rad i a t . R e s . , 6 6 , 4 0 1 . D u g l e , D . L . g G i l l e s p i e , C . J . ( 1 9 7 4 ) K i n e t i c s o f t h e s i n g l e - s t r a n d r e p a i r m e c h a n i s m i n mamma 1 i a n c e 1 1 s . I n : M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f DNA, ( P . C . H a n a w a l t g R . B . S e t l o w e d s . ) , P l e n u m P r e s s , New Y o r k , 685 . D u g l e , D . L . , G i l l e s p i e , C . J . g C h a p m a n , J . D . ( 1 9 7 6 ) DNA s t r a n d b r e a k s , r e p a i r a n d s u r v i v a l i n x - i r r a d i a t e d mamma 1 i a n c e 1 1 s . P r o c . N a t l . A c a d . S c i . U S A , 7 3 , 8 0 9 . D u n n , W . C . g R e g a n , J . D . ( 1 9 7 9 ) I n h i b i t i o n o f DNA e x c i s i o n r e p a i r i n human c e l l s by a r a b I n o f u r a n o s y 1 c y t o s i ne : . E f f e c t on n o r m a l a n d x e r o d e r m a p i g m e n t o s u m c e l l s . M o l . P h a r m a c o l . , ' 1 5 , 3 6 7 . E l k i n d , M . M . g S u t t o n , H . ( 1 9 6 0 ) R a d i a t i o n r e s p o n s e o f m a m m a l i a n c e l l s g r o w n i n c u l t u r e . I. R e p a i r o f x - r a y d a m a g e i n s u r v i v i n g C h i n e s e h a m s t e r c e l l s . R a d i a t . R e s . , 1 3 , 5 5 6 . E l k i n d , M . M . g W h i t m o r e , G . ( 1 9 6 7 ) The R a d i o b i o l o g y o f C u l t u r e d M a m m a l i a n C e l l s , G o r d o n a n d B r e a c h , New Y o r k . E l k i n d , M . M . g K a m p e r , C . ( 1 9 7 0 ) Two f o r m s o f r e p a i r o f DNA i n m a m m a l i a n c e l l s f o l l o w i n g I r r a d i a t i on . B i o p h y s . J . , 1 0 6 , 2 3 7 . 85 E l k i n d , M . M . ( 1 9 7 9 ) DNA r e p a i r a n d c e l l r e p a i r : A r e t h e y r e l a t e d ? I n t . J . R a d i a t ' . O n e . B i o l . P h y s . , 5_, 1 0 8 9 . F l e t c h e r , G . H . ( 1 9 7 3 ) C l i n i c a l d o s e r e s p o n s e c u r v e s o f human m a l i g n a n t e p i t h e l i a l t u m o r s . B r . J . R a d i o l . , 46 , 1 . F o x , M . 5 F o x , B . W . ( 1 9 7 3 ) R e p a i r r e p l i c a t i o n i n x - i r r a d i a t e d Lymphoma c e l l s 1 n - v i t r o I n t . J . R a d i a t . B i o l . , 2 3 , 3 3 3 . F r a n k e n b e r g , D. S F r a n k e n b e r g - S c h w a g e r , M . ( 1 9 8 1 ) I n t e r p r e t a t i o n o f t h e s h o u l d e r o f d o s e r e s p o n s e c u r v e s w i t h i m m e d i a t e p l a t i n g i n t e r m s o f r e p a i r o f p o t e n t i a l l y l e t h a l l e s i o n s d u r i n g a r e s t r i c t e d t i m e p e r i o d . I n t . J . Rad i a t . B i o l . , 3 9 , 6 1 7 . F r a n k e n b e r g - S c h w a g e r , M . , F r a n k e n b e r g , D . , B l o c h e r , D. S A d a m a z y c k , C . ( 1 9 8 0 ) R e p a i r o f DNA DSB i n i r r a d i a t e d y e a s t c e l l s u n d e r n o n g r o w t h c o n d i t i o n s . R a d i a t . R e s . , 8 2 , 4 9 8 . F r a n k e n b e r g - S c h w a g e r , M . , F r a n k e n b e r g , D . , B l o c h e r , D. £ A d a m c z y c k , C . ( 1 9 8 1 ) E f f e c t o f d o s e r a t e o n t h e I n d u c t i o n o f DNA d o u b l e - s t r a n d b r e a k s In e u k a r y o t i c c e l l s . R a d i a t . R e s . , 37 , 2 0 7 . F r e i f e l d e r , D. ( 1 9 6 9 ) M a t c h i n g o f s i n g l e - s t r a n d b r e a k s t o f o r m d o u b l e - s t r a n d b r e a k s i n DNA. B 1 o p o l y m e r s , ]_, 6 8 1 . G a r r e t t , W . R . £ P a y n e , M . G . ( 1 9 7 8 ) A p p l i c a t i o n s o f m o d e l s f o r c e l l s u v i v a l : The f i x a t i o n t ime p i c t u r e . R a d i a t . R e s . , 7 3 , 2 0 4 . G i l l e s p i e , C . J . , C h a p m a n , J . D . , R e u v e r s , A . P . £ D u g l e , D . L . ( 1 9 7 5 ) The i n a c t l v a t i o n o f C h i n e s e h a m s t e r c e l l s by x - r a y s : S y n c h r o n i z e d a n d e x p o n e n t i a l c e l l p o p u l a t i o n s . R a d i a t . R e s . , 6 4 , 3 5 3 . G r a y , L . H . , C o n g e r , A . D . , E b e r t , M , H o r n s e y , S . £ S c o t t , O . C . A . ( 1 9 5 3 ) The c o n c e n t r a t i o n o f o x y g e n d i s s o l v e d In t i s s u e s a t t h e t i m e o f i r r a d i a t i o n a s a f a c t o r i n r a d i o t h e r a p y . B r . J . R a d i o l . , 2 6 , 63 8 . 86 G r e e n , A . E . S . & B u r k f , H . J . ( 1 9 7 2 ) A n o t e on s u r v i v a l c u r v e s w i t h s h o u l d e r s . Rad i a t . R e s . , 60 , 5 3 6 . G r o s s m a n , L . ( 1 9 7 4 ) E n z y m e s i n v o l v e d i n t h e r e p a i r o f DNA. A d v . R a d i a t . B i o l . , 4 , 7 7 . G r o s s m a n , L . , B r a u n , A . , F e l d b e r g , R. & M a h l e r , I. ( 1 9 7 5 ) E n z y m a t i c r e p a i r o f DNA. A n n . R e v . B i o c h e m . , 4 4 , 1 9 . G u n t h e r , K . S S c h u l z , W. ( 1 9 7 2 ) R a d i a t i o n q u a l i t y : A t h e o r y o f a c t i o n o f i o n i z i n g r a d i a t i o n s o n DNA, m i c r o o r g a n i s m s a n d m a m m a l i a n c e l l s . I . G e n e r a l o u t l i n e a n d some r e s u l t s . S t u d . B i o p h y s . , 3_4, 16 5 . H a h n , G . M . S L i t t l e , J . B . ( 1 9 7 2 ) P l a t e a u - p h a s e c u l t u r e s o f m a m m a l i a n c e l l s : An I n - v i t r o m o d e l f o r human c a n c e r . C u r r . t o p . R a d i a t . R e s . Q . , _8, 3 9 . H a h n , G . M . , B a g s h a w , M . A . , E v a n s , G . & G o r d o n , L . F . ( 1 9 7 3 ) R e p a i r o f p o t e n t i a l l y l e t h a l l e s i o n s i n x - i r r a d i a t e d , d e n s i t y - I n h I b I t e d C h i n e s e h a m s t e r c e l l s : M e t a b o l i c e f f e c t s a n d h y p o x i a . R a d I a t . R e s . . 55 , 28 0 . H a l l , E . J . ( 1 9 7 8 ) R a d l o b i o l o g y f o r t h e R a d i o l o g i s t , 2 n d e d . , H a r p e r a n d R o w , New Y o r k . H a n a w a l t , P . C . ( 1 9 6 6 ) The u . v . s e n s i t i v i t y o f b a c t e r i a : I t s r e l a t i o n t o t h e r e p l i c a t i on e y e 1 e . P h o t o c h e m . P h o t o b l o l . , 5_, 1 . H a n a w a l t , P . C , C o o p e r , P . K . , G a n e s a n , A . K . S S m i t h , C . A . ( 1 9 7 9 ) DNA r e p a i r i n b a c t e r i a a n d m a m m a l i a n c e l l s . A n n . R e v . B i o c h e m . , 48 , 78 3 . H a r m , W. ( 1 9 6 8 ) E f f e c t s o f d o s e f r a c t i o n a t i o n o n u l t r a v i o l e t s u r v i v a l o f E s c h e r i c h i a c o 1 i • P h o t o c h e m . P h o t o b l o l . , 7_, 7 3 . H a y n e s , R . H . ( 1 9 6 6 ) The i n t e r p r e t a t i o n o f m i c r o b i a l i n a c t i v a t i o n a n d r e c o v e r y p h e n o m e n a . R a d i a t . R e s , s u p p ! . 6 , 1 . 87 H e r s h e y , A . D . , K a m e n , M . D . , K e n n e d y , J . W . S G e s t , H . ( 1 9 5 7 ) M o r t a l i t y o f b a c t e r i o p h a g e c o n t a i n i n g a s s i m i l a t e d r a d i o a c t i v e p h o s p h o r u s . J . G e n . P h y s i o l . , 3_4, 3 0 5 . H i s s , E . A . S P r e s t o n , R . J . ( 1 9 7 7 ) The e f f e c t o f c y t o s i n e a r a b i n o s i d e on t h e f r e q u e n c y o f s i n g l e - s t r a n d b r e a k s i n DNA o f m a m m a l i a n c e l l s f o l l o w i n g i r r a d i a t i o n o f c h e m i c a l t r e a t m e n t . B i o c h i m . B i o p h y s . A c t a , 47 8 , 1 . H o , K . S . Y . ( 1 9 7 5 ) I n d u c t i o n o f DNA d o u b l e s t r a n d b r e a k s by x - r a y s i n a r a d i o s e n s i t i v e s t r a i n o f t h e y e a s t S a c c h a r o m y c e s  c e r e v i s i a e . M u t a t . R e s . , 3 0 , 3 2 7 . H u m p h r e y , R . M . , S t e w a r d , D . L . S S e d i t a , B . A . ( 1 9 6 8 ) DNA s t r a n d b r e a k s a n d r e j o i n i n g f o l l o w i n g e x p o s u r e o f s y n c h r o n i z e d C h i n e s e h a m s t e r c e l l s t o i o n i z i n g r a d i a t i o n . M u t a t . R e s . , 6 , 45 9 . H u r w i t z , C . S T o l m a c h , L . J . ( 1 9 6 9 ) T i m e - l a p s e c I n e m i c r o g r a p h i c s t u d i e s o f x - i r r a d i a t e d H e L a S3 c e l l s . I. P r o g r e s s i o n a n d c e l l d i s i n t e g r a t i o n . B i o p h y s . J . , 9., 6 07 . H u t c h i n s o n , F . ( 1 9 7 5 ) C u r r e n t k n o w l e d g e o f t h e f o r m a t i o n a n d r e p a i r o f DNA d o u b l e - s t r a n d b r e a k s . I n : M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f DNA, p a r t B , ( P . C . H a n a w a l t S R . B . S e t l o w e d s . ) , P l e n u m P r e s s , New Y o r k , 699 . 1 1 i a k i s , G . ( 1 9 8 0 ) E f f e c t s o f B - a r a b i n o f u r a n o s y 1 a d e n i n e o n t h e g r o w t h a n d r e p a i r o f p o t e n t i a l l y l e t h a l d a m a g e i n E r l i c h a s c i t e s t u m o r e e l I s . R a d I a t . R e s . , 83 , 537 . J a c o b s e n , B . S . ( 1 9 5 7 ) E v i d e n c e f o r r e c o v e r y f r o m x - r a y d a m a g e i n c h 1 a m y d o m o n a s . R a d i a t . R e s . , 7_, 3 9 5 . J u n g , H . ( 1 9 8 2 ) P o s t - i r r a d i a t i o n g r o w t h k i n e t i c s o f v i a b l e a n d n o n - v i a b l e CHO c e l l s . R a d i a t . R e s . , 89 . , 8 8 . K a p l a n , H . S . , S m i t h , K . C . S T o m l i n , P . A . ( 1 9 6 2 ) E f f e c t o f h a l o g e n a t e d p y r i m t d i n e s on r a d i o s e n s i t i v i t y o f E . c o l I. Rad i a t . R e s . , 1 6 , 9 8 . 88 K a p l a n , H . S . & M o s e s , L . E . ( 1 9 6 4 ) B i o l o g i c a l c o m p l e x i t y a n d r a d i o s e n s i t i v i t y . S c i e n c e , 1 4 5 , 2 1 . K a p l a n , H . S . ( 1 9 7 2 ) E n z y m a t i c r e p a i r o f r a d i a t i o n - i n d u c e d s t r a n d b r e a k a g e i n c e l l u l a r . D N A a n d I t s c h e m i c a l i n h i b i t i o n . R a d i o l o g y , 1 0 5 , 1 2 1 . ° K a p p o s , A . & P o h l i t , W. ( 1 9 7 2 ) ••'A c y b e r n e t i c m o d e l f o r r a d i a t i o n r e a c t i o n s i n l i v i n g c e l l s . I . S p a r s e 1 y - I on I z i ng r a d i a t i o n s ; s t a t i o n a r y c e l l s . I n t . J . Rad i a t . B i o l . , 2 2 , 5 1 . K e l l e r e r , A . M . S R o s s i , H . H . ( 1 9 7 2 ) The t h e o r y o f d u a l r a d i a t i o n a c t i o n . C u r r . T o p i c s . R a d i a t . R e s . Q . , 8_, 8 5 . K e l l e r e r , A . M . S R o s s i , H . H . ( 1 9 7 8 ) A g e n e r a l i z e d f o r m u l a t i o n o f d u a l r a d i a t i o n a c t i o n . R a d i a t . R e s . , 7 5 , 4 7 1 . K i e f e r , J . ( 1 9 7 1 ) T a r g e t t h e o r y a n d s u r v i v a l c u r v e s . J . T h e o r . B i o l . , 30 , 30 7 . K o c h , C . J . ' ( 1 9 7 5 ) M e a s u r e m e n t o f v e r y l o w o x y g e n t e n s i o n s i n l i q u i d s : D o e s t h e e x t r a p o l a t i o n n u m b e r f o r m a m m a l i a n s u r v i v a l c u r v e s d e c r e a s e a f t e r x - i r r a d i a t i o n u n d e r a n o x i c c o n d i t i o n s ? I n : C e l l S u r v i v a l a f t e r Low D o s e s o f R a d i a t i o n :  T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , ( T . A l p e r e d . ) , J o h n W i l e y S S o n s , L o n d o n , 1 6 7 . . K o h n , K . W . S E w i g , R . A . G . ( 1 9 7 3 ) A l k a l i n e e l u t i o n a n a l y s i s , a new a p p r o a c h t o t h e s t u d y o f DNA s i n g l - e s t r a n d i n t e r r u p t i o n s i n c e l l s . C a n c e r R e s . , 3 3 , 1 8 4 9 . K o h n , K . W . , E r i k s o n , L . C . , E w i g , R . A . G . S F r i e d m a n , C . A . ( 1 9 7 6 ) F r a c t i o n a t i o n o f DNA f r o m m a m m a l i a n c e l l s by a l k a l i n e e 1 u t i o n . B 1 o c h e m . , 1 5 , 4 6 2 9 . L a n g e , C . S . ( 1 9 7 4 ) The r e p a i r o f DNA d o u b l e - s t r a n d b r e a k s i n m a m m a l i a n c e l l s a n d t h e o r g a n i z a t i o n o f t h e DNA i n t h e i r c h r o m o s o m e s . I n : M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f DNA, ( P . C . H a n a w a l t & R . B . S e t l o w e d s . ) , P l e n u m P r e s s , New Y o r k , 677 . 89 L a p e y r e , J . M . £ B e k h o r , I. ( 1 9 7 4 ) E f f e c t s o f 5 b r o m o - 2 ' d e o x y u r i d i n e a n d d i m e t h y l s u l p h o x i d e on p r o p e r t i e s a n d s t r u c t u r e o f c h r o m a t i n . J . M o l . B i o l . , 8 9 , 1 3 7 . L e a , D . E . , H a i n e s , R . B . & C o u l s o n , C . A . ( 1 9 3 6 ) The m e c h a n i s m o f t h e b a c t e r i o c i d a l a c t i o n o f r a d i o a c t i v e r a d i a t i o n s . P r o c . R o y . S o c . B , 1 2 0 , 4 7 . L e a , D . E . ( 1 9 5 5 ) A c t i o n o f R a d i a t i o n s on L i v i n g C e l l s , C a m b r i d g e U n i v e r s i t y P r e s s , New Y o r k . L e e , Y . C . , B e n n e t , L . R . & B y f i e l d , J . E . ( 1 9 7 2 ) I n h i b i t i o n o f r e p a i r o f DNA s i n g l e - s t r a n d b r e a ' k s i n m o u s e l e u k a e m i a c e l l s by a c t i n o m y c i n D. B i o c h e m . B l o p h y s . R e s . C o m m u n . , 4 9 , 7 5 8 . L e e n h o u t s , H . A P . S C h a d w i c k , K . H . ( 1 9 7 8 ) The c r u c i a l r o l e o f DNA d o u b l e - s t r a n d b r e a k s i n c e l l u l a r r a d i o b i o l o g i c a l e f f e c t s . A d v . R a d i a t . B i o l . , 7_, 5 5 . L e h m a n n , A . R . S O r m e r o d , M . G . ( 1 9 7 0 a ) D o u b l e - s t r a n d b r e a k s i n t h e DNA o f a m a m m a l i a n c e l l a f t e r x - i r r a d I a t i o n . . B i o c h i m . B i o p h y s . A c t a , 2 1 7 , 2 6 8 . L e h m a n n , A . R . £ O r m e r o d , M . G . ( 1 9 7 0 b ) The r e p l i c a t i o n o f DNA i n M u r i n e l y m p h o m a c e l l s ( L 5 1 7 8 y ) . I. R a t e o f r e p l i c a t i o n . B i o c h i m . e t B i o p h y s . A c t a , 2 0 4 , 1 2 8 . L e h m a n n , A . R . ( 1 9 7 2 ) E f f e c t o f c a f f e i n e o n DNA s y n t h e s i s i n m a m m a l i a n c e l l s . B i o p h y s . J . , 1 2 , 1 3 1 6 . L e n n a r t z , M . , C o q u e r e l l e , T . S H a g e n , U . ( 1 9 7 3 ) E f f e c t o f o x y g e n o n DNA s t r a n d b r e a k s i n i r r a d i a t e d t h y m o c y t e s . I n t . J . R a d i a t . B i o l . , 2 4 , 6 2 1 . L e t t , J . T . , C a l d w e l l , I . , D e a n , C . J . £ A l e x a n d e r , P . ( 1 9 6 7 ) R e j o i n i n g o f x - r a y I n d u c e d b r e a k s i n t h e DNA o f l e u k a e m i c c e l l s . N a t u r e , 2 1 4 , 7 9 0 . L e t t , J . C . , K l u c i s , E . S . & S u n , On t h e s i z e o f t h e DNA S t r u c u r a l s u b u n i t s . B i o p h y s . J . , 10 , 2 7 7 . C . ( 1 9 7 0 ) i n t h e m a m m a l i a n c h r o m o s o m e : 90 L i t t b r a n d , B . S R e v e s z , L . ( 1 9 6 9 ) T h e e f f e c t o f o x y g e n o n c e l l u l a r s u r v i v a l a n d r e c o v e r y a f t e r r a d i a t i o n . B r . J . Rad i o l . , 4 2 , 9 1 4 . L i t t l e , J . B . ( 1 9 7 3 ) F a c t o r s i n f l u e n c i n g t h e r e p a i r o f p o t e n t i a l l y l e t h a l r a d i a t i o n d a m a g e i n g r o w t h - i n h i b i t e d human c e l l s . R a d i a t . R e s . , 5 6 , 3 2 0 . L o n g u e t - H i g g i n s , H . C . & Z i m m , B . ( I 9 6 0 ) C a l c u l a t i o n o f t h e r a t e o f u n c o i l i n g o f t h e DNA m o l e c u l e . J . M o l . B i o l . , 2./ 1 . L y m a n , G . H . , P r e i s l e r , H . D . £ P a p a h a d j o p o u l o s , D. ( 1 9 7 6 ) M e m b r a n e a c t i o n o f DMSO a n d o t h e r c h e m i c a l i n d u c e r s o f F r i e n d l e u k a e m i c c e l l d i f f e r e n t i a t i o n . N a t u r e ( L o n d o n ) , 2 6 2 , 3 6 0 . M c B r i d e , O . W . & P a t e r s o n / E . A . ( 1 9 7 0 ) S e p a r a t i o n o f n u c l e i r e p r e s e n t i n g d i f f e r e n t p h a s e s o f t h e g r o w t h c y c l e f r o m u n s y n c h r o n I z e d m a m m a l i a n c e l l c u l t u r e s . J . C e l l B i o l . , k7_, 1 3 2 . M c G r a t h , R . A . £ W i l l i a m s , R . W . ( 1 9 6 6 ) R e c o n s t r u c t i o n In v i v o o f i r r a d i a t e d E . c o l i d e o x y r i b o n u c l e i c a c i d . " The r e j o i n i n g o f b r o k e n p i e c e s . N a t u r e ( L o n d o n ) , 2 1 2 , 5 3 4 . M c N a l l y , N . J . ( 1 9 7 5 ) T h e e f f e c t o f r e p e a t e d s m a l l d o s e s o f r a d i a t i o n o n r e c o v e r y f r o m s u b l e t h a l d a m a g e by C h i n e s e h a m s t e r c e l l s i r r a d i a t e d i n o x i c o r h y p o x i c c o n d i t i o n s i n t h e p l a t e a u p h a s e o f g r o w t h . I n : C e l l S u r v i v a l a f t e r Low D o s e s o f R a d i a t i o n :  T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , ( T . A l p e r e d . ) , J o h n W i l e y a n d S o n s , L o n d o n , 1 1 9 . M a r i n , G . £ B e n d e r , M . A . ( 1 9 6 3 ) ^ c o m p a r i s o n o f m a m m a l i a n c e l l k i l l i n g by i n c o r p o r a t e d H - t h y m i d i n e a n d H - u r i d i n e . I n t . J . Rad i a t . B i o l . , 7., 22 1 . M a r k s , P . A . £ R i f k l n d , R . A . ( 1 9 7 8 ) E r y t h r o 1 e u k e m 1 c d i f f e r e n t i a t i o n . A n n . R e v . B i o c h e m . , 47 . , 4 1 9 . M a r t i n s o n , H . G . ( 1 9 7 3 ) T h e b a s i s o f f r a c t i o n a t i o n o f s i n g l e - s t r a n d e d n u c l e i c a c i d s o n h y d r o x y 1 a p a t i t e . B I o c h e m . , 1 2 , 2 7 3 1 . 91 M a r t i n s o n , H . G . S W a g e n a a r , E . B . ( 1 9 7 4 ) The e f f e c t o f t h e a m o u n t o f n u c l e i c a c i d l o a d on h y d r o x y 1 a p a t i t e c h r o m a t o g r a p h y . C a n . J . B i o c h e m . , 52 , 26 7 . i M e n e g h i n i , R . , M e n c k , C . F . M . & S c h u m a c h e r , R . I . ( 1 9 8 1 ) M e c h a n i s m s o f t o l e r a n c e t o DNA l e s i o n s i n m a m m a l i a n e e l I s . Q u a r t . R e v . B i o p h y s . , 1 4 , 3 8 1 . M i l l e r , D . R . ( 1 9 7 0 ) 1 T h e o r e t i c a l s u r v i v a l c u r v e s f o r r a d i a t i o n d a m a g e i n b a c t e r i a . J . T h e o r . B i o l . , 2 6 , 38 3 . M i l l e r , D . R . , G l a z e b r o o k , A . D . , M u l l e n , B . M . S B r a m a l l , L . ( 1 9 7 5 ) P r e d i c t i o n o f b a c t e r i a l s u r v i v a l a f t e r u l t r a v i o l e t r a d I a t i o n . I n : C e l l S u r v i v a l a f t e r Low D o s e s o f R a d i a t i o n :  T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , ( T . A l p e r e d . ) , J o h n W i l e y a n d S o n s , L o n d o n , 1 7 9 . M u n r o , T . R . ( 1 9 7 0 ) The r e l a t i v e r a d i o s e n s i t i v i t y o f t h e n u c l e u s a n d c y t o p l a s m o f C h i n e s e h a m s t e r f i b r o b l a s t s . R a d i a t . R e s . , 4 2 , 4 5 1 . N a k a t s u g a w a , K . , I s h i z a k i , K. & S u g a h a r a , T . ( 1 9 7 8 ) The r e d u c t i o n i n f r e q u e n c y o f x - r a y - I n d u c e d s i s t e r c h r o m a t i d e x c h a n g e s i n c u l t u r e d m a m m a l i a n c e l l s d u r i n g p o s t - i r r a d i a t i o n i n c u b a t i o n i n H a n k s ' b a l a n c e d s a l t s o 1 u t i o n . I n t . J . R a d I a t . R e s . , 3 4 , 4 8 9 . N e a r y , G . J . ( 1 9 6 5 ) C h r o m o s o m e a b e r r a t i o n s a n d t h e t h e o r y o f R B E . I. G e n e r a l c o n s i d e r a t i o n s . I n t . J . R a d i a t . B i o l . , 9., 4 7 7 . ' N i e d e r e r , J . & C u n n i n g h a m , J . R . ( 1 9 7 6 ) T h e r e s p o n s e o f c e l l s i n c u l t u r e t o f r a c t i o n a t e d r a d i a t i o n : A t h e o r e t i c a l a p p r o a c h . P h y s . M e d . B i o l . , 2 1 , 82 3 . P a i n t e r , R . B . ( 1 9 7 0 ) R e p a i r o f DNA i n m a m m a l i a n c e l l s . C u r r . T o p . R a d i a t . R e s . Q . , ]_,. 4 5 . P a l c i c , B . S S k a r s g a r d , L . D . ( 1 9 7 2 ) The e f f e c t o f o q y g e n o n DNA s i n g l e - s t r a n d b r e a k s p r o d u c e d by i o n i z i n g r a d i a t i o n i n m a m m a l i a n c e l l s . I n t . J . R a d i a t . B i o l . , 2 1 , 4 1 7 . 92 P a l c i c , B . , B r o s i n g , J . S S k a r s g a r d , L . D . ( 1 9 8 2 ) S u r v i v a l m e a s u r e m e n t s a t l o w d o s e s : O x y g e n e n h a n c e m e n t r a t i o . B r . J . C a n c e r , i n p r e s s . P a r k e r , L . , S k a r s g a r d , L . D . & E m m e r s o n , P . T . ( 1 9 6 9 ) S e n s i t i z a t i o n o f a n o x i c m a m m a l i a n c e l l s t o x - r a y s by t r i a c e t o n e a m i n e N - o x y l . S u r v i v a l a n d t o x i c i t y s t u d i e s . R a d i a t . R e s . , 3 8 , 4 9 3 . P a t e r s o n , M . C . S S e t l o w , R . B . ( 1 9 7 2 ) E n d o n u c 1 e o l y t i c a c t i v i t y f r o m M i c r o c o c c u s l u t e u s t h a t a c t s o n y - r a y i n d u c e d d a m a g e i n p l a s m i d DNA o f E s c h e r i c h i a c o l i m i n i c e l l s . P r o c . N a t l . A c a d . S c i . U S A , 6 9 , 2 9 2 7 . P a t e r s o n , M . C . ( 1 9 7 5 ) U s e o f p u r i f i e d 1 e s i o n - r e c o g n i z i n g e n z y m e t o a s s a y DNA r e p a i r In c u l t u r e d a n i m a l c e l l s . I n : R a d i a t i o n R e s e a r c h : B i o m e d i c a l , C h e m i c a l a n d P h y s i c a l  P e r s p e c t i v e s , ( O . F . N y g a a r d , H . I . A d l e r & W . K . S i n c l a i r e d s . ) , A c a d e m i c P r e s s , New Y o r k , 7 1 8 . P a t e r s o n , M . C , S m i t h , B . P . , L o h m a n , P . H . M , A n d e r s e n , A . F . S F i s h m a n , L . ( 1 9 7 6 ) D e f e c t i v e e x c i s i o n r e p a i r o f gamma r a y - d a m a g e d DNA i n human ( A t a x i a T e 1 a n g I a c t a s i a ) F i b r o b l a s t s . N a t u r e , 2 6 0 , 4 4 4 . P a y n e , M . G . & G a r r e t t , W . R . ( 1 9 7 5 ) Some r e l a t i o n s b e t w e e n c e l l s u r v i v a l m o d e l s h a v i n g d i f f e r e n t i n a c t i v a t i o n m e c h a n i s m s . R a d I a t . R e s . , 62 , 38 8 . P e t t e r s e n , E . O . , W i l b e , E . , L ^ v h a u g , D . , O f t e b r o , R. S B r u s t a d , T . ( 1 9 7 5 ) E f f e c t s o f o x y g e n a n d TMPN on t h e i n i t i a l p a r t o f t h e d o s e - e f f e c t c u r v e s o f human c e l l s i n c u l t u r e . I n : C e l l S u r v i v a l a f t e r Low D o s e s o f R a d i a t o n :  T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , ( T . A l p e r e d . ) , J o h n W i l e y a n d S o n s , L o n d o n , 1 1 9 . P o h l i t , W. ( 1 9 7 5 ) . The s h a p e o f d o s e - e f f e c t c u r v e s f o r d i p l o i d y e a s t c e l l s i r r a d i a t e d w i t h i o n i z i n g p a r t i c l e s . I n : C e l l S u r v i v a l a f t e r Low D o s e s o f R a d i a t o n :  T h e o r e t i c a l a n d C l i n i c a l I m p l i c a t i o n s , ( T . A l p e r e d . ) , J o h n W i l e y a n d S o n s , L o n d o n , P o h l i t , W. S H e y d e r , I . R . ( 1 9 8 1 ) The s h a p e o f d o s e s u r v i v a l c u r v e s f o r m a m m a l i a n c e l l s a n a l y z e d by h y p e r t o n i c t r e a t m e n t . R a d i a t . R e s . , 8 7 , 6 1 3 . 93 P o w e r s , E . L . ( 1 9 6 2 ) C o n s i d e r a t i o n s o f s u r v i v a l c u r v e s a n d t a r g e t t h e o r y . P h y s . M e d . B i o l . , 7_, 3 . P o w e r s , W . E . S T o l m a c h , L . J . ( 1 9 6 3 ) A m u 1 t i c o m p o n e n t x r a y s u r v i v a l c u r v e f o r m o u s e l y m p h o s a r c o m a c e l l s i r r a d i a t e d i n v i v o . N a t u r e , 1 9 7 , 7 1 0 . P r e s c o t t , D . M . S B e n d e r , M . A . ( 1 9 6 3 ) A u t o r a d i o g r a p h i c s t u d y o f c h r o m a t i d d i s t r i b u t i o n o f l a b e l l e d DNA i n t w o t y p e s o f m a m m a l i a n c e l l s i n v ? t r o . E x p . C e l 1 R e s . , 2 9 , 4 3 0 . P u c k , T . T . , M a r c u s , P . I . S C i e c i u r a , S . J . ( 1 9 5 6 ) C l o n a l g r o w t h o f m a m m a l i a n c e l l s In v i t r o . J . E x p t l . M e d . , 1 0 3 , 2 7 3 . P u c k , T . T . & M a r c u s , P . I . ( 1 9 5 6 ) A c t i o n o f x - r a y s on mamma 1 i a n c e 1 1 s . J . E x p t l . M e d . , 1 0 3 , 65 3 . R a a p h o r s t , G . P . S D e w e y , W . C . ( 1 9 7 9 a ) A l t e r a t i o n s i n t h e r a d I o s e n s i t i v i t y o f CHO c e l l s by a n i s o t o n i c t r e a t m e n t s : C o r r e l a t i o n s b e t w e e n c e l l l e t h a l i t y a n d c h r o m o s o m a l a b e r r a t i o n s . R a d i a t . R e s . , 7 9 , 40 3 . R a a p h o r s t , G . P . S D e w e y , W . C . ( 1 9 7 9 b ) A s t u d y o f r e p a i r o f p o t e n t i a l l y l e t h a l a n d s u b l e t h a l r a d i a t i o n d a m a g e In C h i n e s e h a m s t e r c e l l s e x p o s e d t o e x t r e m e l y h y p o o r h y p e r t o n i c N a C l s o l u t i o n s . Rad I a t . R e s . , 77 , 3 2 5 . R a a p h o r s t , G . P . S A z z a m , E . I . ( 1 9 8 1 F i x a t i o n o f p o t e n t i a l l y l e t h a l r a d i a t i o n d a m a g e i n C h i n e s e h a m s t e r c e l l s by a n i s o t o n i c s o l u t i o n s , p o l y a m i n e s a n d DMSO. R a d i a t . R e s . , 8 6 , 5 2 . R e g a n , J . D . S S e t l o w , R . B . ( 1 9 7 4 ) Two f o r m s o f r e p a i r i n t h e DNA o f human c e l l s d a m a g e d by c h e m i c a l s , c a r c i n o g e n s a n d m u t a g e n s . C a n c e r R e s . , 3 4 , 3 3 1 8 . R o b b i n s , E . P e d e r s o n , T . S K l e i n , P . ( 1 9 7 0 ) C o m p a r i s o n o f m i t o t i c p h e n o m e n a a n d e f f e c t s i n d u c e d by h y p e r t o n i c s o l u t i o n s i n H e L a c e l l s . J . C e l 1 B i o l . , 4 4 , 4 0 0 . 94 R o b e r t s o n , M . C . £ B e r m e k , E . ( 1 9 7 7 ) S t u d i e s o f t h e p r o t e i n s y n t h e t i c a c t i v i t y o f l y s a t e s f r o m H e L a c e l l s i n c u b a t e d i n h y p e r t o n i c m e d i u m . B i o c h i m . B i o p h y s . A c t a , 4 7 6 , 8 8 . R i t t e r , M . A . , C l e a v e r , J . E . £ T o b i a s , C . A . ( 1 9 7 7 ) H i g h LET r a d i a t i o n s i n d u c e a l a r g e p r o p o r t i o n o f n o n - r e j o i n i n g DNA b r e a k s . N a t u r e , 2 6 6 , 6 5 3 . R o o t s , R. £ S m i t h , K . C . ( 1 9 7 5 ) f.-' R e j o i n i n g o f DNA s i n g l e - s t r a n d b r e a k s i n m a m m a l i a n c e l l s j i n c u b a t e d i n b u f f e r o r i n m e d i u m a f t e r a e r o b i c o r a n a e r o b i c x - I r r a d . i a t i on . I n t . J . Rad i a t . B i o l . . 27 , 595 . R y d b e r g , B . ( 1 9 7 5 ) The r a t e o f s t r a n d s e p a r a t i o n i n a l k a l i o f DNA o f i r r a d i a t e d m a m m a l i a n c e l l s . Rad i a t . R e s . , 6 1 , 2 7 4 . R y d b e r g , B . ( 1 9 8 0 ) D e t e c t i o n o f i n d u c e d DNA s t r a n d b r e a k s w i t h i m p r o v e d s e n s i t i v i t y i n human" c e l l s . R a d I a t • R e s . , 81 , 4 9 2 . S a h a s r a b u d d h e , C . G . £ S a u n d e r s , G . F . ( 1 9 7 7 ) S a l t - I n d u c e d s t r u c t u r a l c h a n g e s i n n u c l e o s o m e s . N u c l e i c A c i d s R e s . , 4., 85 3 . S a w a d a , S . £ O k a d a , S . ( 1 9 7 2 ) E f f e c t s o f B U d R - 1 a b e 1 1 i n g on r a d i a t i o n - i n d u c e d DNA b r e a k a g e a n d s u b s e q u e n t r e j o i n i n g i n c u l t u r e d m a m m a l i a n c e l I s . I n t . J . R a d i a t . B i o l . , 2 1 , 5 9 9 . S c h e r , W. £ F r i e n d , C . ( 1 9 7 8 ) B r e a k a g e o f DNA a n d a l t e r a t i o n s i n f o l d e d g e n o m e s by I n d u c e r s o f d i f f e r e n t i a t i o n i n F r i e n d e r y t h r o l e u k e m i c c e l I s . C a n c e r R e s . , 38 , 8 4 1 . S i n c l a i r , W . K . ( 1 9 6 6 ) The s h a p e o f r a d i a t i o n s u r v i v a l c u r v e s o f m a m m a l i a n c e l l s c u l t u r e d i n v ? t r o . I n : B i o p h y s i c a l A s p e c t s o f R a d i a t i o n Q u a l i t y T e c h n i c a l R e p o r t S e r i e s N o . 5 8 , I . A . E . A . , V i e n n a , 2 1 . S i n c l a i r , W . K . ( 1 9 6 8 ) C y c l i c x - r a y r e s p o n s e s t o m a m m a l i a n c e l l s i n - v 1 1 r o . Rad ? a t . R e s . , 33 , 62 0 . 95 S k a r s g a r d , L . D . , K i h l m a n , B . A . , P a r k e r , L . , P u j a r a , C M . S R i ' c h a r d s o n , S . ( 1 9 6 7 ) S u r v i v a l , c h r o m o s o m e a b n o r m a l i t i e s ' a n d r e c o v e r y i n h e a v y - i o n a n d x - i r r a d i a t e d m a m m a l i a n c e l l s . R a d i a t . R e s . , s u p p 1 . 7 , 2 0 8 . S k o v , K . A . , P a l c i c , B . S S k a r s g a r d , L . D . ( 1 9 7 9 ) R a d i o s e n s i t i z a t i o n o f m a m m a l i a n c e l l s by m i s o a n d o x y g e n : DNA d a m a g e e x p o s e d by M . 1 u t e u s e n z y m e s . r a d i a t . R e s . , 7 9 , 5 9 1 . S k o v , K . A . , B e r g e r , S . , A d o m a t , K . , P a l c i c , B . S S k a r s g a r d , L . D . ( 1 9 8 2 ) C e l l u l a r r e p a i r o f r a d i a t I o n - i n d u c e d b r e a k s i n DNA. Rad i a t . R e s . , i n p r e s s , ( a b s t . ) S p a r r o w , A . H . , U n d e r b r o o k , A . G . & S p a r r o w , R . C ( 1 9 6 7 ) C h r o m o s o m e s a n d c e l l u l a r r a d i o s e n s i t i v i t y . I. The r e l a t i o n s h i p o f Dg t o c h r o m o s o m e v o l u m e a n d c o m p l e x i t y i n s e v e n t y - n i n e d i f f e r e n t o r g a n i s m s . R a d i a t . R e s . , 3 2 , 9 1 5 . S t r y e r , L . ( 1 9 8 1 ) B1 o c h e m l s t r y , 2 n d e d . , W . H . F r e e m a n a n d C o . , S a n F r a n s i s c o . S z y b a l s k i , W. ( 1 9 6 7 ) M o l e c u l a r e v e n t s r e s u l t i n g i n r a d i a t i o n i n j u r y , r e p a i r a n d s e n s i t i z a t i o n o f DNA. R a d i a t . R e s . , s u p p 1 . 7 , 1 4 5 . T h o m l i n s o n , R . H . & G r a y , L . H . ( 1 9 5 5 ) The h i s t o l o g i c a l s t r u c t u r e o f some human l u n g c a n c e r s a n d t h e p o s s i b l e I m p l i c a t i o n s f o r r a d i o t h e r a p y . B r . J . C a n c e r , 9., 5 3 9 . T h o m p s o n , L . H . S S u i t , H . D . ( 1 9 6 9 ) P r o l i f e r a t i o n k i n e t i c s o f x - i r r a d i a t e d m o u s e L c e l l s s t u d i e d w i t h t i m e l a p s e p h o t o g r a p h y I I . I n t . J . R a d i a t . B i o l . , 1 5 , 3 4 7 . T o b i a s , C . A . , B l a k e l y , E . A . , N g o , F . H . S Y a n g , T . H . ( 1 9 8 0 ) The r e p a i r - m I s r e p a i r m o d e l o f c e l l s u r v i v a l . I n : R a d i a t i o n B i o l o g y i n C a n c e r R e s e a r c h , ( R . E . M e y n S H . R . W i t h e r s e d s . ) , R a v e n P r e s s , New Y o r k , 1 9 5 . T o m u r a , T . S V a n L a n c k e r , J . L . ( 1 9 7 5 ) The e f f e c t o f a m a m m a l i a n r e p a i r e n d o n u c l e a s e o n x - i r r a d I a t e d DNA. B i o c h i m . B i o p h y s . A c t a , 4 0 2 , 3 4 3 . 96 V a n d e r S c h a n s , G . P . ( 1 9 7 8 ) G a m m a - r a y i n d u c e d d o u b l e s t r a n d b r e a k s i n DNA f r o m r a n d o m l y - i n f 1 i c t e d s i n g l e - s t r a n d b r e a k s : l o c a l d e n a t u r a t i o n , a new r a d i a t i o n p h e n o m e n o n ? I n t . J . R a d I a t . B I o l . , 33 , 1 0 5 . V o n S o n n t a g , C . , H a g e n , U . , S c h o n - B o p p , A . S S c h u l t e - F r o h l i n d e , D. ( 1 9 8 1 ) R a d i a t i o n - i n d u c e d s t r a n d b r e a k s i n DNA: C h e m i c a l a n d e n z y m a t i c a n a l y s i s o f e n d g r o u p s a n d m e c h a n i s t i c a s p e c t s . A d v . R a d 1 a t . B i o l . , 9., 1 0 9 . W a r d , J . F . ( 1 9 7 5 ) M o l e c u l a r m e c h a n i s m s o f r a d i a t I o n - 1 n d u c e d d a m a g e t o n u c 1 e I c a c i d s . A d v . R a d i a t . B i o l . , 5_, 1 8 1 . W a r d , J . F . , B l a k e l y , W . F . S J o i n e r , E . I . ( 1 9 8 2 ) E f f e c t s o f i n h i b i t o r s o f DNA s t r a n d b r e a k r e p a i r o n H e L a c e l l r a d i o s e n s I t i v t y . I n t . J . R a d i a t . O n e . B i o l . P h y s . , In p r e s s , ( a b s t . ) W a t s o n , J . D . ( 1 9 7 7 ) M o l e c u l a r B i o l o g y o f t h e G e n e , 3 r d e d . , W . A . B n j a m i n , I n c . , Don M i l l s , O n t a r i o . W e i b e z a h n , K . F . , S e x a u e r , C . 6 C o q u e r e l l e , T . ( 1 9 8 0 ) N e g a t i v e p i o n i r r a d i a t i o n o f m a m m a l i a n c e l l s . I I I . A c o m p a r a t i v e a n a l y s i s o f DNA s t r a n d b r e a k a g e r e p a i r a n d c e l l s u r v i v a l a f t e r e x p o s u r e t o IT - m e s o n s a n d x - r a y s . I n t . J . R a d i a t . B i o l . , 38 , 36 5 . W e i b e z a h n , K . F . £ C o q u e r e l l e , T . ( 1 9 8 1 ) D o u b l e - s t r a n d b r e a k s a r e r e j o i n e d by a l i g a t i o n a n d r e c o m b i n a t i o n p r o c e s s . N u c l e i c A c i d s R e s . , ^9, 3 1 3 9 . W h i l l a n s , D .W. S R a u t h , A . M . ( 1 9 8 0 ) An e x p e r i m e n t a l a n d a n a l y t i c a l s t u d y o f o x y g e n d e p l e t i o n i n s t i r r e d c e l l s u s p e n s i o n s . R a d i a t . R e s . , 8 4 , 9 7 . W i e r o w s k i , J . V . £ W h e e l e r , K . T . ( 1 9 8 2 ) I n s i t u r e p a i r i n l e s s a c e s s l b l e r e g i o n s _ o f m a m m a l i a n DNA. B i o p h y s . J . , 37 , 299 . ( a b s t . ) W i t h e r s , H . R . ( 1 9 7 5 a ) C e l l c y c l e r e d i s t r i b u t i o n a s a f a c t o r i n m u 1 t i f r a c t i o n i r r a d I a t i o n . Rad i o l o g y , 1 1 4 , 1 9 9 . r e s u l t i n g T e m p o r a 1 97 W i t h e r s , H . R . ( 1 9 7 5 b ) The f o u r R ' s o f r a d i a t h e r a p y . A d v . R a d i a t . B i o l . , 5, 2 4 1 . Z i r k l e , R . E . ( 1 9 5 7 ) P a r t i a l c e l l i r r a d i a t i o n . M e d . P h y s . , 5 , 1 0 3 . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095183/manifest

Comment

Related Items