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Effect of intercellular contact on radiation-induced DNA damage MacPhail, Susan Helen 1988

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EFFECT OF INTERCELLULAR CONTACT ON RADIATION-INDUCED DNA DAMAGE By SUSAN HELEN MACPHAIL B. Sc., The U n i v e r s i t y o f B r i t i s h C o lumbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f P a t h o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d Dr. P. L. Olive Dr. R. Durand Dr. B. Palcic Dr. S. Dedhar THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © Susan H e l e n M a c P h a i l , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia Vancouver, Canada Date A p r i l 28. 1988  DE-6 (2/88) ABSTRACT Chin e s e hamster V79-171B c e l l s grown f o r about 24 hours i n s u s p e n s i o n c u l t u r e d i s p l a y i n c r e a s e d r e s i s t a n c e t o c e l l k i l l i n g by i o n i z i n g r a d i a t i o n compared w i t h c e l l s grown as m o n o l a y e r s , an o b s e r v a t i o n o r i g i n a l l y termed t h e " c o n t a c t e f f e c t " . More r e c e n t l y , development of t h a t r e s i s t a n c e was shown t o be accompanied by changes i n t h e c o n f o r m a t i o n o f t h e DNA w h i c h reduce i t s d e n a t u r a t i o n r a t e i n h i g h s a l t / w e a k a l k a l i . These changes i n DNA c o n f o r m a t i o n , m e d i a t e d by t h e c e l l u l a r m i c r o -e n v i r o n m e n t , appear t o be r e s p o n s i b l e f o r t h e c o n t a c t e f f e c t . The c o n d i t i o n s n e c e s s a r y f o r t h e development o f t h e e f f e c t a r e n o t , however, c o m p l e t e l y u n d e r s t o o d . I n p a r t i c u l a r , when c e l l s grown as monolayers on p e t r i p l a t e s a r e suspended i n s p i n n e r c u l t u r e f l a s k s , t h e i r growth c h a r a c t e r i s t i c s change i n t h r e e d i s t i n c t ways. F i r s t , c e l l s i n s u s p e n s i o n no l o n g e r have a s o l i d s u b s t r a t e , so t h e y remain round. Second, a f t e r s e v e r a l h o u r s , t h e y b e g i n t o aggregate t o form " s p h e r o i d s " , so t h a t t h r e e - d i m e n s i o n a l i n t e r c e l l u l a r c e l l c o n t a c t d e v e l o p s . T h i r d , c e l l s i n t h e s t i r r e d s u s p e n s i o n c u l t u r e s a r e not s u b j e c t e d t o h i g h l o c a l c o n c e n t r a t i o n s o f m e t a b o l i c b y - p r o d u c t s o r s u r r o u n d e d by a zone d e p l e t e d o f n u t r i e n t s , as a r e c e l l s i n monolayer c u l t u r e . The s t u d i e s d e s c r i b e d h e r e were d e s i g n e d t o d e t e r m i n e how each o f t h e s e f a c t o r s i n f l u e n c e changes i n DNA c o n f o r m a t i o n , as a s s a y e d u s i n g t h e a l k a l i u n w i n d i n g t e c h n i q u e . Our r e s u l t s i n d i c a t e d t h a t a round shape may not be an e s s e n t i a l r e q u i r e m e n t , s i n c e c e l l s s p r e a d out on t h e s u r f a c e of c y t o d e x beads i n s u s p e n s i o n c u l t u r e , and s p a r s e l y - s e e d e d c e l l s i n monolayer c u l t u r e d emonstrated a t l e a s t a p a r t i a l c o n t a c t e f f e c t . T h r e e - d i m e n s i o n a l i n t e r c e l l u l a r c o n t a c t does not always seem n e c e s s a r y f o r t h e development of t h e c o n t a c t e f f e c t . C e l l s grown i n a m e t h y l c e l l u l o s e m a t r i x d e v e l o p e d r a d i o r e s i s t a n c e , even though t h e c e l l s formed o n l y s m a l l c l u s t e r s o f l e s s t h a n f i v e c e l l s . S i m i l a r l y , s u s p e n s i o n c u l t u r e c e l l s w h i c h were p r e v e n t e d from a g g r e g a t i n g by f r e q u e n t exposure t o t r y p s i n , a l s o d e v e l o p e d t h e c o n t a c t e f f e c t . There was no e v i d e n c e t h a t n u t r i e n t d e p l e t i o n p l a y s a r o l e i n t h e f a i l u r e o f c e l l s grown as monolayers t o d e v e l o p a c o n t a c t e f f e c t . However, c e l l s grown as s p h e r o i d s i n t h e p r e s e n c e of monolayer c e l l s , o r i n monolayer c e l l - c o n d i t i o n e d medium, d i d not d i s p l a y a f u l l c o n t a c t e f f e c t . T h i s i n d i c a t e s a r o l e f o r monolayer c e l l - p r o d u c e d f a c t o r s ( p o s s i b l y e x t r a c e l l u l a r m a t r i x p r o t e i n s ) i n p r e v e n t i n g t h e development o f t h e c o n t a c t e f f e c t . We c o n c l u d e t h a t changes i n DNA c o n f o r m a t i o n and t h e i n c r e a s e i n r a d i a t i o n r e s i s t a n c e , seen i n V79-171b c e l l s grown as s p h e r o i d s , a r e not t h e r e s u l t of i n t e r c e l l u l a r c o n t a c t o r round shape of t h e c e l l s . T h i s r a d i o r e s i s t a n c e appears t o be t h e r e s u l t o f an absence of monolayer c e l l - p r o d u c e d f a c t o r s w h i c h c o u l d c o n t r o l b o t h c e l l shape and DNA c o n f o r m a t i o n . i i i TABLE OF CONTENTS Page A b s t r a c t i i L i s t o f T a b l e s v i i L i s t o f F i g u r e s v i i i Acknowledgements x I . I n t r o d u c t i o n 1 A. The Problem 1 B. The R a t i o n a l e 2 C. H i s t o r y of t h e C o n t a c t E f f e c t 3 D. DNA as a T a r g e t o f R a d i a t i o n Damage 10 E. A R o l e f o r DNA C o n f o r m a t i o n i n t h e C o n t a c t E f f e c t . 14 F. The Approach 17 I I . M a t e r i a l s and Methods 20 A. C e l l L i n e s and C u l t u r e Techniques 20 a. V79-171b C e l l L i n e 20 b. SCCVII C e l l L i n e 21 c. RIF-1 C e l l L i n e 22 d. WiDr C e l l L i n e 22 e. C-4 I , C-4 I I C e l l L i n e s 23 B. P r o c e d u r e s 23 a. P r e p a r a t i o n of Samples 23 b. DNA S t r a n d Breakage A n a l y s i s 25 c. H y d r o x y a p a t i t e Chromatography 26 d. C a l c u l a t i n g DNA Damage 27 i v C. Treatments 28 a. C e l l Crowding and Density 29 b. Medium Flow 30 c. Conditioned Medium 31 d. Poly (HEMA) 32 e. Trypsinization 33 f. Methyl Cellulose 3 4 I I I . Results 35 A. Culture Conditions Under Which the Contact E f f e c t i s Observed 35 a. C e l l Crowding i n Monolayer Cultures 38 b. Medium Replenishment i n Monolayer Cultures.... 40 c. Growth of C e l l s on Microcarrier Beads 44 d. Growth of C e l l s i n Suspension Culture 48 e. E f f e c t s of T r y s i n i z i n g C e l l Cultures 51 f. Growth of C e l l s i n Methyl Cellulose 53 g. Growth of C e l l s with Poly (HEMA) 56 B. Factors I n h i b i t i n g Development of the Contact E f f e c t 58 a. I n h i b i t i o n of C e l l Growth and D i v i s i o n 58 b. Growth of C e l l s i n Monolayer Culture 58 c. Use of Conditioned Medium 59 C. Other C e l l Lines 62 IV. Discussion 73 A. I n t e r c e l l u l a r Contact and Communication 7 3 B. DNA Conformation and C e l l Shape 76 C. C e l l Attachment and Spreading Factors 7 8 v D. R o l e of C e l l Shape and E x t r a c e l l u a r M a t r i x 81 V. Summary and C o n c l u s i o n 88 V I . R e f e r e n c e s 90 v i LIST OF TABLES T a b l e T i t l e Page I . C e l l L i n e s Examined f o r t h e C o n t a c t E f f e c t 6 I I . Summary of R e s u l t s from V79 C e l l L i n e E x p e r i m e n t s . . . 36 I I I . Summary o f R e s u l t s from Tumor C e l l L i n e E x p e r i m e n t s . 72 v i i LIST OF FIGURES F i g u r e T i t l e Page 1. The C o n t a c t E f f e c t 4 2. Mechanisms o f DNA S t r a n d Breakage by t h e H y d r o x y l R a d i c a l 12 3. Schematic R e p r e s e n t a t i o n o f t h e A l k a l i Unwinding Assay 16 4. Proposed Model f o r t h e P r e s e n c e o f C o n s t r a i n t s t o DNA Unwinding i n C e l l s Grown as S p h e r o i d s 18 5. M i c r o s c o p i c Appearance o f C e l l s U s i n g D i g i t i z e d Images 37 6. E f f e c t o f C e l l D e n s i t y - V a r y i n g C e l l Number 39 7. E f f e c t o f C e l l Crowding on Monolayer C e l l s 41 8. E f f e c t of C e l l D e n s i t y - X-ray Dose Response 42 9. E f f e c t o f C e l l D e n s i t y - DNA Unwinding K i n e t i c s 43 10. Monolayer C u l t u r e s i n Moving Medium o f S p i n n e r F l a s k s 45 11. Monolayer C u l t u r e s i n Renewed Medium - X-ray Dose Response 46 12. Monolayer C u l t u r e s i n Renewed Medium - DNA Unwinding K i n e t i c s 47 13. Monolayer C u l t u r e s on M i c r o c a r r i e r Beads 49 14. E f f e c t of C e l l Crowding on S p h e r o i d C e l l s 50 15. T r y p s i n Treatment o f M o n o l a y e r s 52 16. T r y p s i n Treatment of S p h e r o i d s 54 v i i i 17. Monolayer C u l t u r e s Grown i n M e t h y l C e l l u l o s e 5 5 18. C e l l s Grown w i t h Poly(HEMA) 57 19. Monolayer C u l t u r e s i n Moving Medium on Moving P l a t f o r m I n c u b a t o r 60 20. C e l l s Grown i n C o n d i t i o n e d Medium - X - r a y Dose Response 61 21. C o n d i t i o n e d Medium C o n t r o l - Growth on G l a s s P e t r i P l a t e s 63 22. C o n d i t i o n e d Medium C o n t r o l - Medium C o n d i t i o n e d i n G l a s s o r P l a s t i c 64 23. SCCVII C e l l L i n e and t h e C o n t a c t E f f e c t - X - r a y Dose Response 65 24. SCCVII C e l l L i n e and t h e C o n t a c t E f f e c t - DNA Unwinding K i n e t i c s 66 25. RIF-1 C e l l L i n e and t h e C o n t a c t E f f e c t - X-ray Dose Response 68 26. RIF-1 C e l l L i n e and t h e C o n t a c t E f f e c t - DNA Unwinding K i n e t i c s 69 27. WiDr C e l l L i n e and t h e C o n t a c t E f f e c t 70 28. C-4 I and C-4 I I C e l l L i n e s and t h e C o n t a c t E f f e c t . . 71 i x ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h er g r a t i t u d e t o Dr. Peggy L. O l i v e o f t h e Departments of P a t h o l o g y and P h y s i c s . T h i s r e s e a r c h was a c c o m p l i s h e d under h er d i r e c t i o n and o n l y h e r p a t i e n c e and sense o f humor made i t p o s s i b l e . Acknowledgements must a l s o be made t o Dr. R a l p h Durand and Dr. Branko P a l c i c , a l s o o f t h e Departments o f P a t h o l o g y and P h y s i c s , who a c t e d as a d v i s o r s f o r t h i s p r o j e c t . T h i s work was s u p p o r t e d by t h e M e d i c a l R e s e a r c h C o u n c i l and was c a r r i e d o u t i n t h e M e d i c a l B i o p h y s i c s U n i t o f t h e B r i t i s h Columbia Cancer R e s e a r c h C e n t r e . x INTRODUCTION A. The Problem Chinese hamster V79 l u n g f i b r o b l a s t s , grown f o r j u s t 24 hours i n s u s p e n s i o n c u l t u r e , a r e more r e s i s t a n t t o damage by i o n i z i n g r a d i a t i o n t h a n a r e t h e same c e l l s grown as monolayers. The i n c r e a s e d s u r v i v a l o f t h e c e l l s grown i n s u s p e n s i o n s u g g e s t s an enhanced c a p a c i t y f o r a c c u m u l a t i o n and f o r r e p a i r o f s u b l e t h a l r a d i a t i o n damage (17). I t was th o u g h t t h a t t h e t h r e e - d i m e n s i o n a l c o n t a c t w h i c h d e v e l o p s between c e l l s grown i n s u s p e n s i o n i n some way a f f e c t s r a d i a t i o n s e n s i t i v i t y , and t h i s i n c r e a s e d r a d i o -r e s i s t a n c e was t h e r e f o r e termed t h e " c o n t a c t e f f e c t " . P r e l i m i n a r y e x p e r i m e n t s , done i n our l a b o r a t o r y u s i n g s p a r s e l y - s e e d e d monolayer c u l t u r e s , s u g g e s t e d t h a t t h r e e -d i m e n s i o n a l c o n t a c t was not t h e o n l y f a c t o r c o n t r i b u t i n g t o development o f t h e c o n t a c t e f f e c t . Hence, u s i n g t h e t e c h n i q u e of a l k a l i u n w i n d i n g of t h e DNA t o i n d i c a t e t h e p r e s e n c e of the c o n t a c t e f f e c t , my i n t e n t i o n s were t o d e t e r m i n e : a) whether changes i n c e l l shape, p r e c e d i n g X - r a y t r e a t m e n t , c o n t r i b u t e t o t h e r a d i o r e s i s t a n c e . b) whether m a n i p u l a t i o n o f growth environment a l t e r s 1 c e l l u l a r r a d i o s e n s i t i v i t y . c) whether o t h e r c e l l t y p e s d i s p l a y a c o n t a c t e f f e c t i n terms of DNA unwind i n g k i n e t i c s . The s p e c i f i c g o a l s o f t h i s u n d e r t a k i n g were t o examine t h e r e l a t i v e i m p o r t a n c e of c e l l - t o - c e l l c o n t a c t , c e l l shape and m e t a b o l i t e exchange i n t h e development of t h e c o n t a c t e f f e c t . B. The R a t i o n a l e Human c a n c e r c e l l s grown i n v i t r o have been used t o p r e d i c t tumor r a d i o s e n s i t i v i t y t o a n t i c a n c e r t h e r a p y , b u t t h e r e s u l t s do not always t a k e i n t o account t h e r o l e of t h e t h r e e - d i m e n s i o n a l growth t h a t o c c u r s i n s o l i d tumors. C e l l s grown i n c l o s e t h r e e - d i m e n s i o n a l c o n t a c t o f t e n d i f f e r i n r a d i a t i o n s e n s i t i v i t y , perhaps as a r e s u l t of a change i n t h e a b i l i t y o f t h e c e l l s t o r e p a i r DNA damage (17, 24, 2 9 ) . The r a d i o c u r a b i l i t y o f human tumors v a r i e s w i d e l y and some o f t h i s v a r i a b i l i t y may be r e l a t e d t o t h e a b i l i t y o f d i f f e r e n t tumors and normal t i s s u e s t o r e p a i r r a d i a t i o n damage, and not m e r e l y t o the i n h e r e n t r a d i o s e n s i t i v i t y of t h e v a r i o u s tumors ( 5 8 ) . T h e r e f o r e , t h e r a t i o n a l e f o r t h i s p r o j e c t was t o d e v e l o p a g r e a t e r u n d e r s t a n d i n g o f t h e f a c t o r s i n f l u e n c i n g tumor response t o t h e r a p y and t o d e v e l o p s u i t a b l e models t o s t u d y t h e s e f a c t o r s . 2 The role of DNA conformation i n radiation s e n s i t i v i t y i s also emphasized i n t h i s thesis. The idea that c e l l s e n s i t i v i t y to damage i s the r e s u l t of the action of DNA repair genes i s an obvious o v e r s i m p l i f i c a t i o n . Increased radioresistance may be due to differences i n DNA conformation (43). Changes i n DNA conformation i n response to changes i n c e l l shape and growth substratum are believed to occur, but d i r e c t evidence l i n k i n g DNA conformation to c e l l shape or e x t r a c e l l u l a r environment i s lacking. C. History of the Contact E f f e c t More than a decade ago, Durand and Sutherland (17) f i r s t reported that Chinese hamster V79 c e l l s , grown i n suspension culture, form c l u s t e r s of c e l l s which are more r e s i s t a n t to k i l l i n g by i o n i z i n g radiation than the same c e l l s grown on p e t r i plates as monolayers. Since the c e l l s i n i t i a l l y aggregate and then grow as "spheroids" i n suspension culture, contact between the c e l l s was presumed to play a role i n the development of t h i s increased resistance. Therefore, t h i s resistance was termed the "contact e f f e c t " . After 24 hours i n suspension culture, there i s no i n d i c a t i o n of differences, between spheroid and monolayer c e l l s , i n c e l l cycle d i s t r i b u t i o n or rate of DNA synthesis, yet there i s an increase i n the f r a c t i o n of c e l l s which survive to form colonies a f t e r radiation exposure (see Figure 1). Thus, c e l l cycle and DNA synthesis cannot account for the increased radia t i o n resistance of the spheroid c e l l s . The small size 3 0 5 10 15 R a d i a t i o n D o s e (Gy) Figure 1. The Contact E f f e c t . Survival curves of c e l l s from day-old spheroid and monolayer cultures. These c e l l s were grown as usual, then try p s i n i z e d to single c e l l suspensions and i r r a d i a t e d at room temperature i n spinner f l a s k s . The c e l l s were then plated at pre-determined d i l u t i o n s , i n ten ml of growth medium and incubated for seven days, after which the surviving c e l l s , which had formed colonies were counted. This figure displays the increased s u r v i v a l of the spheroid c e l l s as compared with the monolayer c e l l s . As evidenced by the wider shoulder, the spheroid c e l l s are capable of increased accumulation and repair of sublethal radiation damage. 4 (20 to 50 c e l l s i n a cluster) of the spheroids eliminates com-pl i c a t i o n s that could be caused by hypoxic and non-cycling c e l l s present i n the larger spheroids. An increase i n the shoulder of the radiation s u r v i v a l curve for spheroid c e l l s i s correlated with enhanced a b i l i t y to repair sublethal c e l l damage. The role of c e l l - c e l l contact at the time of i r r a d i a t i o n i s unclear however. The spheroid c e l l s demonstrate increased radio-resistance even when separated before i r r a d i a t i o n and exposed as single c e l l s , and for several hours after d i s s o c i a t i o n . The ef f e c t i s l o s t gradually over the time i t takes the c e l l s to spread out afte r p l a t i n g as monolayers (17, 42). It seems that the h i s t o r y of growth of the c e l l s i s more c r i t i c a l to radio-resistance than contact during r a d i a t i o n exposure. Other investigators have since reported a si m i l a r "contact e f f e c t " , i n s o l i d tumors as well as spheroids, that enhances resistance to radiati o n (see Table I ) . However, the e f f e c t varies considerably between c e l l l i n e s and does not appear to be universal. Many d i f f e r e n t c e l l l i n e s , including human (HeLa), hamster (V79, B14-FAF28), mouse (3T3, L) and rat (BICR/MlR-K), were used by Dertinger and Heulser (13). Their r e s u l t s indicated that the contact e f f e c t was a property acquired during three-dimensional growth under the influence of i n t e r c e l l u l a r communication, at least i n these c e l l l i n e s . Guichard et a l . (24) used four human tumor c e l l l i n e s ( B e l l melanoma, HCT-8, HRT-18 and HT-29) growing as xenografts i n athymic mice. C e l l s were i s o l a t e d , plated as monolayers, and i r r a d i a t e d i n v i t r o . 5 TABLE I: CELL LINES EXAMINED FOR THE "CONTACT EFFECT" Contact Eff e c t E l e c t r i c a l l y Coupled Ref. Chinese hamster V79-171b V79-M4 V79 B14-FAF28 CHO Mouse 3T3 L KHT Si mammary Ca JM1 mammary Ca Embryo c e l l s EMT6-RW ++ + ++ ++ + + + 7 7 + + 9 + ? ? ? 7 Durand (17) Dertinger (13) n Du r and, unpub. Dertinger (13) H i l l (29) Durand, unpub. II Rockwell (47) Rat BICR/MIR-K P19 9L Mammary Thyroid ++ + + + + ? ? Dertinger (13) Olive, unpub. Wallen (55) Cathers (9) Mulcahy (39) Human Hela + HT-29 colon Ca ++ HRT-18 colon Ca ++ B e l l melanoma ++ Na i l melanoma HCT-8 colon Ca + Mammary + Skin f i b r o b l a s t s ++ + ? 7 7 7 7 Dertinger (13) Guichard (24) Cathers (9) Durand, unpub. 6 These were compared w i t h tumors i r r a d i a t e d i n v i v o . Three ( a l l b u t HCT-8) showed i n c r e a s e d r a d i o r e s i s t a n c e i n v i v o w h i c h c o u l d not be a c c o u n t e d f o r s o l e l y by the f a c t t h a t t h e c e l l s were h y p o x i c - U s i n g EMT6-RW mouse mammary c e l l s , grown as s o l i d tumors i n BALB/c mice and as monolayers, t h e n i r r a d i a t e d under c o n d i t i o n s o f u n i f o r m , maximal h y p o x i a , R o c k w e l l (47) f a i l e d t o f i n d e v i d e n c e f o r a c o n t a c t e f f e c t . A l s o , no d i f f e r e n c e was ob s e r v e d i n t h e r a d i o r e s i s t a n c e of c e l l s p l a t e d from t h e s o l i d tumors and i r r a d i a t e d a t v a r i o u s t i m e s a f t e r p l a t i n g . T h i s i n d i c a t e d t h a t t h e r e was no i n c r e a s e of r a d i o s e n s i t i v i t y w i t h time as would be seen w i t h a c o n t a c t e f f e c t . T h i s was a l s o t r u e of t h e two human melanoma x e n o g r a f t s used by R o f s t a d and B r u s t a d ( 4 8 ) . A l t h o u g h some form o f c e l l - t o - c e l l communication may have been p r e s e n t , i t d i d not enhance t h e r a d i o r e s i s t a n c e o f t h e s e melanomas. C u r t i s and Tenf o r d e (12) d e v e l o p e d a model f o r t h e r a d i a t i o n r e s p o n s e o f t h e rhabdomyosarcoma tumor system i n r a t s , u s i n g an i n v i t r o system. C e l l s u r v i v a l d a t a i n d i c a t e d t h a t o xygenated tumor c e l l s i n s i t u were l e s s r a d i o s e n s i t i v e ( o r more r e a d i l y r e p a i r e d ) than the same c e l l s i r r a d i a t e d i n s u s p e n s i o n , i n d i c a t i n g t h e pr e s e n c e of some ty p e of c o n t a c t e f f e c t . I n a s t u d y of t h e r a d i o s e n s i t i v i t y of r a t 9L subcutaneous tumor c e l l s from tumors of v a r i o u s s i z e s , W a l l e n e t a l . ( 5 5 ) r e p o r t e d a c o n t a c t e f f e c t when t h e c e l l s were i r r a d i a t e d i n v i v o compared t o exposure i n v i t r o as s i n g l e c e l l s u s p e n s i o n s . T h i s tumor does not d e v e l o p h y p o x i a as do some o t h e r tumors and s p h e r o i d s . H y p o x i a causes r a d i o r e s i s t a n c e and can be a 7 c o m p l i c a t i n g f a c t o r i n c o n t a c t e f f e c t s t u d i e s . C e l l s i r r a d i a t e d as s i n g l e c e l l s u s p e n s i o n s showed o n l y a s l i g h t i n c r e a s e i n r a d i o r e s i s t a n c e compared w i t h t h o s e grown as monolayer c u l t u r e s , and t h i s d i d not change w i t h t i m e . I f t h e c o n t a c t e f f e c t i s d e f i n e d as a r i s i n g i n c e l l s grown t o g e t h e r t h r e e - d i m e n s i o n a l l y and r e t a i n i n g a r e s i d u a l o f t h e p r o p e r t y o f r a d i o r e s i s t a n c e f o r s e v e r a l h o u r s , whether i r r a d i a t e d i n c o n t a c t o r n o t , t h e e f f e c t i s m i n i m a l i n t h e s e c e l l s . KHT sarcoma c e l l s were i r r a d i a t e d , i n c o n t a c t as s m a l l l u n g c o l o n i e s i n s i t u and as i n d i v i d u a l c e l l s i n v i t r o , by H i l l e t a l . ( 2 9 ) . The i n v i t r o e x p e r i m e n t s were performed u s i n g c e l l s u s p e n s i o n s w h i c h were i r r a d i a t e d as a f u n c t i o n o f time a f t e r e x c i s i o n of t h e tumors from t h e h o s t a n i m a l s . I n agreement w i t h r e s u l t s r e p o r t e d by Durand and S u t h e r l a n d ( 1 7 ) , c e l l s u r v i v a l d e c r e a s e d d u r i n g t h e f i r s t c e l l c y c l e . I n c o n t r a s t w i t h r e s u l t s r e p o r t e d by Durand and S u t h e r l a n d , t h e r e was not a l a r g e i n i t i a l d i f f e r e n c e i n c e l l s u r v i v a l between t h e c e l l s i r r a d i a t e d i n c o n t a c t as l u n g c o l o n i e s and t h o s e i r r a d i a t e d w i t h i n two hours a f t e r e x c i s i o n and s e p a r a t i o n i n t o s i n g l e c e l l s . A n o ther e f f e c t t h a t was seen w i t h some c e l l t y p e s i s t h a t o f i n c r e a s e d c e l l s u r v i v a l i f t h e c e l l s a r e l e f t i n c o n t a c t f o r some time a f t e r i r r a d i a t i o n . C a t h e r s and Gould (9) measured t h i s e f f e c t w i t h i r r a d i a t e d r a t mammary g l a n d s . Mulcahy e t a l . (39) used normal r a t t h y r o i d c e l l s and found t h a t t h e r e was no d i f f e r e n c e i n c e l l s u r v i v a l between c e l l s i r r a d i a t e d i n v i v o and 8 removed i m m e d i a t e l y f o r a s s a y , and tho s e i r r a d i a t e d as s i n g l e c e l l s i n v i t r o . However, an i n c r e a s e i n c e l l s u r v i v a l was seen i f t h e c e l l s were i r r a d i a t e d and t h e n l e f t i n s i t u f o r 24 hours b e f o r e a s s a y i n g . T h i s i n s i t u r e p a i r i n v o l v e s an i n c r e a s e i n the s h o u l d e r o f t h e s u r v i v a l c u r v e , w i t h no change i n t h e s l o p e . T h i s i s analogous t o r e s u l t s o b t a i n e d by Durand and S u t h e r l a n d (18) w i t h V79 s p h e r o i d s w h i c h were i r r a d i a t e d and t h e n p l a t e d i n t a c t . S u r v i v a l was g r e a t e r t h a n f o r t h o s e d i s s o c i a t e d and p l a t e d as s i n g l e c e l l s i m m e d i a t e l y a f t e r i r r a d i a t i o n . S e v e r a l o t h e r o b s e r v a t i o n s have been made w h i c h may h e l p e l u c i d a t e t h e mechanism(s) o f t h e c o n t a c t e f f e c t . Durand and S u t h e r l a n d (18) found t h a t exposure o f V79 c e l l s t o t h e c h e m i c a l mutagen N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) r e s u l t e d i n the c l o n i n g o f a c e l l s u b l i n e w h i c h d i d not d i s p l a y a c o n t a c t e f f e c t , s u g g e s t i n g t h a t t h e c o n t a c t e f f e c t may be m e d i a t e d by g e n e t i c e v e n t s . H i l l e t a l . (29) o b s e r v e d t h a t , w i t h KHT sarcoma c e l l s , t h e l o s s o f t h e c o n t a c t e f f e c t as a f u n c t i o n o f time a f t e r the tumors were d i s s o c i a t e d d i d not r e q u i r e c e l l d i v i s i o n . C e l l s u r v i v a l d e c l i n e d a t t h e same r a t e i f t h e c e l l s were h e l d a t 37°C or 22°C (when movement t h r o u g h t h e c e l l c y c l e would be s l o w e d ) , b u t d i d not d e c l i n e i f t h e c e l l s were h e l d a t 4°C between p l a t i n g and i r r a d i a t i o n . T h i s s u g g e s t e d t h a t a m e t a b o l i c p r o c e s s was r e s p o n s i b l e . A l o n g t h i s same l i n e , Edgren ( 1 9 ) , u s i n g an e x p e r i m e n t a l system o f g l u t a t h i o n e - d e f i c i e n t and - p r o f i c i e n t human f i b r o b l a s t s , o b s e r v e d t h a t some p a r t of t h e c e l l u l a r r e j o i n i n g p r o c e s s o f DNA s i n g l e - s t r a n d b r e a k s r e q u i r e d t h e 9 presence of glutathione or some other aminothiol. Sweigert and Alpen (51) reported that 9L rat gliosarcoma spheroid c e l l s displayed an increased radiation-induced d i v i s i o n delay that was s i g n i f i c a n t l y longer than for monolayer c e l l s . They speculated that, l i k e the contact e f f e c t , t h i s d i v i s i o n delay could be a consequence of growth of the c e l l s i n three-dimensional contact, or at least of the spherical shape. They suggested that enhanced rad i a t i o n s u r v i v a l seen with spheroid c e l l s might be a d i r e c t r e s u l t of increased d i v i s i o n delay, although other studies, using the V79 c e l l l i n e , do not support t h i s hypothesis (14, 37). There are many c e l l types, normal and transformed, where three-dimensional growth r e s u l t s i n greater c e l l s u r v i v a l at a given radiat i o n dose. This radioresistance, or contact e f f e c t , i s not universal. I t manifests i t s e l f i n various ways i n d i f f e r e n t c e l l l i n e s and the mechanism(s) i s s t i l l unknown. D. DNA as a Target of Radiation Damage The b i o l o g i c a l e f f e c t s of i o n i z i n g r a d i a t i o n are caused by the absorption of the radiation energy i n c e l l s . Absorption may lead to e x c i t a t i o n , or at higher energies, to i o n i z a t i o n when one or more o r b i t a l electrons are ejected from the atom. Free r a d i c a l s and excited molecules can r e s u l t . Free radicals are e l e c t r i c a l l y neutral atoms or molecules having an unpaired electron i n t h e i r outer o r b i t a l s . They are very reactive and can be electron acceptors or donors. In l i v i n g material, which 10 c o n s i s t s of 70 - 90 p e r c e n t w a t e r , most of t h e ab s o r b e d energy w i l l be t a k e n up by water m o l e c u l e s . H y d r a t e d e l e c t r o n s , h y d r o x y l r a d i c a l s and hydrogen atoms a r e formed, w h i c h a r e a l l h i g h l y r e a c t i v e . T h i s i n d i r e c t a c t i o n of i o n i z i n g r a d i a t i o n uses aqueous f r e e r a d i c a l s as i n t e r m e d i a r i e s i n t h e t r a n s f e r o f r a d i a t i o n energy t o b i o l o g i c a l m o l e c u l e s . I n t e r a c t i o n w i t h a water m o l e c u l e produces an i o n r a d i c a l and an e l e c t r o n : H 20 > H 2 0 + + e" The i o n r a d i c a l i s h i g h l y r e a c t i v e , b u t s h o r t - l i v e d and r e a c t s w i t h a n o t h e r w a t e r m o l e c u l e t o produce an i o n and a f r e e r a d i c a l : H 2 0 + + H 20 } H 3 0 + + *OH T h i s h y d r o x y l r a d i c a l i s r e a c t i v e and a l s o has a r e l a t i v e l y l o n g l i f e t i m e and so i s t h e major m e d i a t o r o f m a c r o m o l e c u l a r damage i n the c e l l . Perhaps 7 5 p e r c e n t o f a l l X - r ay damage i s me d i a t e d by the h y d r o x y l r a d i c a l and damage o c c u r s i n a l l b i o l o g i c a l macro-m o l e c u l e s . L e s s f r e q u e n t i s t h e d i r e c t 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 , w h i c h i n v o l v e s t h e s i m p l e i n t e r a c t i o n between t h e i o n i z i n g r a d i a t i o n and c r i t i c a l b i o l o g i c a l m o l e c u l e s . The e v e n t s of g r e a t e s t b i o l o g i c a l s i g n i f i c a n c e p r o b a b l y i n v o l v e damage t o DNA. H y d r o x y l r a d i c a l s cause s t r a n d b r e a k s by a t t a c k i n g t h e sugar m o i e t y o f t h e p h o s p h o d i e s t e r backbone. T h i s r e a c t i o n p r o b a b l y r e s u l t s i n two t y p e s o f b r e a k s ( F i g u r e 2 ) ; e i t h e r t h e complete e l i m i n a t i o n o f t h e sugar and a s s o c i a t e d b a se, l e a v i n g 1 1 Figure 2. Mechanisms of DNA Strand Breakage by the Hydroxyl Radical (31). DNA + •OH ^/OP0CH2 ^ Base H 0. ' Abstraction 0 "0—P—0/v 0 0 II /^/0P0CH2 Base Elimination © H 2 0 H+ Rapid 0 I /^/OPO + ® C H 2 Base /V/0P0CH2 « Base 0. | 2nd £ I - 0 - ' Elimination 0_ HO HO • RH —R O II ^ / 0 P 0 C H 2 ^ , , _ Base Disproportionation Reaction 0 " Base / A ^ 0 P 0 C H 2 0, O II r x / 0 P 0 - C H 2 0_ 1 1 H -f Base 12 p h o s p h o r y l groups a t b o t h the 51 and 3' t e r m i n i , or a b r eak w h i c h l e a v e s a p h o s p h o r y l group a t t h e 5' end, b u t an open r i n g s t r u c t u r e o f t h e sugar a t the 3' end. S e v e r a l t y p e s of DNA damage can r e s u l t from i o n i z i n g r a d i a t i o n , i n c l u d i n g base damage, base l o s s , DNA-DNA and DNA-p r o t e i n c r o s s l i n k s and damage t o t h e sugar-phosphate backbone, r e s u l t i n g i n DNA s i n g l e - and d o u b l e - s t r a n d b r e a k s . F o r low LET ( l i n e a r energy t r a n s f e r ) r a d i a t i o n l i k e X - r a y s , d o u b l e - s t r a n d b r e a k s o c c u r much l e s s f r e q u e n t l y t h a n s i n g l e - s t r a n d b r e a k s and when u n r e p a i r e d , a r e thought t o be t h e " l e t h a l " l e s i o n i n some r e p a i r models (8, 6 1 ) . Two s i n g l e - s t r a n d b r e a k s , v e r y c l o s e t o g e t h e r on o p p o s i t e DNA s t r a n d s , can a l s o g i v e r i s e t o d o u b l e -s t r a n d b r e a k s . T h e r e f o r e , th e measurement of DNA s t r a n d breakage by i o n i z i n g r a d i a t i o n s h o u l d p r o v i d e i n f o r m a t i o n t h a t i s r e l e v a n t t o c e l l s u r v i v a l . The r e p a i r of r a d i a t i o n - i n d u c e d DNA damage i s assumed t o be p e r f o r m e d by e n z y m a t i c r e p a i r complexes. R e p a i r may r e q u i r e s e v e r a l enzyme complexes t h a t b i n d , o p e r a t e on t h e t e m p l a t e , and d i s s o c i a t e s e q u e n t i a l l y b e f o r e t h e l e s i o n ( s ) i s removed and t h e c h r o m a t i n s t r u c t u r e r e s t o r e d . A t a h i g h e r l e v e l of o r g a n i z a t i o n , t h e p a c k i n g of DNA o r c h r o m a t i n must a l s o be r e t u r n e d t o i t s o r i g i n a l c o n f i g u r a t i o n . W h i l e r e c e n t emphasis on DNA r e p a i r c a p a c i t y has been d i r e c t e d towards the r e p a i r enzymes p r e s e n t w i t h i n c e l l s , i t i s i m p o r t a n t t o remember t h a t t h e r e may a l s o be a c o m p l i c a t i o n o f a c c e s s i b i l i t y o f the damage t o the r e p a i r 13 p r o c e s s (26, 4 0 ) . Some models of r a d i a t i o n a c t i o n i n mammalian c e l l s make the assumption t h a t t h e r e p a i r p r o c e s s e s a r e dose-dependant. I t i s h y p o t h e s i z e d t h a t t h e shape of t h e mammalian c e l l s u r v i v a l c u r v e i s c a u s a l l y r e l a t e d t o t h e s a t u r a t i o n of a DNA r e p a i r p r o c e s s (28, 6 1 ) . Recent i n t e r e s t i n i s o l a t i o n and c h a r a c t e r i z a t i o n o f DNA-r e p a i r d e f i c i e n t mutants (52, 62) as w e l l as c l o n i n g o f a human DNA r e p a i r gene ( 6 0 ) , a r e t h e r e s u l t o f emphasis on r e p a i r enzyme a c t i v i t y as a prime c o n t r o l l e r o f c e l l u l a r r e p a i r c a p a c i t y . However, the c o n f o r m a t i o n of t h e t e m p l a t e i s a l s o o f c r i t i c a l i m p o r t a n c e i n DNA r e p a i r . DNA s e q u e n c i n g methods have r e v e a l e d t h a t m u t a t i o n " h o t - s p o t s " o r g e n e t i c a l t e r a t i o n s do n o t always c o r r e s p o n d t o t h e a r e a s where most c h e m i c a l damage o c c u r s , i n d i c a t i n g t h e i m p o r t a n c e of what has been termed DNA "secondary" s t r u c t u r e . R e p a i r i s a l s o more e f f i c i e n t i n t r a n s c r i b e d as compared t o n o n - t r a n s c r i b e d DNA s t r a n d s and i n p r o t e i n - m a t r i x a s s o c i a t e d v e r s u s n o n - a s s o c i a t e d DNA ( 4 6 ) . The n a t u r e of t h e damage appears t o d e t e r m i n e the t y p e of m u t a t i o n w h i c h r e s u l t s ( f o r example, b u l k y l e s i o n s l e a d t o f r a m e s h i f t m u t a t i o n s , w h i l e minor l e s i o n s cause base damage). W h i l e the complement of r e p a i r enzymes i s c r i t i c a l t o r e s i s t a n c e t o DNA damage, the s t r u c t u r e of the t a r g e t s h o u l d not r e c e i v e l e s s a t t e n t i o n t h a n i s m e r i t e d . E. A R o l e f o r DNA C o n f o r m a t i o n i n t h e C o n t a c t E f f e c t When c e l l s a r e l y s e d i n a m i l d a l k a l i s o l u t i o n (pH 1 2 . 3 ) , 14 t h e DNA undergoes d e n a t u r a t i o n and s l o w u n w i n d i n g a t s i t e s of DNA s t r a n d - b r e a k a g e . Most a s s a y s w h i c h measure s i n g l e - s t r a n d b r e a k s a l s o measure d o u b l e - and a l k a l i - l a b i l e - s t r a n d b r e a k s . Unwinding g e n e r a l l y o c c u r s a t a c o n s t a n t r a t e f o r a g i v e n t e m p e r a t u r e and s a l t c o n c e n t r a t i o n . Under a p p r o p r i a t e c o n d i t i o n s , t h e amount of r e s u l t a n t d o u b l e - s t r a n d e d DNA i s b e l i e v e d t o be p r o p o r t i o n a l t o t h e number of s t r a n d b r e a k s p r e s e n t . The a s s a y was o r i g i n a l l y d e v e l o p e d by Ahnstrom and E r i x o n (3) and i s now i n s t a n d a r d use t o measure DNA s t r a n d breakage by i o n i z i n g r a d i a t i o n and g e n o t o x i c d r u g s . F i g u r e 3 d e p i c t s t h e method of use i n our l a b o r a t o r y . U s i n g t h i s a s s a y , O l i v e and Durand (42) r e p o r t e d a d i f f e r e n c e i n t h e p a t t e r n of DNA u n w i n d i n g i n s p h e r o i d c e l l s l y s e d i n a l k a l i a f t e r i r r a d i a t i o n ; DNA u n w i n d i n g f o r monolayer c e l l s was e x p o n e n t i a l w h i l e t h e r a t e of u n w i n d i n g of s p h e r o i d c e l l DNA was e x p o n e n t i a l o n l y f o r t h e f i r s t t e n m i n u t e s . E x p l a n a t i o n s f o r t h i s may i n c l u d e t h e p r e s e n c e of r a d i a t i o n -i n d u c e d l e s i o n s w h i c h i n h i b i t u n w i n d i n g , d i f f e r e n c e s i n t h e i n t e r a c t i o n between DNA and o t h e r c e l l c o n s t i t u e n t s o r a d i f f e r e n c e i n DNA c o n f o r m a t i o n between t h e monolayer and s p h e r o i d DNA. U s i n g t h e u n w i n d i n g a s s a y , t h e r e j o i n i n g of s t r a n d b r e a k s was o b s e r v e d t o be 25 - 50 p e r c e n t f a s t e r i n s p h e r o i d c e l l s ( 4 3 ) , w h i c h may e x p l a i n why t h e s p h e r o i d c e l l s seem t o have an advantage i n the s u r v i v a l of r a d i a t i o n damage. A d i f f e r e n c e i n DNA c o n f o r m a t i o n was a l s o r e v e a l e d u s i n g t h e 15 F i g u r e 3. Schematic R e p r e s e n t a t i o n of the A l k a l i Unwinding Assay. Alkaline solution pH r12 Suspension of labelled and irradiated cells at 0* C separation on column of hydroxylapatite s i n g l e -st ra nded ON A double -stranded ON A I S C 16 n u c l e o i d s e d i m e n t a t i o n a s s a y i n w h i c h p r o t e i n - and RNA-depleted n u c l e i , w i t h t h e DNA s t i l l t i g h t l y a s s o c i a t e d , were sedimented on n e u t r a l s u c r o s e g r a d i e n t s . These s t r u c t u r e s were more compact, and so sediment f u r t h e r , when l e s s DNA damage was p r e s e n t . N u c l e o i d s from s p h e r o i d c e l l s sedimented t w i c e as r a p i d l y as t h o s e from monolayer c e l l 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 i s was t h a t t h e s p h e r o i d DNA was more s u p e r c o i l e d t h a n monolayer DNA, but t h i s was shown t o be u n t r u e when e t h i d i u m bromide, a DNA i n t e r c a l a t o r , was used ( 4 3 ) . A more l i k e l y e x p l a n a t i o n was t h a t t h e " p a c k a g i n g " o f t h e DNA d i f f e r e d . O l i v e e t a l . (43) s u g g e s t e d t h a t when c e l l s a r e grown i n s u s p e n s i o n , t h e p a c k a g i n g o f t h e DNA i s a l t e r e d by t h e p r e s e n c e of c o n s t r a i n t s a t r e g u l a r i n t e r v a l s . A s c h e m a t i c o f t h i s model i s shown i n F i g u r e 4. There may be s i m i l a r c o n s t r a i n t s i n monolayer c e l l s , b u t t h e y would be s e p a r a t e d by much g r e a t e r d i s t a n c e s . These c o n s t r a i n t s may r e p r e s e n t s i t e s of c o v a l e n t attachment o f DNA t o t h e n u c l e a r m a t r i x . I f t h e c o n s t r a i n t s a r e c l o s e r t o g e t h e r i n s p h e r o i d c e l l DNA, i t c o u l d e x p l a i n t h e d i f f e r e n c e i n u n w i n d i n g k i n e t i c s and would a l l o w t h e DNA t o r e j o i n more q u i c k l y and perhaps more a c c u r a t e l y a f t e r s t r a n d breakage. I f DNA r e p a i r o c c u r s i n a s s o c i a t i o n w i t h t h e n u c l e a r m a t r i x as do r e p l i c a t i o n and t r a n s c r i p t i o n ( 7 , 45, 5 6 ) , more f r e q u e n t attachment s i t e s c o u l d i n c r e a s e DNA r e p a i r c a p a c i t y . F. The Approach The main o b j e c t i v e of t h i s p r o j e c t was t o i n v e s t i g a t e t h e 17 F i g u r e 4. P r o p o s e d M o d e l f o r t h e P r e s e n c e o f C o n s t r a i n t s t o DNA U n w i n d i n g i n C e l l s Grown a s S p h e r o i d s ( 4 3 ) . X - R A Y S ALKALI TREATMENT NEUTRALIZATION, SONICATION <r ^ <f ^ H Y D R O X Y A P A T I T E C H R O M A T O G R A P H Y 18 mechanism of t h e c o n t a c t e f f e c t , s p e c i f i c a l l y t h e r o l e o f c e l l shape and c e l l environment w i t h r e l a t i o n t o r a d i o r e s i s t a n c e . S i n c e t h e s p h e r o i d c e l l s grow i n c o n t a c t and r e m a i n round, t h e f i r s t t a s k was t o s e p a r a t e the two p r o p e r t i e s . To a c c o m p l i s h t h i s , c e l l s were grown on m e t h y l c e l l u l o s e w h i c h a l l o w e d them t o remain r o u n d , b u t d i d not a l l o w them t o a g g r e g a t e . A n o t h e r method of p r e v e n t i n g c e l l attachment and f l a t t e n i n g was a l s o used. P o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) , a h y d r o p h i l i c h y d r o g e l of n e u t r a l c h a r g e , was a p p l i e d t o n o n - t i s s u e c u l t u r e d i s h e s . T h i s polymer has been shown t o c o n t r o l t h e e x t e n t o f c e l l s p r e a d i n g and attachment (21) so t h a t c e l l s can be grown i n monolayer c o n d i t i o n s , b u t cannot d e v e l o p t h e u s u a l f l a t o r extended shape and so remain e i t h e r more s p h e r i c a l o r i n s u s p e n s i o n . I t was p r e v i o u s l y o b s e r v e d t h a t s p a r s e l y seeded monolayer c e l l s d i s p l a y a r e l a t i v e r a d i o r e s i s t a n c e and t h a t t h e i r shape i s somewhat d i f f e r e n t from c o n f l u e n t monolayer c u l t u r e s ( 4 1 ) . To i n v e s t i g a t e a p o s s i b l e r o l e f o r t h e growth e n v i r o n m e n t , t h e e f f e c t s o f d i f f e r e n t growth media and c o n d i t i o n s were s t u d i e d . 19 MATERIALS AND METHODS A. C e l l L i n e s and C u l t u r e Techniques A v a r i e t y o f c e l l l i n e s , o t h e r t h a n t h e V79 l i n e , was chosen t o o b s e r v e t h e g e n e r a l i t y of t h e c o n t a c t e f f e c t as d e f i n e d by DNA u n w i n d i n g . C e l l s were s e l e c t e d f o r t h e i r a b i l i t y t o grow i n monolayer as w e l l as s u s p e n s i o n c u l t u r e s , e i t h e r as ag g r e g a t e s o r n o t . a. V79-171B C e l l L i n e V79 i s an e s t a b l i s h e d mammalian c e l l l i n e o f C h i n e s e hamster l u n g f i b r o b l a s t s . I t i s an e x t e n s i v e l y used l i n e because o f i t s h i g h p l a t i n g e f f i c i e n c y , r a p i d d o u b l i n g t i m e , d i s c r e t e c o l o n y f o r m a t i o n and ease o f o b t a i n i n g s i n g l e c e l l s u s p e n s i o n s . T h i s l i n e grows w e l l i n monolayer c u l t u r e on p e t r i p l a t e s , as w e l l as i n s u s p e n s i o n , where many of i t s s u b l i n e s grow as a g g r e g a t e s . The s p h e r o i d - f o r m i n g s u b l i n e , V79-171b ( o r i g i n a l l y o b t a i n e d from Dr. Warren S i n c l a i r a t Argonne N a t i o n a l L a b o r a t o r i e s ) , was r o u t i n e l y grown i n our l a b o r a t o r y and m a i n t a i n e d as monolayer c u l t u r e s , i n e x p o n e n t i a l growth, by s u b c u l t u r i n g e v e r y two o r t h r e e days on 100 mm p l a s t i c t i s s u e c u l t u r e p l a t e s ( F a l c o n ) , i n E a g l e s M i n i m a l E s s e n t i a l Medium (Gibco) c o n t a i n i n g t e n p e r c e n t (v/v) f e t a l b o v i n e serum ( G i b c o ) . The c e l l s were i n c u b a t e d a t 20 37°C i n an h u m i d i f i e d atmosphere o f f o u r p e r c e n t CO^ i n a i r . S p h e r o i d s were i n i t i a t e d by s e e d i n g 2 x 10 e x p o n e n t i a l l y g r owing c e l l s i n t o 200 ml of t h e above-mentioned medium i n B e l l c o g l a s s s p i n n e r c u l t u r e f l a s k s . The f l a s k s were f l u s h e d w i t h f o u r p e r c e n t CC<2 i n a i r a f t e r a d d i n g t h e medium and were a l l o w e d t o e q u i l i b r a t e a t 37°C b e f o r e t h e c e l l s were added. A f t e r f o u r t o s i x h o u r s , t h e c e l l s a g g r e g a t e d s p o n t a n e o u s l y t o form s m a l l c l u s t e r s and d i v i d e d about t w i c e i n a 24 hour p e r i o d t o produce s p h e r o i d s c o n t a i n i n g 20 - 50 c e l l s each. These c e l l s were s t i l l i n e x p o n e n t i a l growth so t h a t t h e r a t e of DNA s y n t h e s i s and t h e c e l l c y c l e d i s t r i b u t i o n were i d e n t i c a l w i t h t h o s e o f monolayers. The average c e l l c y c l e t i m e f o r t h i s s u b l i n e i s 11 - 12 h o u r s . R o u t i n e passages o f monolayer c e l l s were performed by t w i c e r i n s i n g and l e a v i n g a f i l m o f 0.1 p e r c e n t t r y p s i n (Gibco) i n phosphate b u f f e r e d s a l i n e . The p l a t e s were i n c u b a t e d f o r s e v e r a l m i n u t e s , t h e n t h e c e l l s were resuspended i n medium by p i p e t t i n g v i g o r o u s l y s e v e r a l t i m e s , c o u n t e d and used t o s t a r t s u b c u l t u r e s . b. SCCVII C e l l L i n e These murine squamous c e l l c a r c i n o m a c e l l s grow as t r a n s -p l a n t a b l e tumors i n mice, b u t a l s o adapt w e l l t o c u l t u r e . The c e l l l i n e we i s o l a t e d grows as monolayers and i n s u s p e n s i o n , a l t h o u g h the c e l l s do not a g g r e g a t e t o form s p h e r o i d s . The 21 d o u b l i n g t i m e of t h e s e c e l l s i s l i k e t h a t o f t h e V79 c e l l l i n e . T h i s l i n e was passaged and m a i n t a i n e d i n t h e same manner as the V79 c e l l l i n e and i s r o u t i n e l y h e l d i n our l a b o r a t o r y . c. RIF-1 C e l l L i n e The RIF-1 c e l l l i n e o r i g i n a l l y a r o s e as a murine tumor as a consequence o f i r r a d i a t i o n , and was o b s e r v e d t o grow r e a d i l y i n c u l t u r e ( 5 4 ) . I t was s e l e c t e d f o r i t s a b i l i t y t o grow i n monolayer c u l t u r e as w e l l as i n s u s p e n s i o n as s p h e r o i d s . The d o u b l i n g t i m e f o r t h i s c e l l l i n e i s about 24 h o u r s . The c e l l s were passaged w e e k l y u s i n g e i t h e r 0.1 p e r c e n t t r y p s i n i n c i t r a t e s a l i n e o r 0.25 p e r c e n t t r y p s i n i n phosphate b u f f e r e d s a l i n e . A f t e r t w i c e r i n s i n g and t h e n i n c u b a t i n g t h e c e l l s i n a t r y p s i n f i l m f o r s e v e r a l m i n u t e s , t h e y were resuspended and p l a t e d as u s u a l , i n medium c o n t a i n i n g t e n p e r c e n t f e t a l b o v i n e serum. d. WiDr C e l l L i n e T h i s i s a human c o l o n adenocarcinoma c e l l l i n e , o b t a i n e d from American Type C u l t u r e C o l l e c t i o n , w h i c h grows w e l l i n c u l t u r e ( 5 9 ) . I n o r d e r t o form u n i f o r m s p h e r o i d s i n s p i n n e r c u l t u r e , t h e c e l l s must f i r s t be p e r m i t t e d t o a g g r e g a t e i n u n s t i r r e d medium f o r 24 h o u r s . These c e l l s were passaged and m a i n t a i n e d i n t h e same manner as t h e RIF-1 c e l l l i n e . 22 e. C-4 I , C-4 I I C e l l L i n e s These human c e r v i c a l squamous carcinoma c e l l s o r i g i n a t e d from t h e same tumor b i o p s y and have been m a i n t a i n e d as c e l l l i n e s f o r many y e a r s . The c u l t u r e s were k i n d l y p r o v i d e d by Dr. N e l l y A u e r s p e r g (Dept. o f Anatomy, U. B. C ) . B o t h l i n e s grow as monolayers and i n s u s p e n s i o n . The C-4 I s u b l i n e w i l l grow i n s u s p e n s i o n as a g g r e g a t e s e s p e c i a l l y i f i n i t i a t e d i n u n s t i r r e d medium i n t h e same manner as t h e WiDR c e l l l i n e . The C-4 I I does not a g g r e g a t e w e l l nor grow c o n s i d e r a b l y i n s u s p e n s i o n (4). These c e l l l i n e s were passaged and m a i n t a i n e d i n t h e same manner as t h e RIF-1 c e l l l i n e . B. P r o c e d u r e s a. P r e p a r a t i o n o f Samples There a r e some t e c h n i c a l d i f f i c u l t i e s i n t h e measurement o f damage t o DNA. S t r a n d b r e a k s caused by i o n i z i n g r a d i a t i o n a r e most e a s i l y d e t e c t e d by u s i n g r a d i o i s o t o p e s i n c o r p o r a t e d i n t o t h e DNA as t r a c e r m o l e c u l e s . However, r a d i o i s o t o p e s damage DNA, a l t h o u g h t h i s can be m i n i m i z e d by t h e use o f low c o n c e n t r a t i o n s of t h e i s o t o p e . I n a d d i t i o n , 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 i n mammalian c e l l s a re r a p i d l y r e j o i n e d i n a h a l f - t i m e of o n l y a few m i n u t e s , so t h e c e l l s must be i r r a d i a t e d on i c e o r h a n d l e d q u i c k l y and m i n i m a l l y . 23 For DNA un w i n d i n g e x p e r i m e n t s , the c e l l s were r a d i o -a c t i v e l y l a b e l l e d . T h i s was done by p l a t i n g 2 x 10 e x p o n e n t i a l l y growing c e l l s , w h i c h had been t r y p s i n i z e d and resuspended as p r e v i o u s l y d e s c r i b e d , i n t o t e n ml of f i v e p e r c e n t serum-supplemented medium c o n t a i n i n g a c o n c e n t r a t i o n of 0.74 KBq/ml [ "^4C] t h y m i d i n e ( s p e c i f i c a c t i v i t y , 2.2 GBq/mmole) . These p l a t e s were t h e n i n c u b a t e d a t 37°C f o r 24 hours and t h e c e l l s were a g a i n suspended w i t h t r y p s i n and a l l o w e d t o grow i n u n l a b e l l e d medium, i n t h e c o n d i t i o n s p a r t i c u l a r t o t h e ex p e r i m e n t . 4 2 C e l l s , r a d i o l a b e l l e d as above, were p l a t e d a t 3 x 10 /cm 5 and 2.5 x 10 c e l l s / m l medium i n 100 mm t i s s u e c u l t u r e d i s h e s o r g grown i n s u s p e n s i o n a t 2 - 3 x 10 c e l l s / 2 0 0 ml medium i n s p i n n e r f l a s k s . These p r o p o r t i o n s were used i n a l l e x p e r i m e n t s e x c e p t where s p e c i a l c o n d i t i o n s f o r d e n s i t y and c e l l c r o w d i n g p r e v a i l e d . I m m e d i a t e l y p r i o r t o i r r a d i a t i o n , s p h e r o i d s were c e n t r i f u g e d t o form a p e l l e t , t h e medium was a s p i r a t e d and t h e s p h e r o i d s suspended i n f i v e ml o f t r y p s i n . The c e l l s were i n c u b a t e d f o r f i v e m i n u t es a f t e r w h i c h a t l e a s t f i v e ml o f medium was added t o t h e tube t o s t o p t h e t r y p s i n a c t i v i t y . The c e l l s were a g a i n c e n t r i f u g e d t o a p e l l e t , resuspended i n two ml of c o l d medium, coun t e d and a d j u s t e d t o a c o n c e n t r a t i o n o f 1 - 2 x 10^ c e l l s / 5 0 u l , p l a c e d i n a f i v e ml capped p o l y e t h y l e n e t e s t tube and s e t i n an i c e - w a t e r b a t h . F o r monolayer c u l t u r e s , the medium was a s p i r a t e d , t h e p l a t e s were r i n s e d w i t h f i v e ml o f t r y p s i n and t h e n i n c u b a t e d f o r f i v e minutes i n two ml of f r e s h 24 t r y p s i n t o more c l o s e l y d u p l i c a t e the exposure t o t r y p s i n o f t h e s p h e r o i d c e l l s . As w i t h t h e c e l l s from s p h e r o i d s , medium was added t o t h e p l a t e s c o n t a i n i n g c e l l s and t r y p s i n , the c e l l s p i p e t t e d v i g o r o u s l y , t r a n s f e r r e d t o tubes and c e n t r i f u g e d t o a p e l l e t . The medium p l u s t r y p s i n was removed and t h e c e l l s were resuspended and t r e a t e d i n t h e same manner as the s p h e r o i d c e l l s . b. DNA S t r a n d Breakage A n a l y s i s C e l l s suspended i n medium on i c e , as d e s c r i b e d above, were exposed t o 250 kvp X - r a y s a t a dose r a t e of 7.58 Gy/min. T r i p l i c a t e samples o f 50 u l each were t a k e n and p l a c e d i n f i v e ml t e s t t u b e s f o r each r a d i a t i o n dose exposure. F o r dose r e s p o n s e a n a l y s i s , t h i s was done f o r s u c c e s s i v e e x p o s u r e s o f 2.5 Gy each, up t o an accumulated t o t a l o f t e n Gy. I n t h e s t u d y of DNA u n w i n d i n g k i n e t i c s , a s i n g l e t e n Gy dose was g i v e n . The samples remained on i c e a t a l l t i m e s t o p r e v e n t t h e a c t i o n o f DNA r e p a i r enzymes. The 50 u l samples were l y s e d i n one ml o f f r e s h l y p r e p a r e d , i c e - c o l d s o l u t i o n c o n t a i n i n g 1 M N a C l and 0.03 M NaOH. The samples were t h e n p l a c e d i n the d a r k , t o p r e v e n t subsequent UV l i g h t damage, and t h e DNA a l l o w e d t o unwind. F o r dose r e s p o n s e a n a l y s i s , t h e samples were removed a f t e r 60 m i n u t e s , n e u t r a l i z e d w i t h 0.03 M NaH 2P0 4 t o s t o p t h e u n w i n d i n g p r o c e s s , and i m m e d i a t e l y s o n i c a t e d f o r t e n seconds u s i n g a Heat Systems U l t r a s o n i c s o n i c a t o r w i t h m i c r o t i p . T h i s b r e a k s DNA i n t o s m a l l s i n g l e - and d o u b l e - s t r a n d fragments (10 d a l t o n s ) . 25 A p p r o x i m a t e l y 50 u l of sodium d o d e c y l s u l p h a t e (SDS) was t h e n added t o each tube t o reduce t h e a s s o c i a t i o n o f DNA w i t h p r o t e i n . When DNA unwind i n g k i n e t i c s were a n a l y s e d , t h e c e l l s were a l l o w e d t o l y s e and t h e DNA t o unwind f o r i n c r e a s i n g amounts o f ti m e from 0.1 t o 130 m i n u t e s , t h e n t r e a t e d as above. c. H y d r o x y a p a t i t e Chromatography H y d r o x y a p a t i t e chromatography was used t o s e p a r a t e t h e r e s u l t a n t s i n g l e - and d o u b l e - s t r a n d e d DNA. S m a l l p o l y e t h y l e n e columns, c o n t a i n i n g 150 mg of h y d r o x y a p a t i t e c r y s t a l s ( B i o - G e l HTP, B i o r a d L a b o r a t o r i e s ) , were l o a d e d i n t o a column h o l d e r . The column h o l d e r and a l l s o l u t i o n s were h e l d a t 65°C t o m e l t i m p e r f e c t l y matched b a s e - p a i r e d r e g i o n s o f t h e DNA. A l l washing and e l u t i n g s o l u t i o n s c o n t a i n e d 0.40 p e r c e n t SDS, t o reduce t h e a s s o c i a t i o n o f DNA w i t h c e l l u l a r p r o t e i n . The columns were r e h y d r a t e d w i t h t h r e e ml of 0.012 M sodium phosphate b u f f e r (pH 6.8) and were a l l o w e d t o d r a i n . The samples, warmed b r i e f l y t o 65°C, were p o u r e d onto t h e columns. A f t e r t h e s e had d r a i n e d , the columns were once a g a i n washed w i t h 0.012 M b u f f e r . S i n g l e -s t r a n d e d DNA was e l u t e d w i t h t h r e e ml o f 0.12 M b u f f e r (pH 7.4) and c o l l e c t e d i n l i q u i d s c i n t i l l a t i o n v i a l s ( F i s h e r S c i e n t i f i c ) . The d o u b l e - s t r a n d e d DNA was e l u t e d , u s i n g 1.5 ml of 0.4 M b u f f e r (pH 7.4), i n t o l i q u i d s c i n t i l l a t i o n v i a l s c o n t a i n i n g 1.5 ml o f d i s t i l l e d w a t e r , t o b r i n g the volume t o t h r e e ml. F i v e ml o f s c i n t i l l a t i o n c o c k t a i l ( H y d r o f l u o r , N a t i o n a l D i a g n o s t i c s ) were added t o each v i a l . The v i a l s were t h e n c o u n t e d f o r two 26 m i n u t e s u s i n g a Beckman l i q u i d s c i n t i l l a t i o n c o u n t e r w i t h a 14 window s e t f o r C. The c o u n t i n g e f f i c i e n c y o f t h e s i n g l e - and d o u b l e - s t r a n d e d f r a c t i o n s was d e t e r m i n e d , and found t o be i d e n t i c a l . R e s u l t s were e x p r e s s e d as c o u n t s p e r mi n u t e . d. Q u a n t i f y i n g DNA Damage R e s u l t s were e x p r e s s e d as t h e p e r c e n t a g e o f DNA i n d o u b l e -s t r a n d e d form. The average and s t a n d a r d d e v i a t i o n were c a l c u l a t e d f o r t h e t r i p l i c a t e samples and t h i s i s e x p r e s s e d a s : P = cpm d o u b l e - s t r a n d e d DNA x 100 cpm d o u b l e - s t r a n d e d DNA + cpm s i n g l e - s t r a n d e d DNA Dose r e s p o n s e d a t a were p l o t t e d on a l o g a r t h m i c s c a l e o f p e r c e n t a g e on t h e o r d i n a t e and a l i n e a r s c a l e o f dose on t h e a b s c i s s a . A l l e x p e r i m e n t s were r e p e a t e d s e v e r a l t i m e s and p r e s e n t e d one of two ways; (a) t h e mean and s t a n d a r d d e v i a t i o n of t r i p l i c a t e samples from one r e p r e s e n t a t i v e s e t o f r e s u l t s i s shown, o r (b) r e s u l t s from two o r t h r e e r e p e a t s o f an expe r i m e n t a r e r e p r e s e n t e d by one c u r v e ( i n a l l c a s e s , t r i p l i c a t e samples were used f o r each d a t a p o i n t i n each s e p a r a t e e x p e r i m e n t ) . The s t a n d a r d e r r o r f o r t h e s e combined e x p e r i m e n t s was c a l c u l a t e d by d i v i d i n g t h e s t a n d a r d d e v i a t i o n of each d a t a p o i n t by t h e square r o o t o f n, t h e number of e x p e r i m e n t s p e r c u r v e . I n al m o s t a l l c a s e s , t h e s t a n d a r d e r r o r b a r s were s m a l l e r t h a n t h e graph symbol s i z e s . C l a r i t y was the d e t e r m i n a n t i n c h o o s i n g the 27 method used f o r p r e s e n t i n g r e s u l t s from an i n d i v i d i a l e x p e r i m e n t . R e s u l t s were n o r m a l i z e d f o r each e x p e r i m e n t so t h a t the u n i r r a d i a t e d c e l l samples r e p r e s e n t e d 90 p e r c e n t d o u b l e -s t r a n d e d DNA. T h i s v a l u e was chosen s i n c e t h e average v a l u e f o r t e n e x p e r i m e n t s was found t o be 90 + 0.5 p e r c e n t . S i g n i f i c a n t s t a t i s t i c a l d i f f e r e n c e s were d e t e r m i n e d by c a l c u l a t i n g t h e 95 p e r c e n t c o n f i d e n c e l i m i t s f o r t h e s l o p e o f each c u r v e d e f i n i n g l o g p e r c e n t d o u b l e - s t r a n d e d DNA as a f u n c t i o n o f r a d i a t i o n dose. I f t h e c o n f i d e n c e l i m i t s o f two s l o p e s d i d not o v e r l a p , t h e r e s u l t s were c o n s i d e r e d t o be s i g n i f i c a n t l y d i f f e r e n t . DNA u n w i n d i n g k i n e t i c s a n a l y s i s was p l o t t e d on a l o g / l o g s c a l e o f p e r c e n t a g e d o u b l e - s t r a n d e d DNA v e r s u s t i m e o f l y s i s . A g a i n , t h e e x p e r i m e n t s were r e p e a t e d s e v e r a l t i m e s and a r e p r e s e n t a t i v e s e t of d a t a p r e s e n t e d . C. Treatments U n l e s s o t h e r w i s e i n d i c a t e d : 1) c o n t r o l s f o r a l l t r e a t m e n t s c o n s i s t e d o f the same c e l l s as used i n t h e t r e a t m e n t s , grown as monolayers and s p h e r o i d s , seeded and a s s a y e d as p r e v i o u s l y mentioned. 2) c e l l s were i n c u b a t e d i n m i n i m a l e s s e n t i a l medium supplemented w i t h f i v e p e r c e n t f e t a l b o v i n e serum. 28 3) X - r ay dose r e s p o n s e e x p e r i m e n t s were performed. a. C e l l Crowding and D e n s i t y Monolayer and s p h e r o i d c e l l s were compared f o r r e s p o n s e s t o i o n i z i n g r a d i a t i o n when grown a t d i f f e r e n t c e l l p a c k i n g d e n s i t i e s . To o b s e r v e t h e e f f e c t s o f c r o w d i n g , th e same number 4 o f c e l l s (5 x 10 ) were p l a t e d i n i n c r e a s i n g l y l a r g e r a r e a s (1 -80 square cm) on 100 mm p l a s t i c p e t r i p l a t e s . The c e l l s were a l l o w e d t o a t t a c h f o r two h o u r s i n an i n c u b a t o r . Medium was added t o each p l a t e t o b r i n g t h e t o t a l volume t o t e n m l , and t h e p l a t e s were i n c u b a t e d f o r 24 h o u r s . The c e l l s were t h e n t r y p s i n i z e d , c o u n t e d f o r y i e l d and a s s a y e d f o r p e r c e n t d o u b l e - s t r a n d e d DNA as p r e v i o u s l y d e s c r i b e d . V a r y i n g numbers of c e l l s were a l s o seeded i n t o t h e same s i z e d a r e a , i n 100 mm p e t r i p l a t e s . The c e l l s were i n c u b a t e d o v e r n i g h t and t r e a t e d as above. To f u r t h e r examine t h e d i f f e r e n c e s between c e l l s grown a t h i g h and low d e n s i t i e s , t h e DNA u n w i n d i n g k i n e t i c s were a n a l y s e d as p r e v i o u s l y d e s c r i b e d . D e n s i t y e f f e c t s were a l s o a n a l y z e d f o r s p h e r o i d s by s e e d i n g 2 x 10^ c e l l s i n t o 20 ml o f medium i n a s p i n n e r f l a s k and a l l o w i n g t h e c e l l s t o i n c u b a t e . The same number of c e l l s was a l s o p l a t e d i n t o 20 ml o f medium i n a p e t r i p l a t e and a n a l y s e d as a c o n t r o l . 29 b. Medium Flow I n s p h e r o i d c u l t u r e s , the medium i s c o n s t a n t l y moving, w h i l e i n m o n o l a y e r s , i t i s r e l a t i v e l y s t a t i c . To t e s t t h e e f f e c t s o f c o n t i n u a l l y moving medium on monolayer c u l t u r e s , s e v e r a l e x p e r i m e n t s were d e v i s e d . M i c r o c a r r i e r beads (Cytodex 1, Pharmacia) were p r e p a r e d as pe r m a n u f a c t u r e r ' s i n s t r u c t i o n s by a u t o c l a v i n g one gram o f d r y beads i n 50 ml o f phosphate b u f f e r e d s a l i n e . A volume o f beads and c e l l s were t h e n added t o 200 ml of medium i n a s p i n n e r f l a s k , so as t o p r o v i d e a bead s u r f a c e a r e a t o c e l l r a t i o t h a t 4 2 was e q u i v a l e n t t o 3 x 10 c e l l s / c m ( t h e same d e n s i t y as c e l l s p l a t e d on p e t r i p l a t e s ) . These beads p r o v i d e d t i n y monolayer c u l t u r e s w h i c h t r a v e l l e d t h r o u g h the moving medium o f t h e s p i n n e r f l a s k . As w i t h p l a t e d c u l t u r e s , c e l l c r o w d i n g on t h e beads c o u l d be c o n t r o l l e d by s e e d i n g f l a s k s w i t h v a r y i n g c e l l c o n c e n t r a t i o n s . The c e l l s were i n c u b a t e d f o r 24 hours and as s a y e d f o r DNA damage caused by i o n i z i n g r a d i a t i o n . A v a r i a t i o n o f t h i s was t o p l a t e c e l l s i n 60 mm p e t r i p l a t e s and a l l o w them t o a t t a c h f o r s e v e r a l h o u r s , a f t e r w h i c h t h e p l a t e s were p l a c e d i n s p i n n e r f l a s k s . Then, 200 ml o f medium was c a r e f u l l y added t o t h e f l a s k s , w h i c h had been gass e d w i t h f o u r p e r c e n t C 0 2 i n a i r , and t h e y were p e r m i t t e d t o s p i n i n an i n c u b a t o r f o r 24 h o u r s . T h i s p r o v i d e d a c o n t i n u a l movement of medium o v e r t h e monolayer c e l l s . 30 C e l l s were a l s o p l a c e d i n 60 mm t i s s u e c u l t u r e p l a t e s and a l l o w e d t o a t t a c h f o r s e v e r a l h o u r s . The p l a t e s were t h e n p l a c e d i n a moving p l a t f o r m i n c u b a t o r s e t a t such a speed as t o a l l o w t h e medium t o move g e n t l y back and f o r t h o v e r t h e monolayers. As a c o n t r o l , d u p l i c a t e p l a t e s were p l a c e d i n a non-moving i n c u b a t o r f o r 24 h o u r s . B o t h were i n c u b a t e d and as s a y e d f o r DNA damage f o l l o w i n g r a d i a t i o n e x p o s u r e . Another v a r i a t i o n o f t h i s was t o p l a t e c e l l s on 100 mm p e t r i p l a t e s , a l l o w them t o a t t a c h f o r s e v e r a l h o u r s , t h e n t o r e p l a c e t h e medium e v e r y hour f o r 24 h o u r s , w h i l e i n c u b a t i n g . c. C o n d i t i o n e d Medium To f u r t h e r t e s t t h e e f f e c t s o f medium, c e l l s were grown as 3 2 4 mo n o l a y e r s , low (3 x 10 c e l l s /cm ) and h i g h (3 x 10 2 c e l l s / c m ) d e n s i t y and as s p h e r o i d s , i n medium w h i c h had been " c o n d i t i o n e d " . T h i s medium was o b t a i n e d from monolayer c u l t u r e s w h i c h had been growing i n i t f o r 24 h o u r s . The purpose o f t h i s was t o o b s e r v e whether medium w i t h m e t a b o l i c b y - p r o d u c t s o r n u t r i e n t d e p l e t i o n had any e f f e c t on r a d i o r e s i s t a n c e . To ensure t h a t any e f f e c t s seen were n o t due t o some f a c t o r r e l e a s e d i n t o t h e medium from t h e p l a s t i c p e t r i p l a t e s , medium was a l s o c o n d i t i o n e d i n g l a s s p e t r i p l a t e s and t h e two compared. C e l l s were a l s o grown i n g l a s s p e t r i p l a t e s and a s s a y e d f o r t h e c o n t a c t e f f e c t , as a c o n t r o l . 31 d. Poly(HEMA) P o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) o r poly(HEMA) i s a polymer, w h i c h when a p p l i e d t o t i s s u e c u l t u r e d i s h e s , r educes t h e i r a d h e s i v e n e s s and t h u s t h e e x t e n t t h a t c e l l s can s p r e a d o r even a t t a c h . Poly(HEMA) c r y s t a l s (Hydron L a b o r a t o r i e s , New J e r s e y ) were d i s s o l v e d i n 95 p e r c e n t e t h a n o l t o a s t o c k concen-t r a t i o n o f 12 p e r c e n t (w/v). T h i s m i x t u r e was t u r n e d s l o w l y o v e r n i g h t a t 37°C and t h e n c e n t r i f u g e d f o r 30 mi n u t e s t o remove u n d i s s o l v e d p a r t i c l e s . The s t o c k was t h e n d i l u t e d t o o n e - t e n t h th e c o n c e n t r a t i o n w i t h 95 p e r c e n t e t h a n o l , and two ml each were p l a t e d on 100 mm b a c t e r i a l p e t r i p l a t e s . Depending on t h e t h i c k n e s s o f t h e poly(HEMA), t h e V79 c e l l l i n e w i l l adhere t o a c e r t a i n e x t e n t o v e r a 24 hour p e r i o d , w i t h many c e l l s l e f t f l o a t i n g , a l t h o u g h t h e r e were no c e l l s a t t a c h e d a t t h e c o n c e n t r a t i o n used i n t h e s e e x p e r i m e n t s . These p l a t e s were a l l o w e d t o d r y o v e r n i g h t on a l e v e l bench f r e e from v i b r a t i o n s . Growth medium and c e l l s were added t o t h e poly(HEMA)-coated p l a t e s . The p l a t e s were t h e n i n c u b a t e d f o r 24 h o u r s , and t h e c e l l s w h i c h remained f l o a t i n g i n t h e medium as a g g r e g a t e s above the poly(HEMA) were examined f o r DNA damage f o l l o w i n g exposure t o i o n i z i n g r a d i a t i o n . The t h i c k n e s s of t h e polymer was such t h a t no c e l l s a t t a c h e d t o t h e s u r f a c e o f t h e p l a t e s . C e l l s a t t a c h e d t o u n c o a t e d b a c t e r i a l p l a t e s and t h e c e l l s w h i c h f l o a t e d above them were a l s o a s s a y e d f o r co m p a r i s o n . 32 e. T r y p s i n i z a t i o n To d i f f e r e n t i a t e between t h e r o l e s o f c e l l shape and c e l l c o n t a c t , exposure of the c e l l s t o t r y p s i n was used t o keep them from a d h e r i n g t o each o t h e r f o r any l e n g t h o f t i m e , i n t h e ca s e o f s p h e r o i d s , o r from f u l l y e l o n g a t i n g , i n t h e case o f mo n o l a y e r s . C e l l s were seeded i n t o s p i n n e r f l a s k s w i t h medium and i n c u b a t e d as u s u a l e x c e p t t h a t t h e y were removed from t h e f l a s k s e v e r y f o u r h o u r s and c e n t r i f u g e d . The medium was a s p i r a t e d and p u t back i n t o t h e f l a s k s . The c e l l s were t r y p s i n i z e d by p i p e t t i n g g e n t l y i n t h r e e ml of 0.1 p e r c e n t t r y p s i n and i n c u b a t e d f o r f o u r minutes i n a 37°C water b a t h , a f t e r w h i c h medium was added and t h e c e l l s c e n t r i f u g e d a g a i n . The s i n g l e c e l l s were t h e n resuspended i n f i v e ml o f medium and added t o t h e s p i n n e r f l a s k s c o n t a i n i n g t h e p r e v i o u s l y a s p i r a t e d medium. Monolayer c u l t u r e s were t r y p s i n i z e d e v e r y f o u r hours by t w i c e r i n s i n g w i t h 0.1 p e r c e n t t r y p s i n and i n c u b a t e d a t 37°C f o r f o u r m i n u t e s i n a f i l m o f t r y p s i n . The c e l l s were t h e n suspended i n t e n ml of medium by v i g o r o u s p i p e t t i n g . As c o n t r o l s , one p l a t e r e c e i v e d new medium e v e r y f o u r hours b u t no t r y p s i n , one r e c e i v e d the t r y p s i n t r e a t m e n t p l u s f r e s h medium, and a n o t h e r r e c e i v e d the t r y p s i n t r e a t m e n t b u t t h e same medium. C e l l s were t h e n resuspended i n medium, i r r a d i a t e d and examined f o r DNA damage. 33 f . M e t h y l C e l l u l o s e To f u r t h e r s t u d y the e f f e c t s of shape, c e l l s were grown as monolayers i n m e t h y l c e l l u l o s e . T h i s v i s c o u s m a t e r i a l p r e v e n t s t h e c e l l s from s p r e a d i n g and does not encourage a g g r e g a t i o n , e x c e p t as a r e s u l t o f c e l l d i v i s i o n . E l e v e n grams o f m e t h y l c e l l u l o s e powder (4000 c e n t i p o s e ) were added t o 250 ml of d i s t i l l e d w a ter (80 - 100°C) and t h e m i x t u r e was s t i r r e d o v e r n i g h t a t 4°C. I t was a u t o c l a v e d and c o o l e d s l o w l y t o 4°C, a f t e r w h i c h 250 ml o f d o u b l e - s t r e n g t h m i n i m a l e s s e n t i a l medium was added. T h i s m i x t u r e was s t i r r e d o v e r n i g h t a t 4°C. Medium, c o n t a i n i n g t e n p e r c e n t f e t a l b o v i n e serum, was added t o b r i n g t h e c o n c e n t r a t i o n s t o 1.1 p e r c e n t m e t h y l c e l l u l o s e and f i v e p e r c e n t f e t a l b o v i n e serum. A s i n g l e c e l l s u s p e n s i o n (2 x 10 c e l l s i n one ml) was added t o t e n ml o f t h e m e t h y l c e l l u l o s e m i x t u r e , w h i c h had been warmed t o 37°C, i n a 13 ml t e s t tube and t h e tube g e n t l y r o c k e d s e v e r a l t i m e s t o d i s t r i b u t e t h e c e l l s . T h i s was poured i n t o a n o n - t i s s u e c u l t u r e p l a t e , w h i c h was used t o ensure t h a t t h e c e l l s c o u l d not s p r e a d o u t b e n eath t h e m e t h y l c e l l u l o s e medium, and i n c u b a t e d f o r one, two o r t h r e e days. C e l l s were a l s o i n c u b a t e d i n the u s u a l growth medium, i n b o t h t y p e s o f p l a t e s , as c o n t r o l s . 34 RESULTS The c l o s e t h r e e - d i m e n s i o n a l c e l l c o n t a c t w h i c h d e v e l o p s when Ch i n e s e hamster V79-171B c e l l s a re grown i n s u s p e n s i o n has been assumed t o be a r e q u i r e m e n t f o r t h e development of t h e c o n t a c t e f f e c t . However, p r e l i m i n a r y e x p e r i m e n t s s u g g e s t e d t h a t a s i m i l a r e f f e c t c o u l d be o b s e r v e d i n s p a r s e l y seeded monolayer c u l t u r e s o f t h e s e c e l l s and i n two o t h e r n o n - f i b r o b l a s t c e l l l i n e s grown as m o nolayers. These o b s e r v a t i o n s i n d i c a t e d t h a t c e l l s may n o t need i n t e r c e l l u l a r c o n t a c t t o d e v e l o p t h e c o n t a c t e f f e c t . Two o t h e r l i k e l y e x p l a n a t i o n s f o r t h e d i f f e r e n c e s i n DNA u n w i n d i n g k i n e t i c s a r e t h e changes i n c e l l shape w h i c h accompany growth i n s u s p e n s i o n and changes i n n u t r i e n t d e l i v e r y . I n an a t t e m p t t o d i s c r i m i n a t e between t h e s e t h r e e p o s s i b l e c o n t r i b u t o r s t o t h i s e f f e c t , t h e e x p e r i m e n t s d e s c r i b e d below d e f i n e t h e c u l t u r e c o n d i t i o n s under w h i c h t h e c o n t a c t e f f e c t i s o b s e r v e d . I n a l l c a s e s , t h e c o n t a c t e f f e c t has been d e f i n e d i n terms o f DNA u n w i n d i n g k i n e t i c s u s i n g t h e a l k a l i u n w i n d i n g a s s a y . A. C u l t u r e C o n d i t i o n s Under Which th e C o n t a c t E f f e c t i s Observed R e s u l t s o b t a i n e d u s i n g V79 c e l l s a re summarized i n T a b l e I I . T h i s p r o v i d e s a g u i d e f o r f o l l o w i n g t h e e x p e r i m e n t s and i n t e r p r e t i n g t h e d a t a . 35 TABLE I I . SUMMARY OF RESULTS FROM V79 CELL LINE EXPERIMENTS a b C e l l C e l l : c e l l Medium Contact Fi g . Experiment Shape Contact Exchange E f f e c t 6. Low density monolayers f l a t - +/- ++ 8. Monolayers i n c spinner flasks f l a t - + +/-9. Dense monolayers with hourly medium renewal f l a t - + . ++ 11. Microcarrier beads c,d i n spinner flasks f l a t - + +/-12. Spheroids i n spinner flasks round + + ++ 13. Dense monolayers trypsinized every 4 hr round + ++ 14. Spheroids trypsinized every 4 hr round - + ++ 15. C e l l s i n methyl c e l l u l o s e round +/- - ++ 16. C e l l s f l o a t i n g above poly (HEMA) round + - ++ 16. C e l l s f l o a t i n g above d monolayers round + - + 18. Spheroids i n d conditioned medium round + - + 18. Sparse monolayers i n d conditioned medium f l a t - - + a, refe r s to growth of c e l l s i n a large volume of medium or regula r l y renewed medium b, measured using a l k a l i unwinding assay c, density dependent d, p a r t i a l contact e f f e c t , s i g n i f i c a n t l y d i f f e r e n t from monolayer or spheroid response at the 95% confidence l e v e l . 36 46' • i 4 F i g u r e 5. M i c r o s c o p i c a p p e a r a n c e o f c e l l s u s i n g d i g i t i z e d i m a g e s . L e s s e x t e n d e d c e l l s a r e m o r e r a d i o -r e s i s t a n t . W i D r c e l l s : ( a ) h i g h d e n s i t y , ( b ) l o w d e n s i t y -e q u a l l y r a d i o r e s i s t a n t . V79 c e l l s : l o w d e n s i t y ( c ) c o n t r o l , ( e ) c o n d i t i o n e d m e d i u m d i f f e r e n c e i n s h a p e , c o n t r o l m o r e r a d i o r e s i s t a n t . V79 c e l l s : h i g h d e n s i t y ( d ) c o n t r o l , ( f ) c o n d i t i o n e d m e d i u m n o d i f f e r e n c e i n s h a p e o r r a d i o -r e s i s t a n c e . 37 a. C e l l Crowding i n Monolayer C u l t u r e s C h inese hamster V79 c e l l s were p l a t e d on t h e same s u r f a c e a r e a a t v a r i o u s c e l l c o n c e n t r a t i o n s . C e l l s p l a t e d a t 4 2 c o n c e n t r a t i o n s of 3 - 6 x 10 c e l l s / c m and grown f o r 24 h o u r s , r e s u l t e d i n t h e same r a d i o s e n s i t i v i t y , i . e. a monolayer r e s p o n s e ( F i g u r e 6 ) . T h i s c o n c e n t r a t i o n was used f o r t h e " h i g h " d e n s i t y p l a t i n g i n a l l subsequent e x p e r i m e n t s . A t e n f o l d d e c r e a s e i n d e n s i t y was used f o r "low" d e n s i t y p l a t i n g , as i t r e s u l t e d i n a s p h e r o i d - l i k e , r a d i o r e s i s t a n t r e s ponse when examined. A response i n t e r m e d i a t e between t h a t o f monolayers and s p h e r o i d s was o b s e r v e d f o r c e l l s p l a t e d a t i n t e r m e d i a t e d e n s i t i e s . R e s u l t s i n F i g u r e 6 might be e x p l a i n e d by d i f f e r e n c e s i n th e degree o f medium d e p l e t i o n , so t o t e s t t h i s h y p o t h e s i s , t h e same number o f c e l l s were p l a t e d i n t o i n c r e a s i n g l y l a r g e r a r e a s . The d e n s i t y o f c e l l s was chosen t o a v o i d problems w i t h s e v e r e o v e r c r o w d i n g , w h i c h would have o c c u r r e d i n t h e s m a l l e s t a r e a , and p r e v e n t e d t h e c e l l s from d i v i d i n g . The c e l l s were seeded onto t h e p l a t e s and t h e n medium was added t o a l l o w them t o d i s t r i b u t e t o a p r e d e t e r m i n e d a r e a . The c e l l s were a l l o w e d t o a t t a c h f o r two h o u r s , t h e n medium was added t o b r i n g t h e t o t a l i n each p l a t e t o t e n ml. The c e l l s remained i n the a r e a t o which t h e y had a t t a c h e d . A f t e r 24 hours of i n c u b a t i o n , t h e c e l l s were t r y p s i n i z e d t o s i n g l e c e l l 38 100 I 1 ' ' ' < 1 ' 1 • r—T 1 I 1 , , , 1 1 , , , , R 0 5 10 0 5 10 R a d i a t i o n D o s e (Gy) F i g u r e 6. E f f e c t of C e l l D e n s i t y - V a r y i n g C e l l Number. X-ray dose response of monolayer c e l l s grown at v a r y i n g c e l l c o n c e n t r a t i o n s i n the same s i z e d area of 80 square cm. The d o t t e d l i n e r e p r e s e n t s the response seen w i t h the lowest c o n c e n t r a t i o n . 39 s u s p e n s i o n s , c o u n t e d f o r y i e l d , i r r a d i a t e d and a s s a y e d u s i n g t h e DNA un w i n d i n g a s s a y . C e l l s i n t h e more crowded c o n d i t i o n s d i s p l a y e d a monolayer r e s p o n s e , w h i l e as the c e l l s became l e s s crowded, more r a d i o r e s i s t a n c e , i n terms of DNA u n w i n d i n g k i n e t i c s , was ap p a r e n t ( F i g u r e 7 ) . F i g u r e 8 i s t h e low d e n s i t y c o n t r o l g raph, w i t h c e l l s grown and p l a t e d as mentioned above. As t h e more s p a r s e l y seeded c e l l s d i s p l a y e d a more r a d i o r e s i s t a n t dose r e s p o n s e , i t seemed pr u d e n t t o examine a n o t h e r a s p e c t o f t h e c o n t a c t e f f e c t , i . e. the DNA u n w i n d i n g k i n e t i c s i n a l k a l i s o l u t i o n . A d i s t i g u i s h i n g t r a i t o f s p h e r o i d c e l l s i s t h a t t h e DNA, once i r r a d i a t e d and l e f t t o unwind, w i l l cease t o do so a f t e r a p p r o x i m a t e l y t e n m i n u t e s , w h i l e t h e d e n s e l y g r o w i n g monolayer c e l l s w i l l c o n t i n u e t o unwind w i t h t ime ( 4 3 ) . T h i s i s one o f t h e c h a r a c t e r i s t i c s o f s p h e r o i d c e l l s t h a t has l e d t o s p e c u l a t i o n t h a t t h e r e a r e some ty p e o f c o n s t r a i n t s w i t h i n t h e DNA which w i l l n o t p e r m i t f u r t h e r u n w i n d i n g . F i g u r e 9 i n d i c a t e s t h a t s p a r s e l y grown c e l l s show t h i s same p r o p e r t y . I t was o b s e r v e d t h a t low d e n s i t y monolayer c e l l s show a l e s s e l o n g a t e d shape t h a n c o n t r o l c e l l s , when o b s e r v e d w i t h a m i c r o s c o p e a f t e r 24 hours i n c u l t u r e ( F i g u r e 5 ) . b. Medium Rep l e n i s h m e n t i n Monolayer C u l t u r e s To t e s t the e f f e c t of t h e exchange r a t e of medium on d e n s e l y p l a t e d monolayer c u l t u r e s , c e l l s were p e r m i t t e d t o a t t a c h i n f i v e ml o f medium, t o t i s s u e c u l t u r e p l a t e s . These 40 100 1 — i — • — • — 1 — 1 — i — i — 1 — ' — i — i — i I—i—i—i—i—i—i—>—'—1 i—r 0 5 10 0 5 10 R a d i a t i o n D o s e (Gy) F i g u r e 7. E f f e c t of C e l l Crowding on Monolayer C e l l s . X-ray dose response of monolayer c u l t u r e s p l a t e d a t the same c e l l number i n d i f f e r e n t s i z e d areas. The do t t e d l i n e r e p r e s e n t s the response of the c e l l s i n the l a r g e s t area. The sl o p e s of the curves i n panels (b) , (c) and (d) are s i g n i f i c a n t l y d i f f e r e n t (at the 9 5 p e r c e n t confidence l e v e l ) from the slope of the curve i n panel ( a ) . ^ c e l l number = 5 x 10 41 F i g u r e 8. E f f e c t of C e l l D e n s i t y - X-ray Dose Response. C e l l s grown as spheroids, low and hig h d e n s i t y monolayers, low (A), high ( o ) , spheroids (•) high = 3 x 10 4/cm 2 low = 3 x 10 3/cm 2 42 < z a TJ a i TD c ro c_ 4-» 01 I QJ D O a 100 100 1 1 1 1 1 i i i 1 1 1 1 1 1 i i i i i i n | i i i 1 1 u 11 i 1 1 1 1 1 1 1 80 -60 sphero ids 1 1 1 i i i i i 1 1 1 n l i i i 1 1 1 n l i 1 1 1 1 1 M 1 1 1 1 1 1 1 1 1 100 i i i i i i i 1 1 1 1 1 1 | i i 1 1 1 i i | i i i i i i H I i 1 1 1 1 I I 80 -60 -monolayers 3 x M i l l 1 1 1 1 1 1 1 l l 1 10 3 i 1 1 1 u l i i 1 1 1 1111 1 1 1 1 1 1 1 60 40 M i l l 1 1 1 1 1 1 111 1 1 1 1 1 1 1 11 1 i i 111111 i i 1 1 1 1 n -- o -4 monolayers 3 x 10 ~ i i n l i i i 1 1 1 1 1 1 i I I i i 1 1 1 1 1 n i i I l i l i r 0 . 1 1 .0 10 100 Time of L y s i s (min) 1000 F i g u r e 9 . E f f e c t o f C e l l D e n s i t y - D N A U n w i n d i n g K i n e t i c s . C e l l s g r o w n a s s p h e r o i d s , l o w a n d h i g h d e n s i t y m o n o l a y e r s ( c e l l s / s q u a r e c m ) . T h e d o t t e d l i n e r e p r e s e n t s t h e d e n s e l y - g r o w n m o n o l a y e r r e s p o n s e . 43 were p l a c e d i n s p i n n e r f l a s k s t o w h i c h 200 ml o f medium was c a r e f u l l y added- The c u l t u r e s were a l l o w e d t o s p i n o v e r n i g h t . These c e l l s a l s o d i s p l a y e d r a d i o r e s i s t a n c e ( F i g u r e 10) and when obs e r v e d under a m i c r o s c o p e , had a more s p h e r i c a l shape t h a n c e l l s grown i n s t a t i c medium as c o n t r o l s . Thus i t would seem t h a t t h e r e a r e two e f f e c t s ; t h e p h y s i c a l movement o f t h e medium and t h e sweeping away o f by-p r o d u c t s and r e p l e n i s h m e n t o f n u t r i e n t s . To d i s t i n g u i s h between t h e two, d e n s e l y seeded monolayers were grown i n t e n ml o f medium w h i c h was renewed e v e r y hour d u r i n g a 24 hour i n c u b a t i o n p e r i o d . F i g u r e 11 shows t h a t t h i s r e s u l t e d i n r e l a t i v e r a d i o r e s i s t a n c e . T h i s p o p u l a t i o n o f c e l l s was a s s a y e d f o r DNA u n w i n d i n g k i n e t i c s and t h e r e s u l t s a r e shown i n F i g u r e 10. A g a i n t h e DNA u n w i n d i n g k i n e t i c s a r e s i m i l a r t o t h o s e o f s p h e r o i d c e l l s , i n f e r r i n g a s i m i l a r i t y i n DNA c o n f o r m a t i o n . Monolayer c e l l s were a l s o grown as above e x c e p t t h a t t h e medium was renewed e v e r y f o u r hours ( F i g u r e 1 5 ) . R e s u l t s from the a s s a y o f t h i s c u l t u r e i n d i c a t e d an i n t e r m e d i a t e r a d i o -r e s i s t a n c e compared w i t h F i g u r e 11, where the c u l t u r e d i s p l a y e d a f u l l c o n t a c t e f f e c t . c. Growth of C e l l s on M i c r o c a r r i e r Beads U s i n g c e l l s a t t a c h e d t o m i c r o c a r r i e r beads, w h i c h a r e p l a c e d i n s u s p e n s i o n i n s p i n n e r f l a s k s , p r o v i d e d a n o t h e r method 44 F i g u r e 10. Monolayer C u l t u r e s i n Moving Medium of Spinner F l a s k s . X-ray dose response of c e l l s grown on p l a t e s i n s i d e s p i n n e r f l a s k s , bathed by 200 ml of c o n t i n u a l l y moving medium. The d o t t e d l i n e r e p r e s e n t s the s p h e r o i d c o n t r o l response. S = s p h e r o i d s , M = monolayers 4 5 Figure 1 1 . Monolayer Cultures i n Renewed Medium - X-ray Dose Response. C e l l s grown i n medium which was replaced hourly for 24 hours. The dotted l i n e represents the spheroid control response. S = spheroids, M = monolayers 46 < z Q TD QJ TJ C ro c_ 4-> en l cu r—I n a a 100 80 6 0 rmT| 1 1—I I Mill 1 1—I I I I ll| I I I I -TTTTJ I 1 r u i n Sphero i d s nnl "•' i i i 111nl i i 1 1 1 n l i i i 1 1 1 1 1 1 0 0 11111 1 1 1 11 1 11| i 11 • * 11 i i 1 Mill 8 0 A 4& -6 0 -Mono l a y e r s 1 1 1 ii i i i i 11 nl - h o u r l y i i i i 11n l i i i i i I l l l l l 1 0 0 8 0 6 0 40 T I T I l 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 l| 1 1 i i i II II i i 1 Mill 0 0 Q I -'9 -M o n o l a y e r s - c o n t r o l -i 1111 i i i 111111 i i i i 11111 i 1 1 1 1 n l i i i i i i i r 0 . 1 1 . 0 10 1 0 0 T i m e o f L y s i s ( m i n ) 1 0 0 0 F i g u r e 12. Monolayer C u l t u r e s i n Renewed Medium - DNA Unwinding K i n e t i c s . C e l l s grown i n medium which was r e p l a c e d h o u r l y f o r 24 hours. The d o t t e d l i n e s r e p r e s e n t s the monolayer c o n t r o l response. 47 t o p r e v e n t l o c a l g r a d i e n t s from f o r m i n g . When th e c e l l s do not c o m p l e t e l y c o a t t h e beads, the r a d i o r e s i s t a n c e o f t h e s e c e l l s i s l i k e t h a t o f s p h e r o i d c e l l s as can be seen i n F i g u r e 13. However, when t h e beads a r e f u l l y c o a t e d w i t h c e l l s , l e s s r a d i o -r e s i s t a n c e i s o b s e r v e d . I n a l l t h e e x p e r i m e n t s w i t h medium moving o v e r m o n o l a y e r s , t h e medium i t s e l f was assayed f o r c e l l s , as t h e more r a d i o -s e n s i t i v e m i t o t i c c e l l s t e n d t o d e t a c h and t h i s c o u l d i n f l u e n c e t h e r e s u l t s . V e r y few c e l l s were seen when t h i s medium was o b s e r v e d under a m i c r o s c o p e . T h i s medium was i n c u b a t e d i n p l a t e s f o r one week and assay e d f o r c o l o n i e s , w h i c h would have grown from t h e s e c e l l s . V e r y few c o l o n i e s were o b s e r v e d . d. Growth o f C e l l s i n S u s p e n s i o n C u l t u r e S p h e r o i d s a r e u s u a l l y grown i n a much l a r g e r volume of medium t h a n a r e monolayers. To i n v e s t i g a t e t h e p o s s i b i l i t y t h a t t h i s may have some e f f e c t , s p h e r o i d s and monolayers were b o t h grown i n 20 ml o f medium. Bo t h c u l t u r e s showed i n d e n t i c a l c e l l y i e l d s a f t e r 24 h o u r s , so growth was comparable. However, t h e s p h e r o i d c e l l s c o n t i n u e d t o d i s p l a y a c o n t a c t e f f e c t , as seen i n F i g u r e 14. There may be a r o l e f o r movement. Movement may p r e v e n t l o c a l g r a d i e n t s from f o r m i n g , and t h e s t r e s s of medium moving over t h e s u r f a c e of t h e c e l l s may keep them i n a s p h e r i c a l c o n f o r m a t i o n . A l s o , i t i s known t h a t c e l l attachment t o a s u r f a c e i n d u c e s t h e s y n t h e s i s of c e l l s p r e a d i n g f a c t o r s , 48 100 I — i — i — r — i — i — f — i — i — i — i — r 0 5 10 R a d i a t i o n Dose (Gy) F i g u r e 13. Monolayer C u l t u r e s on M i c r o c a r r i e r Beads. X-ray dose response of c e l l s grown at in t e r m e d i a t e d e n s i t y on m i c r o c a r r i e r beads i n spinner f l a s k s . S = sp h e r o i d s , M = monolayers, C = m i c r o c a r r i e r s 49 F i g u r e 14. E f f e c t of C e l l Crowding on Spheroid C e l l s . X-ray dose response of spheroids c e l l s grown at the same d e n s i t y i n e i t h e r 20 or 200 ml of medium. S = s p h e r o i d , M = monolayer 50 and perhaps t h e s e f a c t o r s cause t h e r a d i o s e n s i t i v e response o f the c e l l s grown as monolayers. e. E f f e c t o f T r y p s i n i z i n g C e l l C u l t u r e s Monolayer c e l l s were p l a t e d a t a h i g h d e n s i t y and a l l o w e d t o a t t a c h . E v e r y f o u r h o u r s , t h e y were t r e a t e d , e i t h e r by m e r e l y r e p l a c i n g t h e medium w i t h f r e s h , o r by t r y p s i n i z i n g and r e p l a c i n g t h e medium, o r t r y p s i n i z i n g and a d d i n g t h e same medium back t o t h e p l a t e s . Four hours was chosen as t h e optimum t i m e f o r c e l l s t o r e t a i n most of t h e i r rounded c o n f o r m a t i o n , and y e t be s u b j e c t e d t o as few t r y p s i n t r e a t m e n t s as p o s s i b l e . To p r e v e n t e x c e s s i v e c e l l l o s s due t o c e n t r i f u g a t i o n and h a n d l i n g , t h e c e l l s were t r y p s i n i z e d i n a f i l m r a t h e r t h a n t h e u s u a l two m l , and were resuspended i n t h e same p l a t e i n w h i c h t h e y had been g r o w i n g . The amount o f t r y p s i n used w i l l a f f e c t t h e th e DNA u n w i n d i n g k i n e t i c s o f t h e c e l l s , b u t t h i s i s not a l a r g e e f f e c t ( O l i v e , u n p u b l i s h e d ) . R a d i o r e s i s t a n c e seen i n t h e s e c u l t u r e s i s s l i g h t l y h i g h e r t h a n i f the c e l l s had been t r y p s i n i z e d as u s u a l . F i g u r e 15 seems t o i n d i c a t e t h a t t r y p s i n and new medium r e s u l t i n more r a d i o r e s i s t a n c e t h a n e i t h e r t r e a t m e n t a l o n e . The i n d i v i d u a l t r e a t m e n t s r e s u l t i n an i n t e r -m e diate amount o f r a d i o r e s i s t a n c e . Perhaps t h i s was a r e s u l t of some f a c t o r w h i c h was p r e s e n t i n two forms; s o l u b l e , w h i c h was removed by t h e c h a n g i n g o f t h e medium, and a t t a c h e d t o t h e c e l l s , w h i c h was removed by t h e t r y p s i n t r e a t m e n t . 51 F i g u r e 1 5 . T r y p s i n T r e a t m e n t o f M o n o l a y e r s . X - r a y d o s e r e s p o n s e o f c e l l s t r e a t e d w i t h t r y p s i n t o a l l o w t h e m t o m a i n t a i n a s p h e r i c a l c o n f o r m a t i o n . T h e d o t t e d l i n e r e p r e s e n t s t h e s p h e r o i d c o n t r o l r e s p o n s e . T h e s l o p e s o f t h e c u r v e s i n p a n e l s ( b ) a n d ( c ) a r e s i g n i f i c a n t l y d i f f e r e n t ( a t t h e 9 5 p e r c e n t c o n f i d e n c e l e v e l ) f r o m t h e s l o p e o f t h e s p h e r o i d c o n t r o l r e s p o n s e . S = s p h e r o i d s , M = m o n o l a y e r s 52 To s e p a r a t e t h e e f f e c t of c o n t a c t from t h a t o f shape, s p h e r o i d c e l l s were t r y p s i n i z e d e v e r y f o u r h o u r s . The c u l t u r e s were o b s e r v e d b e f o r e each t r y p s i n t r e a t m e n t and i t was seen t h a t t h e y had not formed many a g g r e g a t e s and t h o s e t h a t had formed were i n groups o f two o r f o u r . Over 60 p e r c e n t o f t h e p o p u l a t i o n was i n s i n g l e c e l l form. The c e l l s i n s u s p e n s i o n t r e a t e d w i t h t r y p s i n m a i n t a i n e d t h e r a d i o r e s i s t a n c e o f c e l l s i n c o n t a c t , and t h e s m a l l d e c r e a s e i n t h e e f f e c t i s p r o b a b l y due t o t o x i c i t y o f t h e t r y p s i n t r e a t m e n t s ( F i g u r e 16). f . Growth of C e l l s i n M e t h y l C e l l u l o s e To f u r t h e r i n v e s t i g a t e t h e r o l e of c e l l shape, c e l l s were grown i n m e t h y l c e l l u l o s e . The v i s c o s i t y o f m e t h y l c e l l u l o s e p r e v e n t s c e l l s from e l o n g a t i n g o r f o r m i n g a g g r e g a t e s o t h e r t h a n t h o s e formed when t h e c e l l s d i v i d e . C e l l s were p l a t e d i n m e t h y l c e l l u l o s e and i n c u b a t e d f o r one, two o r t h r e e days. When ass a y e d u s i n g t h e a l k a l i u n w i n d i n g t e c h n i q u e , i t was o b s e r v e d t h a t t h e s e c e l l s d e v e l o p e d t h e c o n t a c t e f f e c t s l o w l y o ver t h r e e days ( F i g u r e 17). I t has been o b s e r v e d t h a t c e l l s must grow and d i v i d e t o d i s p l a y t h e c o n t a c t e f f e c t , so t h e m e t h y l c e l l u l o s e c u l t u r e s were a s s a y e d f o r r a t e o f growth u s i n g t h e r a d i o i s o t o p e t r i t i a t e d t h y m i d i n e w h i c h i s i n c o r p o r a t e d i n t o t h e DNA of d i v i d i n g c e l l s . The r a t e of growth f o r t h e s e c e l l s was shown t o be a p p r o x i m a t e l y h a l f t h a t of t h e c o n t r o l s p h e r o i d and monolayer c e l l s ( d a t a n ot shown). T h e r e f o r e , t h e s e r e s u l t s may be due t o a d i f f e r e n c e i n growth r a t e . 53 F i g u r e 16. T r y p s i n Treatment of Spheroids. X-ray dose response of s p h e r o i d c e l l s t r e a t e d w i t h t r y p s i n to prevent aggregation. The d o t t e d r e p r e s e n t s the s p h e r o i d c o n t r o l response. S = s p h e r o i d s , M = monolayers 54 < z Q TJ OJ TD C ro c_ - P tn I QJ i—i XD D O • 0 5 10 R a d i a t i o n D o s e ( G y ) Figure 17. Monolayer Cultures Grown i n Methyl Cellulose. X-ray dose response of c e l l s grown for the indicated times i n t h i s medium. The dotted l i n e represents the monolayer control response. The slopes of both curves i n the t h i r d panel are s i g n i f i c a n t l y d i f f e r e n t (at the 95 percent confidence level) from the slope of the monolayer control response. S = spheroids, M = monolayers, M.C. = methyl c e l l u l o s e 55 g. Growth of C e l l s above Poly(HEMA) To u n d e r s t a n d t h e r o l e of c e l l c o n t a c t , a method was d e v i s e d t o grow c e l l s i n s u s p e n s i o n under monolayer c o n d i t i o n s , i . e. i n t e n ml o f u n s t i r r e d growth medium. Thus, the c e l l s c o u l d f l o a t and a g g r e g a t e , b u t t h e medium would n ot be moving. The n a t u r e o f V79 c e l l s i s such t h a t , i n t h e pr e s e n c e o f serum, t h e y w i l l a t t a c h and f l a t t e n even on p e t r i p l a t e s t h a t a r e n o t d e s i g n e d t o a l l o w t h i s . T h e r e f o r e , a r e l a t i v e l y t h i c k l a y e r o f t h e polymer poly(HEMA) was a p p l i e d t o t h e s e p l a t e s t o p r e v e n t any c e l l s from a t t a c h i n g and assuming a f l a t t e n e d c o n f o r m a t i o n . A t t a c h e d and e l o n g a t e d c e l l s exude d i f f e r e n t m e t a b o l i t e s , i n c l u d i n g e x t r a c e l l u l a r m a t r i x p r o t e i n s ( 5 7 ) , i n t o t h e medium and t h e s e would not n e c e s s a r i l y be found i n t h e s p h e r o i d c u l t u r e s w i t h w h i c h t h e s e c e l l s were t o be compared. When t h e c u l t u r e s were a s s a y e d u s i n g t h e a l k a l i u n w i n d i n g a s s a y , t h e r e was a d i f f e r e n c e i n r a d i o r e s i s t a n c e ( F i g u r e 1 8 ) . C o n t r o l c e l l s a t t a c h e d t o p l a t e s d i s p l a y e d a monolayer response as e x p e c t e d , but t h e c e l l s f l o a t i n g s p h e r i c a l and a g g r e g a t e d i n th e same p l a t e s , e x h i b i t e d o n l y a l i t t l e l e s s r a d i o s e n s i t i v i t y . C e l l s f l o a t i n g i n t h e growth medium o f t h e poly(HEMA)-coated p l a t e s though, d i s p l a y e d a l m o s t as much r a d i o r e s i s t a n c e as c o n t r o l s p h e r o i d c e l l s g r o w i n g i n s p i n n e r f l a s k s . There were no a t t a c h e d c e l l s i n t h e poly(HEMA) p l a t e s t o exude monolayer-produced f a c t o r s , s u g g e s t i n g t h a t t h i s may p l a y some r o l e . 56 F i g u r e 18. C e l l s Grown with Poly(HEMA). X-ray dose response of c e l l s grown i n monolayer c o n d i t i o n s without the a b i l i t y to a t t a c h . S = s p h e r o i d s , M = monolayers a t t a c h e d to non-tissue c u l t u r e p l a s t i c p l a t e s A = C e l l s i n suspension above poly(HEMA) B = C e l l s i n suspension above monolayers 5 7 B. F a c t o r s I n h i b i t i n g Development of the C o n t a c t E f f e c t a. I n h i b i t i o n o f C e l l Growth and D i v i s i o n I t i s known t h a t c e l l s r e q u i r e a p p r o x i m a t e l y 24 hours i n s u s p e n s i o n c u l t u r e t o d i s p l a y t h e f u l l c o n t a c t e f f e c t ( 1 7 ) . F o r t h i s r e a s o n i t i s i m p o r t a n t t o ensure t h a t t h e c e l l s a r e g r o w i n g n o r m a l l y , e i t h e r by c o u n t i n g t h e c e l l s f o r y i e l d o r u s i n g t h e i n c o r p o r a t i o n o f t r i t i a t e d t h y m i d i n e i n t o t h e DNA as was done w i t h t h e r e l a t i v e l y s l o w l y g r o w i n g m e t h y l c e l l u l o s e c u l t u r e s . C e l l s i n s p i n n e r f l a s k s , i n c u b a t e d o v e r n i g h t a t room t e m p e r a t u r e , showed no d i v i s i o n and d i d not d i s p l a y a c o n t a c t e f f e c t . These c e l l s were grown under th e same c o n d i t i o n s , e x c e p t t e m p e r a t u r e , t h a t s p h e r o i d s d i s p l a y i n g the c o n t a c t e f f e c t were grown. However, c e l l s grown f o r 24 hours i n s u s p e n s i o n a t 37°C, t h e n i n c u b a t e d o v e r n i g h t a t room t e m p e r a t u r e , d i d not grow but d i d m a i n t a i n t h e r a d i o r e s i s t a n c e . b. Growth o f C e l l s i n Monolayer C u l t u r e I t has been o b s e r v e d t h a t c e l l s i n d e n s e l y seeded monolayer c u l t u r e do n o t d e v e l o p a c o n t a c t e f f e c t . Whether t h i s i s due t o t h e l a c k o f t h r e e - d i m e n s i o n a l c e l l c o n t a c t , c e l l shape o r medium c o n s t i t u e n t s has not been e s t a b l i s h e d . Medium movement o v e r monolayer c e l l s i n s p i n n e r f l a s k s r e s u l t e d i n development of r a d i o r e s i s t a n c e ( F i g u r e 1 0 ) , b u t t h e s e c u l t u r e s were grown i n a 58 l a r g e amount o f medium (200 m l ) . I t was i m p o r t a n t t o d e t e r m i n e i f i t was t h e volume o f medium o r t h e movement o f i t over t h e c e l l s t h a t p e r m i t t e d t h e e x h i b i t i o n o f t h e c o n t a c t e f f e c t . To d i f f e r e n t i a t e between t h e medium and movement, c e l l s were d e n s e l y seeded i n p l a t e s w i t h t e n ml of medium. The c e l l s were a l l o w e d t o a t t a c h and t h e n t h e p l a t e s were p l a c e d on a moving p l a t f o r m i n c u b a t o r . T h i s a l l o w e d t h e medium t o move g e n t l y o v e r t h e c e l l s . The r a d i o s e n s i t i v i t y of t h e s e c e l l s was n o t s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l monolayer c e l l s ( F i g u r e 1 9 ) . These c e l l s were a l s o o b s e r v e d t o be i n t h e more e l o n g a t e d c o n f o r m a t i o n o f t h e c o n t r o l c e l l s . c. Use o f C o n d i t i o n e d Medium To more c l o s e l y o b s e r v e t h e e f f e c t s o f t h e medium, s t u d i e s were done u s i n g c o n d i t i o n e d medium. T h i s was produced by e x p o s i n g t h e u s u a l growth medium t o h i g h d e n s i t y monolayer c u l t u r e s f o r 24 h o u r s . T h i s medium was used t o grow s p h e r o i d , low and h i g h d e n s i t y c u l t u r e s . C o n d i t i o n e d medium d i d not a f f e c t t h e r e s p o n s e o f t h e h i g h d e n s i t y c u l t u r e s , and t h e medium was n o t so d e p l e t e d as t o a f f e c t t h e growth o f c e l l s , b u t i t d i d reduce t h e r a d i o r e s i s t a n c e of t h e s p h e r o i d and low d e n s i t y c u l t u r e s ( F i g u r e 2 0 ) . I t can o n l y be s p e c u l a t e d whether t h i s was t h e r e s u l t o f n u t r i e n t d e p l e t i o n o r t h e p r e s e n c e o f m e t a b o l i c b y - p r o d u c t s w h i c h a f f e c t t h e development o f t h e c o n t a c t e f f e c t . Perhaps some s u b s t a n c e r e l e a s e d from monolayer c e l l s , such as s o l u b l e e x t r a c e l l u l a r m a t r i x p r o t e i n s , would 59 19 Monolayer C u l t u r e s i n Moving Medium on Moving P l a t f o r m Incubator (mixer). X-ray dose response o c e l l grown i n .ten ml of c o n t i n u a l l y moving medium. S = spheroids, M = monolayers 6 0 100 I i i i 1 1 1——i——i 1 1 r 0 5 10 R a d i a t i o n D o s e ( G y ) F i g u r e 20. C e l l s Grown i n C o n d i t i o n e d Medium - X-ray Dose Response. E f f e c t of growing c u l t u r e s i n medium which has been c o n d i t i o n e d by monolayer c e l l s f o r 24 hours. The s l o p e s of the curves i n the top two panels are s i g n i f i c a n t l y d i f f e r e n t (at the 95 percent confidence l e v e l ) from the slopes of t h e i r r e s p e c t i v e c o n t r o l responses. C = cell ' s grown i n normal medium, D = c e l l s grown i n c o n d i t i o n e d medium 61 a f f e c t t h e DNA of t h e s p h e r o i d c e l l s . The low d e n s i t y c e l l s , t r e a t e d w i t h c o n d i t i o n e d medium, were o b s e r v e d t o be l e s s rounded t h a n t h e same c e l l s grown i n c o n t r o l medium ( F i g u r e 5). To ensure t h a t t h e s e r e s u l t s were not caused by t o x i c i t y from t h e p l a s t i c of t h e p e t r i p l a t e s , c e l l s were a l s o grown on g l a s s p l a t e s and assa y e d f o r t h e c o n t a c t e f f e c t . There was no d i f f e r e n c e i n r a d i o s e n s i t i v i t y as compared w i t h c e l l s grown on p l a s t i c p l a t e s ( F i g u r e 2 1 ) . Medium was t h e n c o n d i t i o n e d on p l a s t i c and g l a s s p l a t e s and used t o c u l t u r e s p h e r o i d and monolayer c e l l s . F i g u r e 22 shows t h a t t h e r e was no d i f f e r e n c e r e s u l t i n g from t h e t y p e of c o n d i t i o n e d medium used. The s p h e r o i d c e l l s c o n t i n u e d t o d i s p l a y t h e l o w e r e d r a d i o r e s i s t a n c e seen when t h e c e l l s a r e grown i n c o n d i t i o n e d medium, and t h e r e s u l t s were i d e n t i c a l whether g l a s s - o r p l a s t i c - c o n d i t i o n e d medium was used. C. Other C e l l L i n e s Examined f o r the C o n t a c t E f f e c t To e s t a b l i s h t h e g e n e r a l i t y o f t h e e f f e c t d e s c r i b e d f o r C h i n e s e hamster V79-171b c e l l s , o t h e r c e l l l i n e s were examined f o r t h e p r e s e n c e o f the c o n t a c t e f f e c t . C e l l l i n e s were chosen f o r t h e i r a b i l i t y t o grow i n s u s p e n s i o n , as aggregates o r n o t , as w e l l as i n monolayer c u l t u r e . The SCCVII c e l l l i n e was i n t r i q u i n g because, a l t h o u g h i t grows w e l l i n s u s p e n s i o n , c e l l a g g r e g a t e s do n o t form. When 62 F i g u r e 21. C o n d i t i o n e d Medium C o n t r o l - Growth on Glass P e t r i P l a t e s . X-ray dose response comparison between monolayers grown on g l a s s or p l a s t i c . The d o t t e d l i n e r e p r e s e n t s the monolayer c o n t r o l response. S = s p h e r o i d s , M = monolayers 63 F i g u r e 22. C o n d i t i o n e d Medium C o n t r o l - Medium C o n d i t i o n e d i n Glass or P l a s t i c . Comparison of c u l t u r e s grown i n medium c o n d i t i o n e d by growing monolayer c u l t u r e s f o r 24 hours i n e i t h e r p l a s t i c or g l a s s p l a t e s , c o n t r o l C,0), g l a s s (•,?), p l a s t i c 64 a s s a y e d f o r dose response ( F i g u r e 2 3 ) , and DNA u n w i n d i n g k i n e t i c s ( F i g u r e 2 4 ) , r e s u l t s c h a r a c t e r i s t i c o f V79 c e l l s were ob s e r v e d . T h i s s u p p o r t s the t h e o r y t h a t c e l l c o n t a c t does not p l a y a s i g n i f i c a n t r o l e , a l t h o u g h o t h e r p r o p e r t i e s o f t h i s c e l l l i n e may p r o v i d e t h e e x p l a n a t i o n . The RIF-1 c e l l l i n e grows i n monolayer c u l t u r e and as a g g r e g a t e s i n s u s p e n s i o n much l i k e t h e V79 l i n e , e x c e p t t h a t t h e d o u b l i n g t i m e i s l o n g e r . The X - r a y dose r e s p o n s e and DNA u n w i n d i n g k i n e t i c s i n d i c a t e d t h e p r e s e n c e o f a c o n t a c t e f f e c t ( F i g u r e s 25 and 2 6 ) . The WiDr c e l l l i n e grows as s p h e r i c a l c e l l s i n d i s c r e t e c o l o n i e s i n monolayer c u l t u r e (see F i g u r e 5 ) , and as a g g r e g a t e s i n s u s p e n s i o n . When DNA damage was a s s a y e d ( F i g u r e 27) b o t h monolayers and s p h e r o i d c e l l s were more r a d i o r e s i s t a n t t h a n V79 monolayer c u l t u r e s . T h i s c o u l d s u p p o r t t h e c e l l shape t h e o r y , b u t i t must be n o t e d t h a t u n t i l more s t u d i e s a r e done w i t h t h e s e c e l l s , i t may be a p r o p e r t y of i n h e r e n t r a d i o r e s i s t a n c e and c o u l d r e p r e s e n t a d i f f e r e n t mechanism a l t o g e t h e r . The C-4 I and C-4 I I c e l l l i n e s were a l s o a s s a y e d f o r t h e c o n t a c t e f f e c t ( F i g u r e 2 8 ) . The C-4 I c e l l l i n e grows as s p h e r i c a l c e l l s i n d i s c r e t e c o l o n i e s l i k e t h e WiDR c e l l s , and forms a g g r e g a t e s i n s u s p e n s i o n . An i n t e r m e d i a t e r e s p o n s e was seen f o r t h e monolayer c e l l s and t h e s p h e r o i d r e sponse was l i k e t h a t of t h e V79 c e l l s . The C-4 I I l i n e grows i n a more 65 F i g u r e 23. SCCVII C e l l L i n e and the Contact E f f e c t - X-ray dose response. C e l l s were grown i n suspension c u l t u r e and as monolayer c u l t u r e s f o r 2 4 hours. S = s p h e r o i d s , Sn = i n suspension, M = monolayers 66 4 0 r- i . mil • i i n ml i—i i i mil 1—i i i i I I 0 . 1 1 . 0 10 1 0 0 1 0 0 0 Time of L y s i s (min) F i g u r e 24. S C C V I I C e l l L i n e a n d t h e C o n t a c t E f f e c t - DNA U n w i n d i n g K i n e t i c s . C o m p a r i s o n w i t h V79 c e l l l i n e u n w i n d i n g k i n e t i c s . S = s p h e r o i d s , M = m o n o l a y e r s Sn = i n s u s p e n s i o n 67 < a TD OJ TD C (0 t_ to I QJ i — • £3 D O a 1 0 0 8 0 h 6 0 1 0 0 8 0 6 0 - i 1 1 r V79 C o n t r o l J I I I I L 4 0 r _ ! 1 1— — i L_ 0 5 1 0 Radiation Dose (Gy) F i g u r e 25. RIF-1 C e l l L i n e and the Contact E f f e c t - X-ray Dose Response. C e l l s were grown as monolayers and spheroids f o r two days. S = s p h e r o i d s , M = monolayers 68 0 . 1 1 . 0 10 1 0 0 Time of L y s i s (min) F i g u r e 26. RIF-1 C e l l Line and the Contact E f f e c t - DNA Unwinding K i n e t i c s . Comparison w i t h V79 c e l l l i n e unwinding k i n e t i c s . S = s p h e r o i d s , M = monolayers 69 F i g u r e 27. WiDr C e l l L i n e and the Contact E f f e c t . C e l l s were as monolayers and spheroids f o r two days. S = s p h e r o i d , M = monolayer 70 F i g u r e 28. C-4 I and C-4 II C e l l L i n e s and the Contact E f f e c t . C e l l s were grown as sp h e r o i d s and monolayers f o r two days. The dotted l i n e r e p r e s e n t s the c o n t r o l s p h e r o i d response. S = s p h e r o i d s , M = monolayers 71 flattened conformation and does not aggregate i n suspension, where i t does not grow very well. DNA damage i n the unirradiated c e l l s caused some d i f f i c u l t y i n inte r p r e t i n g the res u l t s . The resultant double-stranded DNA was approximately 7 5 percent i n these c e l l s , as compared with about 90 percent f o r the control V79 c e l l s . So, although both growth conditions produce a p a r t i a l radioresistance, i t i s d i f f i c u l t to ascertain i f t h i s i s a property of contact or not. Table III summarizes the re s u l t s using tumor c e l l l i n e s . TABLE I I I : SUMMARY OF RESULTS FROM TUMOR CELL LINE EXPERIMENTS forms aggregates presence of constraints to unwinding  Fig . c e l l l i n e suspended monolayers Mouse 21. 23. SCCVII Ca RIF-1 Sarcoma + ++ ++ Human 25. 26. 26. WiDR colon Ca C-4 I c e r v i c a l Ca C-4 II c e r v i c a l Ca + + ++ ++ ++ + 72 DISCUSSION The main o b j e c t i v e of t h i s p r o j e c t was t o a s c e r t a i n whether t h r e e - d i m e n s i o n a l c e l l c o n t a c t i s always a r e q u i r e m e n t f o r t h e e x h i b i t i o n o f t h e c o n t a c t e f f e c t , o r i f o t h e r f a c t o r s , such as c e l l shape and growth environment, p l a y a r o l e . T a b l e I I (see R e s u l t s , p. 36) l i s t s t h e key e x p e r i m e n t s and r e s u l t s , w h i c h a r e more f u l l y d i s c u s s e d below. W h i l e i t was n o t p o s s i b l e t o s t a t e c o n c l u s i v e l y t h a t i n t e r c e l l u l a r c o n t a c t was n o t r e q u i r e d f o r t h e c o n t a c t e f f e c t , i t i s c l e a r t h a t f a c t o r s i n a d d i t i o n t o c e l l c o n t a c t c o n t r i b u t e t o , i f not c o n t r o l , t h e development o f r e s i s t a n c e t o DNA u n w i n d i n g . A l i k e l y h y p o t h e s i s i s t h a t c e l l s p r e a d i n g f a c t o r s and e x t r a c e l l u l a r m a t r i x p r o t e i n s c o n t r o l t h e development o f c o n s t r a i n t s t o DNA u n w i n d i n g c h a r a c t e r i s t i c of t h e c o n t a c t e f f e c t . A. I n t e r c e l l u l a r C o n t a c t and C e l l - t o - C e l l Communication C e l l s grown i n c o n t a c t may have t h e advantage o f i n t r a -c e l l u l a r d i f f e r e n c e s t h a t a l l o w f o r g r e a t e r r e p a i r of DNA damage. These d i f f e r e n c e s may i n v o l v e changes i n DNA conform-a t i o n o r i n t h e i n t r a c e l l u l a r environment i t s e l f . Passage o f s m a l l m o l e c u l e s such as g l u c o s e , ATP and o t h e r s , t h r o u g h gap j u n c t i o n s , may a l t e r energy l e v e l s o r m o d i f y r a d i o s e n s i t i v i t y . However, gap j u n c t i o n s a l s o e x i s t between c e l l s grown as monolayers. C e l l s grown i n v i v o o r as s p h e r o i d s i n v i t r o a r e exposed t o a c o n t i n u a l l y renewing environment w h i c h removes 73 m e t a b o l i t e s and r e p l e n i s h e s n u t r i e n t s , whereas monolayer c u l t u r e s a r e n o t . I t has been s u g g e s t e d t h a t communication v i a gap j u n c t i o n s may be i m p o r t a n t i n m o d u l a t i n g th e r a d i a t i o n r e s p o n s e o f c e l l s grown as s p h e r o i d s (13, 17). Gap j u n c t i o n s a r e t h e most common ty p e of c e l l j u n c t i o n and a r e w i d e l y d i s t r i b u t e d i n a n i m a l t i s s u e s . They a l l o w s m a l l (1000 - 1500 d a l t o n s ) w a t e r s o l u b l e m o l e c u l e s ( s u g a r s , i n o r g a n i c i o n s , amino a c i d s , n u c l e o t i d e s , v i t a m i n s ) t o pass d i r e c t l y from th e c y t o p l a s m o f one c e l l t o t h e c y t o p l a s m o f a n o t h e r ( 1 ) . Durand and S u t h e r l a n d (17) i m p l i c a t e d a r o l e f o r b i o e l e c t r i c a l and b i o c h e m i c a l communication between c e l l s , p o s s i b l y w i t h an exchange of r e p a i r m o l e c u l e s . D e r t i n g e r and H u e l s e r (13) o b s e r v e d t h a t , w i t h t h e c e l l l i n e s t h e y examined, o n l y e l e c t r i c a l l y c o u p l e d c e l l s e x h i b i t e d a c o n t a c t e f f e c t ( T a b l e I ) , a l t h o u g h i t was s u g g e s t e d t h a t growth of c e l l s i n t h r e e - d i m e n s i o n a l shape i s a l s o r e q u i r e d . They h y p o t h e s i z e d t h a t t h e c o n t a c t e f f e c t depends on t h e exchange o f c e r t a i n s u b s t a n c e s , p r o b a b l y m o l e c u l e s r e l a t e d t o DNA r e p a i r ( f o r example ATP, cAMP). Though gap j u n c t i o n s may p l a y a r o l e , i t does n o t appear t o be d i r e c t l y r e l a t e d t o t h e r e p a i r p r o c e s s , as s e v e r a l i n v e s t i g a t o r s have s e p a r a t e d s p h e r o i d c e l l s b e f o r e i r r a d i a t i o n and f o u n d t h a t the c e l l s s t i l l r e t a i n e d r a d i o -r e s i s t a n c e (29, 43). I n f a c t , a l l t h e e x p e r i m e n t s r e p o r t e d here used s i n g l e c e l l s u s p e n s i o n s , t r y p s i n i z e d p r i o r t o i r r a d i a t i o n . A l s o , c e l l s grown s p a r s e l y seeded i n monolayer c u l t u r e d i s p l a y e d a c o n t a c t e f f e c t and t h e s e c e l l s c o u l d not be e x c h a n g i n g r e p a i r 74 m o l e c u l e s , t o any g r e a t e x t e n t , v i a gap j u n c t i o n s . I t seems t h a t the c l o s e t h r e e - d i m e n s i o n a l c o n t a c t i s n o t always n e c e s s a r y f o r o b s e r v i n g r a d i o r e s i s t a n c e , as e v i d e n c e d by the e x p e r i m e n t s u s i n g m e t h y l c e l l u l o s e ( F i g . 17) and s p h e r o i d s t r y p s i n i z e d e v e r y f o u r h o u r s ( F i g . 1 6 ) . I n t h e v i s c o u s m e t h y l c e l l u l o s e , t h e c e l l s remained s p h e r i c a l and c o u l d not a g g r e g a t e e x c e p t i n groups o f two o r f o u r p r o b a b l y r e s u l t i n g from c e l l d i v i s i o n . A l s o , s p h e r o i d s w h i c h were t r y p s i n i z e d e v e r y f o u r h o u r s o n l y formed a g g r e g a t e s caused by c e l l d i v i s i o n and even t h e n t h e s e were d i s s o c i a t e d e v e r y f o u r hours by t h e t r y p s i n t r e a t m e n t . Even c e l l s grown as monolayers e x h i b i t e d a c o n t a c t e f f e c t as l o n g as c e r t a i n c o n d i t i o n s p r e v a i l e d . These c o n d i t i o n s i n c l u d e d a c o n s t a n t l y renewed s o u r c e o f growth medium, e i t h e r by r e p l a c e m e n t a t r e g u l a r i n t e r v a l s ( F i g u r e 11) or by m a i n t a i n i n g a h i g h medium t o c e l l r a t i o ( F i g u r e s 8, 10, 13). M o nolayers w h i c h were t r y p s i n i z e d e v e r y f o u r hours a l s o d i s p l a y e d a c o n t a c t e f f e c t ( F i g u r e 15). T h i s may have o c c u r r e d because t h e c e l l s m a i n t a i n e d a more s p h e r i c a l c o n f o r m a t i o n , e i t h e r due t o t h e t r y p s i n t r e a t m e n t i t s e l f o r because t h e t r y p s i n removed any a d h e s i o n - p r o m o t i n g p r o t e i n s , such as f i b r o n e c t i n o r v i t r o n e c t i n , w h i c h were a t t a c h e d t o t h e c e l l s (see s e c t i o n C.). A l t h o u g h SCCVII c e l l s do not aggregate i n s u s p e n s i o n , t h e y d i s p l a y e d t h e same r a d i o r e s i s t a n c e and DNA u n w i n d i n g k i n e t i c s as V79 c e l l s , w h i c h a g g r e g a t e i n s u s p e n s i o n ( F i g u r e s 23, 2 4 ) . W i t h 75 SCCVII c e l l s , t h e p r o p e r t y o f a c o n t a c t e f f e c t may r e p r e s e n t a d i f f e r e n t mechanism from t h a t o f V79 c e l l s . A l t e r n a t e l y , i t s u p p o r t s t h e h y p o t h e s i s t h a t c e l l c o n t a c t i s not n e c e s s a r y f o r the development of t h e c o n t a c t e f f e c t . B. DNA C o n f o r m a t i o n and C e l l Shape I t i s agreed t h a t DNA i s a p r i m a r y t a r g e t f o r r a d i a t i o n -i n d u c e d c e l l k i l l i n g , and t h e c o n t a c t e f f e c t r e p r e s e n t s an i n c r e a s e i n r a d i o r e s i s t a n c e , so t h e e l u c i d a t i o n o f t h e mechanism may be found i n D N A - r e l a t e d e v e n t s . U s i n g two a s s a y s f o r DNA damage, a l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n and a l k a l i e l u t i o n , Durand and O l i v e (16) found no d i f f e r e n c e i n t h e numbers of 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 DNA b r e a k s produced i n monolayer and s p h e r o i d c e l l s , and no d i f f e r e n c e i n DNA r e j o i n i n g k i n e t i c s . However, Koerner e t a l . (33) o b s e r v e d t h a t r e j o i n i n g o f s i n g l e - s t r a n d DNA b r e a k s a f t e r i r r a d i a t i o n , t a k e s p l a c e more q u i c k l y and more c o m p l e t e l y i n s p h e r o i d c e l l s , and u s i n g t h e more s e n s i t i v e a l k a l i u n w i n d i n g a s s a y , O l i v e and Durand (42) r e p o r t e d s i m i l a r f i n d i n g s . One e x p l a n a t i o n f o r t h e s e r e s u l t s may be t h a t t h e r e i s a d i f f e r e n c e i n DNA c o n f o r m a t i o n between monolayer and s p h e r o i d c e l l s . I f t h e DNA c o n f o r m a t i o n i s a f f e c t e d by t h e shape of the n u c l e u s and t h e n u c l e a r shape i s r e l a t e d t o t h e c y t o s k e l e t o n , t h e n i t may f o l l o w t h a t t h e DNA " p a c k a g i n g " o f s p h e r i c a l c e l l s i s d i f f e r e n t from t h a t o f more f l a t t e n e d c e l l s . The d i f f e r e n c e i n DNA 76 u n w i n d i n g c h a r a c t e r i s t i c s does not appear t o be a r e s u l t o f a de c r e a s e i n t h e number o f DNA s t r a n d b r e a k s produced i n t h e s e c e l l s by i o n i z i n g r a d i a t i o n , so t h e r e must be a n o t h e r e x p l a n a t i o n . One p o s s i b i l i t y may be t h a t c r o s s l i n k s , e i t h e r DNA-DNA o r DNA - p r o t e i n , b r o u g h t on by a c o n f o r m a t i o n a l change o f t h e DNA, form c o n s t r a i n t s t o DNA un w i n d i n g . O l i v e e t a l . (43) s u g g e s t e d t h a t i n c e l l s w h i c h show r a d i o r e s i s t a n c e , t h e p a c k a g i n g o f t h e DNA i s a l t e r e d and t h a t t h e r e a r e c o n s t r a i n t s a t r e g u l a r i n t e r v a l s . These c o n s t r a i n t s c o u l d e x p l a i n t h e d i f f e r e n c e seen i n u n w i n d i n g k i n e t i c s a f t e r DNA s t r a n d breakage. They a l s o may a l l o w t h e DNA o f t h e s e c e l l s t o r e j o i n more q u i c k l y and/or more a c c u r a t e l y . E x p r e s s i o n o f p a r t i c u l a r genes a r e s p e c i f i c t o c e l l and DNA c o n f o r m a t i o n s . C r o s s l i n k s i n r a d i o r e s i s t a n t DNA may be th e r e s u l t o f p r o t e i n e x p r e s s e d by t h e s e c e l l s , a l t h o u g h t h e r e i s no e v i d e n c e f o r t h i s . The c o n f o r m a t i o n o f DNA i t s e l f c o u l d r e s u l t i n DNA-DNA c r o s s l i n k s v i a t h e a c t i o n o f t o p o i s o m e r a s e s . T h i s c l a s s o f enzymes can c o n t r o l and m o d i f y t h e t o p o l o g i c a l s t a t e s o f DNA by b r e a k i n g t he s t r a n d ( s ) and c a t a l y z i n g i n t e r -c o n v e r s i o n s between d u p l e x m o l e c u l e s . Topoisomerase I I has been found t o a f f e c t many v i t a l b i o l o g i c a l f u n c t i o n s , i n c l u d i n g t h e r e p l i c a t i o n , and p o s s i b l y t h e r e p a i r , o f DNA (38, 5 6 ) . C e l l shape i s known t o cause n u c l e a r changes. The monolayer c e l l s may produce s u b s t a n c e s , such as e x t r a c e l l u l a r m a t r i x p r o t e i n s w h i c h i n f l u e n c e n u c l e a r shape v i a t h e c y t o -77 s k e l e t o n , and wh i c h i n d i r e c t l y p r e v e n t t h e DNA from f o r m i n g t h e more r a d i o r e s i s t a n t c o n f o r m a t i o n . T h i s was s u g g e s t e d by the d e c r e a s e d r a d i o r e s i s t a n c e of s p h e r o i d s grown e i t h e r i n p l a t e s c o n t a i n i n g a t t a c h e d c e l l s o r i n medium w h i c h had been c o n d i t i o n e d by exposure t o a t t a c h e d c e l l s . I f t h i s s u b s t a n c e was removed, such as when monolayer c e l l s were t r y p s i n i z e d f r e q u e n t l y o r when t h e medium was r e p l e n i s h e d , t h e c e l l s showed th e same DNA response as c o n t r o l s p h e r o i d c e l l s ( F i g u r e 1 2 ) . C. C e l l Attachment and S p r e a d i n g F a c t o r s C e l l u l a r a d h e s i o n t o a s u b s t r a t u m and c e l l s p r e a d i n g a r e th o u g h t t o p l a y an i m p o r t a n t r o l e i n many b i o l o g i c a l p r o c e s s e s . A number o f g l y c o p r o t e i n s have been i s o l a t e d t h a t mediate c e l l a t t a chment and s p r e a d i n g i n c u l t u r e . These i n c l u d e f i b r o n e c t i n , v i t r o n e c t i n , l a m i n i n and o t h e r s . F i b r o n e c t i n i s t h e most t h o r o u g h l y c h a r a c t e r i z e d o f t h e s e . I t e x i s t s as a s o l u b l e p r o t e i n i n plasma and i n i n s o l u b l e form ( a t t a c h e d t o r e c e p t o r s ) a t c e l l s u r f a c e s . V i t r o n e c t i n , w h i c h has been more r e c e n t l y c h a r a c t e r i z e d ( 2 7 ) , has some of t h e same p r o p e r t i e s , s uch as p r o m o t i n g c e l l attachment and i n t e r a c t i n g w i t h g l y c o s a m i n o -g l y c a n s and p r o t e o g l y c a n s , b u t has a somewhat d i f f e r e n t t i s s u e d i s t r i b u t i o n . C e l l u l a r r e c e p t o r s r e c o g n i z e t h e s h o r t amino a c i d sequence - a r g i n i n e - g l y c i n e - a s p a r t i c a c i d - (-Arg-Gly-Asp-) i n f i b r o n e c t i n , v i t r o n e c t i n and some o t h e r c e l l u l a r r e c o g n i t i o n p r o t e i n s , and t h i s sequence i s r e q u i r e d f o r t h e b i n d i n g of c e l l r e c e p t o r s t o t h e p r o t e i n s ( 4 4 ) . D e s p i t e t h i s s i m i l a r i t y , 78 however, the c e l l s u r f a c e r e c e p t o r s w h i c h r e c o g n i z e f i b r o n e c t i n and v i t r o n e c t i n a r e d i f f e r e n t . E x p e r i m e n t s w i t h d i f f e r i n g growth c o n d i t i o n s have shown t h a t t h e growth medium may p l a y a l a r g e r r o l e i n t h e c o n t a c t e f f e c t t h a n p r e v i o u s l y assumed, and t h i s may be due t o c e l l s p r e a d i n g f a c t o r s . C e l l s grown d e n s e l y as m o n o l a y e r s , b u t i n an e nvironment w i t h a l a r g e volume of s t i r r e d medium, such as on m i c r o c a r r i e r beads o r p e t r i p l a t e s i n s p i n n e r f l a s k s , e x h i b i t a t l e a s t a p a r t i a l c o n t a c t e f f e c t . Of c o u r s e i t i s not p o s s i b l e t o d i s t i n g u i s h between some c e l l s showing a f u l l e f f e c t w h i l e some show none, and a l l c e l l s showing a p a r t i a l e f f e c t . H i g h c e l l d e n s i t y c u l t u r e s , a t t a c h e d t o p e t r i p l a t e s and grown i n u n s t i r r e d medium, do not e x h i b i t a c o n t a c t e f f e c t . T h i s may be due t o a h i g h l o c a l c o n c e n t r a t i o n of b y - p r o d u c t s , such as c e l l a t tachment and s p r e a d i n g f a c t o r s , produced by t h e s e c e l l s i n r e s p o n s e t o t h e i r s u b s t r a t u m . F e t a l b o v i n e serum, w h i c h i s u sed i n growth medium, c o n t a i n s s p r e a d i n g f a c t o r s , i n c l u d i n g f i b r o n e c t i n and v i t r o n e c t i n . Some c e l l s may use t h e s e components t o a t t a c h and s p r e a d on t h e s u b s t r a t e . However, V79 c e l l s make t h e i r own f i b r o n e c t i n i n response t o attachment t o t i s s u e c u l t u r e p l a t e s . A f t e r a few hours i n monolayer c u l t u r e , V79 c e l l s appear t o be f u l l y s p r e a d . I t seems l i k e l y t h a t a t tachment f a c t o r s o r e x t r a c e l l u l a r m a t r i x p r o t e i n s made by t h e c e l l s t h e m s e l v e s a r e i n v o l v e d i n t h e development of t h e c o n t a c t e f f e c t . O t h e r w i s e , 79 monolayers grown a t low c e l l p a c k i n g o r i n c o n s t a n t l y renewed medium s h o u l d have b e t t e r a c c e s s t o serum s p r e a d i n g f a c t o r s t h a n monolayers grown a t h i g h d e n s i t y . A h i g h c o n c e n t r a t i o n o f t h e s e f a c t o r s may a l s o p r e v e n t t h e c e l l s from m a i n t a i n i n g t h e c e l l shape t h a t i s c o n d u c i v e t o r a d i o r e s i s t a n c e (see s e c t i o n D.). The r a d i o s e n s i t i v i t y may be t h e r e s u l t o f some su b s t a n c e produced by t h e monolayer c e l l s , and n o t m e r e l y due t o t h e r e l a t i v e l y s m a l l volume of medium i n w h i c h t h e c e l l s a r e grown. T h i s t h e o r y i s s u p p o r t e d by t h e r e s u l t s from e x p e r i m e n t s where c e l l s were grown i n a s m a l l amount of medium, i n s u s p e n s i o n ( F i g u r e 14) o r above poly(HEMA)-c o a t e d d i s h e s ( F i g u r e 1 8 ) . These c e l l s e x h i b i t e d a f u l l c o n t a c t e f f e c t . C e l l s grown a t a d e n s i t y t e n t i m e s s p a r s e r t h a n "normal" do e x h i b i t a c o n t a c t e f f e c t . T h i s may be t h e r e s u l t o f r e d u c e d l e v e l s o f t h e s e f a c t o r s as a l l o f the c e l l s a r e f u r t h e r a p a r t from each o t h e r . C o n d i t i o n e d medium e x p e r i m e n t s seem t o s u p p o r t the t h e o r y t h a t some s u b s t a n c e r e l e a s e d by t h e monolayer c e l l s i n t e r f e r e s w i t h t h e a c q u i s i t i o n of t h e c o n t a c t e f f e c t . V e r y dense monolayer c u l t u r e s i n l a r g e amounts of moving medium may o n l y show p a r t i a l r a d i o r e s i s t a n c e because some of t h i s f a c t o r a t t a c h e s t o t h e c e l l s b e f o r e t h e moving medium can sweep i t away. The a ttachment of t h i s f a c t o r t o c e l l s may t h e n be r e s p o n s i b l e f o r changes i n t h e c y t o s k e l e t o n w h i c h i n t u r n b r i n g about changes i n t h e n u c l e u s . 80 D. R o l e of C e l l Shape and E x t r a c e l l u l a r M a t r i x W i t h the p o s s i b l e e x c e p t i o n s of c e l l shape and c e l l - t o - c e l l communication, no d i f f e r e n c e s have been found i n t h e p h y s i c a l p r o p e r t i e s ( c e l l c y c l e d i s t r i b u t i o n , p l a t i n g e f f i c i e n c y , r a t e o f DNA and RNA s y n t h e s i s ) o f V79 c e l l s grown as s p h e r o i d s and monolayers. One of t h e most i n t r i g u i n g o b s e r v a t i o n s o f t h e c o n t a c t e f f e c t i s t h a t c e l l s from s p h e r o i d s can be d i s a g g r e g a t e d p r i o r t o i r r a d i a t i o n and s t i l l d i s p l a y r e l a t i v e r a d i o r e s i s t a n c e . T h e r e f o r e , t h e h i s t o r y o f t h e c e l l w i t h r e s p e c t t o growth c o n d i t i o n s must p l a y a c r i t i c a l r o l e i n t h e e x h i b i t i o n o f t h e c o n t a c t e f f e c t . S e v e r a l l i n e s o f e v i d e n c e s u g g e s t t h a t t h e sub-s t r a t u m upon w h i c h th e c e l l grows p l a y s an i m p o r t a n t r o l e i n d e t e r m i n i n g c e l l shape, r a t e o f DNA s y n t h e s i s , r e s p o n s e t o growth f a c t o r s and s u s c e p t i b i l i t y t o c y t o t o x i c a gents (21, 3 8 ) . Recent r e s u l t s s u ggest t h a t t h e e x t r a c e l l u l a r m a t r i x formed by a c e l l a f f e c t s t h e number and/or d i s t r i b u t i o n o f e x t r a c e l l u l a r m a t r i x r e c e p t o r s i n t h e plasma membrane. T h i s i n f l u e n c e s t h e i n t e r a c t i o n o f p o l y m e r i z e d a c t i n w i t h t h e membrane, and changes i n t h e a c t i n assembly cause changes i n c e l l shape ( 5 7 ) . I t i s p o s s i b l e t h a t a l t e r a t i o n s i n c e l l shape o r i n t r a c e l l u l a r a r c h i t e c t u r e , r e s u l t i n g from d i f f e r e n c e s i n s u b s t r a t u m , a r e r e s p o n s i b l e f o r t h e i n c r e a s e d r e s i s t a n c e t o i o n i z i n g r a d i a t i o n . The i m p o r t a n c e of c e l l shape t o c e l l f u n c t i o n was examined by Ben-Ze'ev e t a l . ( 6 ) . U s i n g anchorage-dependant mouse f i b r o b l a s t s , attachment t o a s u r f a c e and t h e e f f e c t s of c e l l 81 shape were s t u d i e d . Changes i n c e l l shape and l a c k o f a t t a c h -ment were seen t o cause the c e s s a t i o n o f c e r t a i n m e t a b o l i c f u n c t i o n s i n some c e l l t y p e s . These f u n c t i o n s were r e c o v e r e d when the a p p r o p r i a t e c o n d i t i o n s were r e s t o r e d . The r e l a t i o n s h i p between c e l l shape and r e c o v e r y o f m a c r o m o l e c u l a r m e t a b o l i s m was examined. T h e i r r e s u l t s s u g g e s t t h a t p r o t e i n s y n t h e s i s r e c o v e r y i s i n d u c e d by c e l l attachment t o a s u r f a c e , w h i l e n u c l e a r f u n c t i o n s , s uch as r e c o v e r y o f mRNA p r o d u c t i o n , rRNA and DNA s y n t h e s i s r e q u i r e e x t e n s i v e c e l l s p r e a d i n g . F i b r o b l a s t s r e s pond t o shape changes by r e g u l a t i n g growth, b u t o t h e r c e l l t y p e s have d i f f e r e n t r e s p o n s e s . Chondrocytes and e p i t h e l i a l c e l l s have been shown t o change t h e i r spectrum of s y n t h e s i z e d p r o t e i n s when c e l l shape i s a l t e r e d ( 6 ) . An " a p p r o p r i a t e " c e l l shape appears t o be c r i t i c a l f o r DNA s y n t h e s i s s i n c e , as u n t r a n s f o r m e d c e l l s become round, t h e i r r a t e o f DNA s y n t h e s i s d e c r e a s e s . I n c o n t r a s t , a s p h e r i c a l shape has o n l y m i n i m a l e f f e c t on DNA s y n t h e s i s i n tumor c e l l s . L o t a n e t a l . (38) p l a t e d a v a r i e t y of u n t r a n s f o r m e d (human s k i n f i b r o b l a s t s , mouse embryo Swiss 3T3 f i b r o b l a s t s ) and n e o p l a s t i c (human c e r v i c a l c a r c i n o m a HeLa-S3, osteosarcoma Hs791, and murine melanomas B16-F1, S91-C2, S91-C154) c e l l l i n e s on t i s s u e c u l t u r e p l a t e s c o a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f poly(HEMA). B o t h normal c e l l l i n e s and t h e n e o p l a s t i c Hs791 l i n e showed a marked shape dependant d e c r e a s e i n DNA s y n t h e s i s , whereas the o t h e r n e o p l a s t i c l i n e s were o n l y s l i g h t l y a f f e c t e d . I t i s i n t e r e s t i n g t o note t h a t WiDr human c o l o n c a r c i n o m a c e l l s 82 and C-4 I human c e r v i c a l c a r c i n o m a c e l l s showed c o n s t r a i n t s t o u n w i n d i n g even when grown as monolayers ( F i g u r e s 25, 26). L i k e o t h e r m a l i g n a n t c e l l s , t h e s e c e l l s p r o b a b l y do not s y n t h e s i z e e x t r a c e l l u l a r m a t r i x m o l e c u l e s ( 5 7 ) , and t h i s c o u l d account f o r t h e r e s u l t s . K u l e s h and Greene (20) a l s o o b s e r v e d t h a t c e l l u l a r p r o l i f e r a t i o n i n normal c e l l s c a n be r e g u l a t e d by c e l l shape, but n o t i n m a l i g n a n t c e l l s . However, shape-dependant p r o l i f e r a t i o n i n c e r t a i n m a l i g n a n t c e l l s c o u l d be r e s t o r e d by t r e a t m e n t of t h e c e l l s w i t h s p e c i f i c amounts of i n t e r f e r o n . L o t a n n o t i c e d t h i s same e f f e c t w i t h r e t i n o i c a c i d ( 1 8 ) . T h i s s u g g e s t s t h a t m a l i g n a n t c e l l s r e t a i n t h e p o t e n t i a l f o r shape-dependant p r o l i f e r a t i o n . A l s o , t h e i n d u c t i o n o f t h e shape-dependant r e g u l a t o r y mechanism d i d n o t r e q u i r e t h e c o n t i n u o u s p r e s e n c e o f i n t e r f e r o n , s u g g e s t i n g t h a t an a l t e r a t i o n i n gene e x p r e s s i o n was i n v o l v e d . As shown by t h e e x p e r i m e n t s w i t h t r y p s i n , renewed medium (o r s p a r s e s e e d i n g ) , and medium f l o w , t h e r e appears t o be a r o l e f o r c e l l shape i n t h e f o r m a t i o n o f t h e c o n t a c t e f f e c t . I t appears t h a t t h e c e l l must a c t u a l l y grow and pass t h r o u g h th e c e l l c y c l e a t l e a s t t w i c e f o r t h e e f f e c t t o be o b s e r v e d . Folkman and Moscona (21) o b s e r v e d t h a t f o r n o n t r a n s f o r m e d mammalian c e l l s , t h e shape of t h e c e l l i s t i g h t l y c o u p l e d t o DNA s y n t h e s i s . They used th e h y d r o p h i l i c h y d r o g e l , poly(HEMA), t o 83 reduce th e a d h e s i v e n e s s of p l a s t i c t i s s u e c u l t u r e d i s h e s i n a g raded manner. The e x t e n t of c e l l s p r e a d i n g was c o n t r o l l e d so t h a t t h e c e l l c o u l d be h e l d a t any one o f a g r aded s e r i e s o f 3 . . q u a n t i t a t e d shapes. C e l l i n c o r p o r a t i o n o f H-thymidine was f o u n d t o be i n v e r s e l y p r o p o r t i o n a l t o t h e h e i g h t o f t h e c e l l . Thus, s p r e a d i n g , w h i c h i n v o l v e s r e f o r m a t i o n o f t h e c y t o s k e l e t a l framework composed of m i c r o t u b u l e s , m i c r o f i l a m e n t s and i n t e r m e d i a t e f i l a m e n t s ( 5 7 ) , appears t o be r e q u i r e d f o r t h e s e c e l l s , f o r r e a c t i v a t i n g DNA and RNA s y n t h e s i s , m i t o s i s and c e l l d i v i s i o n . The V79 c e l l s used i n t h i s s t u d y a r e a b l e t o d i v i d e r e g a r d l e s s o f c e l l shape, but s u b s t r a t e c o u l d i n f l u e n c e DNA c o n f o r m a t i o n . I n t h i s l a b o r a t o r y , a t t e m p t s t o grow V79 c e l l s on g r a ded c o n c e n t r a t i o n s o f poly(HEMA) were u n s u c c e s s f u l s i n c e , d u r i n g t h e 24 h o u r s r e q u i r e d f o r the development of t h e c o n t a c t e f f e c t , t h e c e l l s e i t h e r s y n t h e s i z e d s u f f i c i e n t f i b r o n e c t i n o r p a r t i a l l y d i s s o l v e d t h e poly(HEMA) l a y e r and t h i s a l l o w e d a t t achment and s p r e a d i n g of t h e c e l l s . However, a h i g h con-c e n t r a t i o n o f poly(HEMA) was used t o c o a t p e t r i p l a t e s and c o m p l e t e l y p r e v e n t e d c e l l attachment t o t h e s e p l a t e s ( F i g . 1 8 ) . O l i v e e t a l . (43) s u g g e s t e d t h a t t h e r e i s a d i f f e r e n c e i n t h e p a c k a g i n g of DNA i n s p h e r o i d c e l l s . The round shape of the c e l l s , w h i c h b r i n g s about a d i f f e r e n t c o n f o r m a t i o n f o r t h e n u c l e u s and DNA, may a l l o w f o r c o n s t r a i n t s t o form r e l a t i v e l y c l o s e t o g e t h e r on t h e DNA. I t i s a l s o p o s s i b l e t h a t t h e l a c k of a s o l i d s u b s t r a t e means t h a t t h e DNA of c e l l s grown i n s u s p e n s i o n r e q u i r e s a d d i t i o n a l p h y s i c a l s u p p o r t w h i c h i s 84 s u p p l i e d i n t h e form of t h e s e c o n s t r a i n t s . The c e l l s i n c o n t i n u a l l y moving medium may d e v e l o p an a l t e r e d o r g a n i z a t i o n of a c t i n f i b e r s , o r perhaps an i n c r e a s e d number of t h e s e f i b e r s as do t h e e n d o t h e l i a l c e l l s i n h i g h b l o o d f l o w a r e a s ( 2 2 ) , and t h i s may cause n u c l e a r changes. However, t h i s cannot be a f a c t o r i n t h e s p a r s e l y seeded c u l t u r e s , a l t h o u g h when o b s e r v e d under a m i c r o s c o p e , t h e c e l l s a r e much l e s s extended t h a n c o n t r o l monolayers ( 4 1 ) . A t low c e l l p a c k i n g , l e s s s p r e a d i n g c o u l d be due t o a lower l o c a l c o n c e n t r a t i o n o f e x t r a c e l l u l a r m a t r i x p r o t e i n s . The l a c k o f c o n t a c t e f f e c t i n t h e moving p l a t f o r m i n c u b a t o r e x p e r i m e n t s ( F i g u r e 19) c o u l d be due t o t h e f a c t t h a t t h e medium was n o t moving w i t h enough f o r c e t o cause t h i s e f f e c t o r , perhaps because of a h i g h c o n c e n t r a t i o n o f V79 c e l l - p r o d u c e d s p r e a d i n g f a c t o r s i n t h e r e l a t i v e l y s m a l l amount o f medium. Lack o f c e l l s p r e a d i n g f a c t o r s may e x p l a i n t h e r a d i o r e s i s t a n c e o f c e l l s g r o w ing above t h e p o l y(HEMA)-coated p l a t e s i n u n s t i r r e d medium. These c e l l s d i s p l a y e d a c o n t a c t e f f e c t perhaps because no a t t a c h e d c e l l s were p r e s e n t t o produce s p r e a d i n g f a c t o r s . I t may be t h a t one o f two e f f e c t s can dominate. I f movement of t h e l i q u i d medium o v e r th e s u r f a c e o f a c e l l i s s t r o n g enough, s t r e s s may cause an a l t e r a t i o n i n t h e c y t o s k e l e t o n and n u c l e u s . A l t e r n a t e l y , i f t h e medium i s f r e e o f h i g h c o n c e n t r a t i o n s o f monolayer c e l l - p r o d u c e d b y - p r o d u c t s , t h e n t h e c e l l s w i l l grow i n t h e s p h e r i c a l c o n f o r m a t i o n and d i s p l a y r a d i o r e s i s t a n c e . 85 There i s i n c r e a s i n g r e c o g n i t i o n t h a t c y t o s k e l e t a l elements m e d i a t e and r e g u l a t e a v a r i e t y of c e l l u l a r p r o c e s s e s . Stevenson e t a l . (50) o b s e r v e d t h a t c e r t a i n c e l l s grown i n v i t r o as monolayers, were more r a d i o s e n s i t i v e t h a n t h o s e grown i n a rounded c o n f o r m a t i o n . These rounded c e l l s were grown b o t h i n c o n t a c t w i t h o t h e r c e l l s and as i n d i v i d u a l c e l l s . When t h e s e rounded c e l l s were exposed t o s p e c i f i c c y t o s k e l e t a l p o i s o n s , w h i c h a f f e c t e d m i c r o t u b u l e s and m i c r o f i l a m e n t s , t h e r a d i o -s e n s i t i v i t y i n c r e a s e d , whether t h e c e l l s had been grown i n c o n t a c t o r n o t . C e l l s grown i n s p h e r i c a l c o n f o r m a t i o n have a d i f f e r e n t morphology t h a n t h o s e grown as monolayers and t h a t t h e r a d i o r e s i s t a n c e o b s e r v e d was i n t e r p r e t e d t o be due t o "improved" c y t o s k e l e t a l ( i n c l u d i n g t h e n u c l e a r m a t r i x ) f u n c t i o n , and n o t due t o c e l l - t o - c e l l c o n t a c t . R e s u l t s from e x p e r i m e n t s w i t h o t h e r c e l l l i n e s can be seen i n T a b l e I I I . D i f f e r e n c e s i n c e l l shape a r e r e f l e c t e d i n r a d i o -r e s i s t a n c e . The RIF-1 and SCCVII c e l l l i n e s b o t h grow i n an e l o n g a t e d shape as monolayers and y e t remain s p h e r i c a l i n s u s p e n s i o n , much l i k e t h e V79 c e l l s . A l l t h r e e o f t h e s e c e l l l i n e s show q u a n t i t a t i v e l y s i m i l a r r a d i a t i o n r e s p o n s e s ( F i g u r e s 23, 2 5 ) . Even though t h e SCCVII c e l l s do not agg r e g a t e i n s u s p e n s i o n , a c o n t a c t e f f e c t was o b s e r v e d . A l t e r n a t i v e l y , w i t h th e WiDr and C-4 I c e l l l i n e s , t h e c e l l s grow i n a more rounded c o n f o r m a t i o n as monolayers, w h i c h may e x p l a i n why the monolayer c u l t u r e s o f t h e s e c e l l s show more r a d i o r e s i s t a n c e t h a n the V79 monolayer c e l l s . Monolayer c e l l s o f t h e WiDr c e l l l i n e have 86 a l s o been shown t o have a s i m i l a r r a d i o r e s p o n s e t o t h e WiDR s p h e r o i d c e l l s when assayed f o r c e l l s u r v i v a l ( 5 9 ) . The C -4 I I monolayer c e l l s have a more extended c e l l shape, but t h e y do not grow w e l l as s p h e r o i d s so i t i s d i f f i c u l t t o draw c o n c l u s i o n s about them. I n a d d i t i o n t o c e l l growth and d i v i s i o n , c e l l shape seems t o e x e r t a p r o f o u n d i n f l u e n c e on a v a r i e t y o f c e l l u l a r and n u c l e a r f u n c t i o n s , i n c l u d i n g p r o t e i n s y n t h e s i s ( 6 , 2 1 ) , RNA s y n t h e s i s ( 7 ) , and a c t i n gene e x p r e s s i o n ( 2 0 ) . C e l l shape appears t o p l a y a r o l e i n t h e a c q u i s i t i o n o f t h e c o n t a c t e f f e c t . T h i s t h e o r y seems r e a s o n a b l e i n t h a t c e l l shape, v i a t h e c y t o s k e l e t o n , i s i n t i m a t e l y r e l a t e d t o n u c l e a r c o n f o r m a t i o n . 87 SUMMARY AND CONCLUSION The c o n t a c t e f f e c t appears t o be f u n c t i o n a l l y r e l a t e d t o c o n s t r a i n t s t o DNA un w i n d i n g w h i c h d e v e l o p when c e l l s a r e grown as s p h e r o i d s . E l u c i d a t i n g t h e growth c o n d i t i o n s t h a t promote t h i s d i f f e r e n c e was the purpose o f t h i s p r o j e c t . T h r e e - d i m e n s i o n a l c e l l c o n t a c t i s not always n e c e s s a r y f o r th e development o f t h e c o n t a c t e f f e c t i n V79-171B c e l l s . S i m i l a r l y , a "round" shape a l o n e does n ot always d e t e r m i n e r a d i o r e s i s t a n c e , as e v i d e n c e d by t h e f a c t t h a t under c e r t a i n c o n d i t i o n s e l o n g a t e d monolayer c e l l s d i s p l a y e d a c o n t a c t e f f e c t w h i l e round c e l l s d i d n o t . However, c e l l s grown i n s u s p e n s i o n , i n medium w h i c h had been exposed t o a t t a c h e d c e l l s , d i s p l a y e d a d e c r e a s e d r a d i o r e s i s t a n c e . T h i s l e d t o t h e h y p o t h e s i s t h a t t h e monolayer c e l l s were s y n t h e s i z i n g some f a c t o r , p o s s i b l y an e x t r a c e l l u l a r m a t r i x p r o t e i n , w h i c h i n h i b i t e d t h e development of the c o n t a c t e f f e c t . These p r o t e i n s can a f f e c t t h e c y t o s k e l e t o n and i n d i r e c t l y , t h e n u c l e u s . T h i s h y p o t h e s i s c o u l d be f u r t h e r i n v e s t i g a t e d by: a) b l o c k i n g r e c e p t o r s f o r e x t r a c e l l u l a r m a t r i x p r o t e i n s , i n monolayer c e l l s , u s i n g s y n t h e t i c a r g - g l y - a s p p e p t i d e s ( 4 4 ) . b) g r o w i n g V79 c e l l s as s p h e r o i d s i n medium c o n d i t i o n e d by s p h e r o i d s r a t h e r t h a n by monolayers. 88 c) growing WiDR monolayer c e l l s , w h i c h do not produce s p r e a d i n g f a c t o r s and do d i s p l a y r a d i o r e s i s t a n c e , i n medium c o n d i t i o n e d by V79 monolayers, o r on p e t r i d i s h e s c o a t e d w i t h e x t r a c e l l u l a r m a t r i x p r o t e i n s . O v e r a l l , i t would appear t h a t c e l l shape, c e l l - t o - c e l l c o n t a c t and growth medium a r e i n t e r r e l a t e d i n p r o m o t i n g t h e development o f t h e c o n t a c t e f f e c t i n V79 c e l l s . C e l l s grown i n s u s p e n s i o n c u l t u r e may d e v e l o p a d i f f e r e n t c y t o s k e l e t a l s t r u c t u r e w h i c h c o u l d l e a d t o a change i n DNA " p a c k a g i n g " . A change i n DNA c o n f o r m a t i o n c o u l d enhance DNA r e p a i r c a p a b i l i t i e s , perhaps by a l l o w i n g b e t t e r a c c e s s o f r e p a i r enzymes t o damaged s i t e s i n DNA. T h i s g r e a t e r r e p a i r c a p a c i t y would t h e n be r e f l e c t e d i n the h i g h e r c e l l s u r v i v a l o f s p h e r o i d c e l l s exposed t o i o n i z i n g r a d i a t i o n . 89 REFERENCES 1. A l b e r t s , B., B r a y , D., L e w i s , J . , R a f f , M., R o b e r t s , K., Watson, J . D. (1983) M o l e c u l a r B i o l o g y o f t h e C e l l . G a r l a n d P u b l i s h i n g , I n c . , New York and London, pp. 682-691. 2. A c k e r , H., C a r l s s o n , J . , Durand, R. and S u t h e r l a n d , R. E. 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