UBC Theses and Dissertations

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UBC Theses and Dissertations

Aging of mammalian cells in vitro Atchison , Brad 1971

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THE AGING OF MAMMALIAN CELLS in Vitro by BRAD ATCHISON B. S c . , S i r George W i l l i a m s U n i v e r s i t y , 1969 A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t o f t h e Re q u i r e m e n t s f o r t h e Degree o f MASTER OF SCIENCE i n t h e Department of Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a , A p r i l , 1971 In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or p u b l i c a t i o n o f t h i s thesis f o r f i n a n c i a l gain shall not be allowed without my written permission. Department of "ZOOLO&X The University of B r i t i s h Columbia Vancouver 8, Canada Date APRIL 27 \<\7l i . ABSTRACT The p u r p o s e o f t h i s s t u d y was t o examine t h e phenomenon o f a g i n g a t t h e c e l l u l a r l e v e l i n v i t r o . Some o f t h e b i o l o g i c a l mechanisms u n d e r l y i n g t h e a g i n g p r o c e s s , w h i c h may i n v o k e a change a t t h e l e v e l o f t h e DNA were s t u d i e d . M o r p h o l o g i c a l changes were a n a l y s e d w i t h p h a s e - c o n t r a s t m i c r o s c o p y and f u n c t i o n a l changes by t h e use o f t r i t i a t e d t h y m i d i n e i n c o m b i n a t i o n w i t h a u t o r a d i o g r a p h y as w e l l as by c e l l t r e a t m e n t w i t h c o l c h i c i n e . A method i s d e s c r i b e d f o r o b t a i n i n g "aged" o r " o l d " c e l l s i n v i t r o . In t h e human c e l l s (human e m b r y o n i c k i d n e y ) a s w e l l as t h e r a t , mouse, and S y r i a n hamster c e l l s , m o r p h o l o g i c a l changes i n v i t r o a r e b a s i c a l l y t h a same as a g i n g p r o g r e s s e s . These i n c l u d e t r a n s f o r m a t i o n t o a p o l y g o n a l , e p i t h e I i a I - 1 i k e shape; b i n u c I e a t i o n ; an a c c u m u l a t i o n o f "age p i g m e n t s " a round t h e n u c l e u s ; t h e ap p e a r a n c e o f ragged edges o f t h e e e l I membrane; an i n c r e a s e i n t h e o v e r a l l c e l l s i z e ; and a l o s s o f a r e g u l a r ( o f t e n p a r a l l e l ) o r i e n t a t i o n t o a d j a c e n t c e l l s . The m i t o t i c r a t e and D N A - s y n t h e s i z i n g c a p a c i t y i n "young" and "aged" c e l l s were examined u s i n g a u t o r a d i o g r a p h y and c e l l t r e a t m e n t w i t h c o l c h i c i n e . E v i d e n c e i s p r e s e n t e d t h a t D N A - s y n t h e s i z i n g aged c e l l s a r e n o n - p r o l i f e r a t i n g w h i l e D N A - s y n t h e s i z i n g young c e l l s a r e m i t o t i c a l l y a c t i v e . The s i g n i f i c a n c e o f D N A - s y n t h e s i s i n n o n - d i v i d i n g "aged" c e l l s i s d i s c u s s e d . The number o f p o p u l a t i o n d o u b l i n g s ( g e n e r a t i o n t i m e s o r c e l l d i v i s i o n s ) t h a t i t t a k e s hamster and mouse c e l l s t o age i n v i t r o was a l s o i n v e s t i g a t e d . T h i r t e e n and s i x c e l l g e n e r a t i o n t i m e s were found t o c a u s e hamster and mouse c e l l s t o age w i t h a l o s s o f p r o l i f e r a t i v e c a p a c i t y . The e f f e c t o f v a r i o u s m o l a r i t i e s o f 4 - n i t r o q u i n o l i n e - 1 o x i d e (4-NQO), on DNA o f aged e e l I s , w h i c h r e s u l t s i n an u n s c h e d u l e d D N A - r e p a i r s y n t h e s i s , i i . was s t u d i e d u s i n g a u t o r a d i o g r a p h y . I t a p p e a r s t h a t an aged c e l l r e s p o n d s t o t h e s e c o n c e n t r a t i o n s i n much t h e same way as young c e l l s ; however, t h e r e does seem t o be a s l i g h t l y g r e a t e r s e n s i t i v i t y t o t o x i c d oses o f 4-NQO i n aged ceI I s . A u t o r a d i o g r a p h i c s t u d i e s a l s o r e v e a l e d t h a t t h e d u r a t i o n o f DNA-r e p a i r i s t h e same i n b o t h aged and young e e l I s , b u t t h e f o r m e r a p p e a r t o have a d e c r e a s e d c a p a c i t y t o r e p a i r damage t o t h e DNA o f t h e p r e t r e a t e d c e l l s c a u s ed by t h e 4-NQO. The s i g n i f i c a n c e o f t h i s a p p a r e n t d e c r e a s e i n t h e D N A - r e p a i r c a p a c i t y o f "aged" c e l l s i s d i s c u s s e d . Mouse c e l l s , aged i n v i t r o , were exposed t o human a d e n o v i r u s t y p e 12 ( s t r a i n H u i e ) . E v i d e n c e i s p r e s e n t e d t h a t t h i s e x t e r n a l a g e n t s t i m u l a t e d t h e s e aged c e l l s t o i n c r e a s e D N A - s y n t h e s i s and a l s o pushed them i n t o m i t o s i s ( f o r a t l e a s t one c e l l d i v i s i o n ) . The p o s s i b i l i t y t h a t t h i s m i g h t be an a c c o u n t a b l e mechanism f o r t h e o b s e r v a t i o n o f an a c c u m u l a t i o n o f m u t a t i o n s i n aged c e l l s i s e v a l u a t e d . Aged c e l l s were examined f o r f r e q u e n c i e s and t y p e s o f chromosome a b e r r a t i o n s a f t e r e x p o s u r e t o a d e n o v i r u s t y p e 12. Among t h e most common a r e c h r o m a t i d b r e a k s and d o u b l e f r a g m e n t s . As w e l l , o l d c e l l s e x h i b i t e d a much h i g h e r f r e q u e n c y o f chromosome a b e r r a t i o n s t h a n young c e l l s a f t e r v i r a l e x p o s u r e . A c o m p a r i s o n o f t h i s i n v i t r o s y s t e m o f c e l l a g i n g w i t h an i n v i v o s y s t e m i s p r e s e n t e d . The a p p l i c a t i o n o f a l l o f t h e a f o r e m e n t i o n e d r e s u l t s and o b s e r v a t i o n s c o n c e r n i n g t h e c e l l u l a r a g i n g p r o c e s s t o t h e p r o b l e m o f c a r c i n o g e n e s i s and n e o p l a s i a i s e m p h a s i z e d . ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h i s g r a t i t u d e t o P r o f e s s o r H. F. S t i c h under whose d i r e c t i o n t h i s r e s e a r c h was c o n d u c t e d . He i s a l s o g r e a t l y i n d e b t e d t o M r s . H. F. S t i c h , whose t e c h n i c a l a d v i c e and i n s t r u c t i o n have made t h i s t h e s i s p o s s i b l e . I would a l s o l i k e t o t h a n k D r . R. L. N o b l e , C a n c e r R e s e a r c h C e n t e r , U n i v e r s i t y o f B r i t i s h C o l u m b i a , f o r p r o v i d i n g t h e n e c e s s a r y a p p a r a t u s and e q u i p m e n t . The f i n a n c f a l s u p p o r t of a N a t i o n a l C a n c e r I n s t i t u t e S t u d e n t s h i p i s g r a t e f u I Iy a c k n o w l e d g e d . TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION PROBLEM EXPERIMENTAL A. MATERIALS AND METHODS (1) CeI Is (2) V i r u s (3) G e n e r a l C e l l C u l t u r e T e c h n i q u e (4) Embryos P u t I n t o C u l t u r e (5) A u t o r a d i o g r a p h y (6) P h a s e - C o n t r a s t M i c r o s c o p y o f L i v i n g C e l l C u l t u r e s (7) V i r a l I n f e c t i o n o f C e l l C u l t u r e s (8) E x p o s u r e o f C e l l s t o 4NQ0 (9) C y t o l o g i c P r e p a r a t i o n s B. RESULTS (1) M o r p h o l o g i c a l Changes Undergone by A g i n g C e l l s (2) M i t o t i c R a t e and D N A - R e p I i c a t i o n S y n t h e s i s i n O l d C e l l s (3) C e l l G e n e r a t i o n Time f o r A g i n g i n v i t r o (4) D u r a t i o n o f 4NQ0-lnduced DNA-Repair S y n t h e s i s i n O l d C e l l (5) E f f e c t o f V a r i o u s Doses of 4NQ0 on DNA-Repair i n O l d C e l l (6) E f f e c t s o f A d e n o v i r u s 12 (AD 12) on D N A - S y n t h e s i s , C e l l D i v i s i o n , and Chromosome A b e r r a t i o n s i n O l d C e l l s Page DISCUSSION 47 BIBLIOGRAPHY 58 LIST OF TABLES T a b l e No. Page 1 STOCHASTIC CAUSES OF AGING. 3 2 GENETIC CAUSES OF AGING. 4 3 AGING OF MAMMALIAN CELLS. 5 4 SUBSTANCES WHICH MIGHT MODIFY THE RATE OF AGING. 8 5 DNA-SYNTHESIS IN YOUNG AND OLD CELLS. 25 6 MITOTIC RATE IN YOUNG AND OLD CELLS. 25 7 DAYS IN CULTURE FOR VARIOUS CELL STRAINS TO LOSE 32 PROLIFERATIVE CAPACITY. 8 CAPACITY OF AD 12 TO INDUCE CHROMOSOME ABERRATIONS 46 IN YOUNG AND OLD MOUSE CELLS. 9 PARAMETERS DISTINGUISHING AGED CELLS FROM YOUNG 57 CELLS i n v i t r o . V i LIST OF FIGURES F i g u r e Page 1 Young r a t c e l l s ( f i r s t p a s s a g e ) showing f a i r l y r e g u l a r 19 o r i e n t a t i o n t o one a n o t h e r . 2 A p o l y g o n a I Iy-shaped aged r a t c e l l i n v i t r o . 19 3 A b i n u c l e a t e d aged r a t c e l l i n v i t r o . 19 4 A young s t e l l a t e - s h a p e d mouse e e l I. 20 5 O l d mouse c e l l s i n v i t r o . 20 6 Young S y r i a n hamster c e l l s i l l u s t r a t i n g p a r a l l e l o r i e n t a t i o n 21 t o one a n o t h e r . 7 O l d S y r i a n hamster c e l l s w i t h ragged edges o f t h e c e l l 21 membranes. 8 A b n o r m a l l y - s h a p e d n u c l e u s o f an aged S y r i a n hamster c e l l . 21 9 E l o n g a t e young human k i d n e y c e l l s ( f i r s t p a s s a g e ) . 22 10 L a r g e r aged human k i d n e y c e l l ( f i f t h p a s s a g e ) . 22 11 S-phase n u c l e i i n young r a t c e l l s . 24 12 S-phase n u c l e u s i n aged r a t c e l l . 24 13 S-phase n u c l e u s i n aged mouse c e l l . 24 14 N u c l e u s w h i c h a p p e a r s t o be i n t h e p r o c e s s o f s p l i t t i n g 26 from an aged mouse e e l I. 15 N u c l e u s w h i c h a p p e a r s t o be i n t h e p r o c e s s o f s p l i t t i n g 26 from an aged mouse c e l l . 16 B i n u c l e a t e d c o n d i t i o n o f an aged mouse c e l l . 26 17 COMPARISON OF SEEDING EFFICIENCY WITH CLONE-FORMING CAP- 29 ACITY AS MOUSE CELLS ARE PASSAGED i n v i t r o . 18 EFFECT OF PASSAGE NUMBER ON CLONE-FORMING CAPACITY OF 3 0 EMBRYONIC HAMSTER CELLS i n v i t r o . EFFECT OF PASSAGE NUMBER ON CLONE-FORMING CAPACITY OF EM-BRYONIC MOUSE CELLS i n v i t r o . DURATION OF DNA-REPAIR SYNTHESIS IN AGED MOUSE CE L L S , PRETREATED I 1/2 HOURS WITH 4NQO ( 4 X I 0_ 6M ) . DURATION OF DNA-REPAIR SYNTHESIS IN AGED MOUSE CE L L S , PRETREATED I 1/2 HOURS WITH 4NQ0 ( 2 X I 0 ~6M ) . N u c l e i from young mouse c e l l c u l t u r e s exposed t o 4NQO ( 4 x l O- 6M 4NQ0) f o r 7 1/2 h o u r s . N u c l e i f r o m young r a t c e l l s exposed t o 4NQ0 (4x10 ^M) f o r I 1/2 h o u r s . Aged mouse c e l l s exposed t o 4NQ0 (4x10 6M) f o r I 1/2 h o u r s . Aged r a t c e l l exposed t o 4NQ0 ( 4 x l 0_ 6M ) f o r I 1/2 h o u r s . EFFECT OF VARIOUS DOSES OF 4NQ0 ON DNA-REPAIR SYNTHESIS OF AGED RAT CELLS i n v i t r o . EFFECT OF VARIOUS DOSES OF 4NQ0 ON DNA-REPAIR SYNTHESIS OF AGED MOUSE CELLS j n _ v i t r o . EFFECT OF AD 12 ( s t r a i n H u i e ) ON DNA-SYNTHESIS IN YOUNG AND AGED MOUSE CELLS. EFFECT OF ADI2 ( s t r a i n H u i e ) ON CELL DIVISION IN YOUNG AND AGED MOUSE CELLS. Metaphase p l a t e from a young n o n - i n f e c t e d mouse c e l l ( c o n t r o l Metaphase p l a t e w i t h an i s o c h r o m a t i d b r e a k f r o m a young mouse c u l t u r e exposed t o AD 12. Metaphase p l a t e w i t h d o u b l e f r a g m e n t s and a s i n g l e b r e a k from a young mouse c u l t u r e exposed t o AD 12. Metaphase p l a t e w i t h a s i n g l e c h r o m a t i d b r e a k from a young mouse c u l t u r e exposed t o AD 12. Metaphase p l a t e w i t h a c o i l i n g d e f i c i e n c y ( " s t i c k i n e s s ) from a young mouse c u l t u r e exposed t o AD 12. i x . F i g u r e Page 35 Metaphase p l a t e w i t h many s i n g l e c h r o m a t i d b r e a k s from an o l d 44 mouse c u l t u r e exposed t o AD 12. 36 Metaphase p l a t e w i t h i s o c h r o m a t i d and s i n g l e c h r o m a t i d b r e a k s 44 from an o l d mouse c u l t u r e exposed t o AD 12. 37 Metaphase p l a t e w i t h d o u b l e f r a g m e n t s from an o l d mouse c u l t u r e 45 exposed t o AD 12. 38 Metaphase p l a t e w i t h a c o i l i n g d e f i c i e n c y ( " h a z i n e s s " ) and a 45 d o u b l e b r e a k on one c h r o m a t i d from an o l d mouse c u l t u r e exposed 45 t o AD 12. 39 Metaphase p l a t e w i t h a c h r o m a t i d exchange and a s i n g l e b r e a k 45 from an o l d mouse c u l t u r e exposed t o AD 12. 1. INTRODUCTION I t Is a commonly-held m i s c o n c e p t i o n t h a t modern m e d i c i n e has l e n g t h -ened t h e human l i f e s p a n . T h i s i m p r e s s i o n i s not s u p p o r t e d by e i t h e r v i t a l s t a t i s t i c s (11) o r b i o l o g i c a l e v i d e n c e ( 3 1 , 3 7 ) . V e r y l i t t l e , i n d e e d , i s known about t h e p r o c e s s of a g i n g a t t h e c e l l u l a r l e v e l . I t has been d e m o n s t r a t e d t h a t c e l l u l a r a g i n g c o n s i s t s m a i n l y i n a l o s s of a d a p t a b i l i t y t o f l u c t u a t i n g i n t e r - and i n t r a c e l l u l a r changes and a p r o g r e s s i v e d e c r e a s e i n t h e c a p a c i t y o f a c e l l t o m a i n t a i n h o m e o s t a s i s ( 1 , 6 5 , 1 4 3 ) . These same b a s i c phenomena a r e o b s e r v e d a t t h e organ and system l e v e l (11,32,52,60, 85,95,104,123,139). C e l l u l a r a g i n g p r o b a b l y i n v o l v e s a s p e c t r u m of changes r a t h e r t h a n one mechanism i n t h e c a p a c i t y of a c e l l t o respond s w i f t l y and e f f i c i e n t l y t o a n t i - h o m e o s t a t i c s t r e s s e s ( 6 5 ) . A l l t h e o r i e s of a g i n g have t h e b a s i c a s s u m p t i o n t h a t t h i s phenomenon r e p r e s e n t s an i n f o r m a t i o n l o s s ( 3 3 ) . T h i s i n f o r m a t i o n l o s s may i n v o l v e changes a t any o r a l l of t h e f o l l o w i n g , l e v e l s : m o l e c u l a r ( e g . DNA), c hromosomal, o r g a n e l l e , membrane, o r i n t e r c e l l u l a r . Whether t h e a g i n g p r o c e s s i s due t o a random mechanism (32). o r i s due t o an " a g i n g programme" (73,163) i s s t i l l a moot p o i n t , b u t c u r r e n t e v i d e n c e f a v o u r s the- l a t t e r p o s s i b i l i t y , i m p l y i n g a b u i l t - i n o b s o l e s c e n c e o f t h e c e l l ( T a b l e s 1,2). I t i s i n t e r e s t i n g t o n o t e t h a t f o r each one o f t h e a g e n t s , l i s t e d i n t h e s e T a b l e s , which a p p e a r s t o c o n t r i b u t e t o t h e a g i n g of an o r g a n i s m , some compensatory o r h o m e o s t a t i c r e s p o n s e e x i s t s i n t h e l a t t e r . T h u s , i f i t were p o s s i b l e t o i d e n t i f y and p o s t pone o r c o m p l e t e l y e l i m i n a t e t h e b i o -l o g i c a l " a g i n g c l o c k " , one c o u l d make t h e c h o i c e between g e r i a t r i c s and g e r -o n t o l o g y . I t i s a l s o u n c l e a r as t o whether a g i n g i s a m o l e c u I a r (ceI I u l a r ) o r m a c r o m o l e c u I a r ( s y s t e m i c ) phenomenon. The c o n c e i v a b l e l e v e l s o f . 2. i n f o r m a t i o n l o s s i n v o l v e d i n a g i n g a r e summarized i n T a b l e 3. T h e r e have been i n d i c a t i o n s of a p o s s i b l e c o n n e c t i o n o f a g i n g w i t h c a n c e r i g e n e s i s i n terms of a m a r k e d l y i m p a i r e d i m m u n o l o g i c a l r e s p o n s e t o tumors (8,9,61,117) and h e t e r o g r a f t s ( 1 5 3 ) . A s - w e l l , a t o x i c e f f e c t of s e n i l e mouse s p l e e n c e l l s t o young, I i g h t I y - i r r a d i a t e d r e c i p i e n t s p l u s a l o s s i n t h e number of i m m u n o l o g i c a I l y - c o m p e t e n t " p r o g e n i t o r " c e l l s i n o l d a n i m a l s have been d e m o n s t r a t e d ( 2 ) , C o m f o r t s u g g e s t s a t h r e e - c o r n e r e d r e l a t i o n s h i p between age p r o c e s s e s , n e o p l a s t i c p r o c e s s e s , and v a r i o u s autoimmune o r s u p p o s e d l y autoimmune phenomena such as s c l e r o d e r m a , lupus e r y t h e m a t o s u s , and a m y l o i d o s i s ( 3 3 ) . The i n v o l v e m e n t o f n e o p l a s i a w i t h t h e a g i n g phenomenon w i l l now be c o n s i d e r e d . I t i s p o s s i b l e t o imagine t h r e e d i f f e r e n t mechanisms f o r t h e o c c u r r e n c e o f t h e e s s e n t i a l g e n e t i c changes a t t h e chromosome l e v e l , i f t h e chromosomes a r e , i n d e e d , t h e o r g a n e l l e s i m p l i c a t e d i n t h e n e o p l a s t i c s t a t e , t h a t make c e l l s c a n c e r o u s . The f i r s t mechanism i s t h e h y p o t h e s i s t h a t c a n c e r s a r e i n d u c e d by v i r u s e s . T h e r e i s e v i d e n c e t h a t s p e c i f i c c a n c e r s o r n e o p l a s i a i n a n i m a l s r a n g i n g from i n v e r t e b r a t e s ( 2 0 ) , lower v e r t e -b r a t e s (53,92,98,103,165) t o b i r d s and mammals (15,26,42,54,55,96,124,131, 145,156) a r e v i r a I I y - c a u s e d . Even some p l a n t tumors have been a t t r i b u t e d t o v i r u s e s ( 1 8 , 1 2 1 ) . The q u e s t i o n i s whether a s i z a b l e f r a c t i o n o f c a n c e r s a r e v i r a I I y - i n d u c e d ; e v i d e n c e i s a c c u m u l a t i n g r a p i d l y t h a t t h i s may c e r t a i n -l y be t h e c a s e . A v a r i e t y o f v i r u s e s p o s s e s s t h e a b i l i t y t o i n d u c e tumors i n newborn a n i m a I s b e f o r e t h e y can mount an e f f e c t i v e i m m u n o l o g i c a l a s s a u l t on t h e i n c i p i e n t t u m o r s . I t i s s u s p e c t e d t h a t i f no a n t i b o d i e s c o u l d be produced t h e n n e a r l y a l l v i r a l i n f e c t i o n s m i g h t r e s u l t i n h o s t death due t o c a n c e r , w h i c h i s n o t t h e c a s e ( 3 0 , 6 3 ) . TABLE 1: STOCHASTIC CAUSES OF AGING A. MACROACCI DENTS B. MICROACCIDENTS ( e g . m u t a t i o n s , enzyme d e n a t u r a t i o n ) w h i c h r e s u l t from d i s r u p t i o n s o f m o l e c u l a r s t r u c t u r e due t o l o c a l l y h i g h c o n c e n t r a t i o n s o f en e r g y a r i s i n g f r o m P h y s i c a l Agents •Chemical A g e n t s -1— -P h y s i c a l A g e n t s i H e a t , c o l d , c u t s , b r u i s e s , b r e a k s , f r e e z i n g , w e a r - a n d - t e a r B a c t e r i a l , f u n g a I , v i r a I i n f e c t i o n s • R a d i a t i o n • N u t r i t i o n A n o x i a T o x i n s • LocaI h e a t f l u c t u a t i o n s • A b s o r p t i o n o f r a d i a n t e n e r g y o r h i g h -e n e r g y p a r t i c l e s . , Heat l i b e r a t e d l o c a l l y Chemical A g e n t s — 7 a s a r e s u l t o f e x o -] t h e r m i c c h e m i c a I r e a c t i o n s * M o d i f i e d a f t e r S t r e h l e r , B. L. T i m e , C e l l s , and A g i n g . N. Y., Academic P r e s s (1970)1 A. CELLULAR-CHANGES P h y s i c a l -•Chemi caI • B. SUPRACELLULAR-CHANGES TABLE 2: GENETIC CAUSES OF AGING — C y t o p l a s m i c v i s c o s i t y • C e l l s i z e ( S u r f a c e / V o l u m e ) •Geometric arrangement o f c e l l s •Loss o f p l a s t i c i t y -i — Di f f e r e n t i a t i o n Chemi caI o v e r s p e c i a l i z a t i o n — — L o s s o f a b i I i t y . t o r e p I ace s u b c e l I u l a r e I emesnts : - S i z e L i m i t a t i o n s Growth c e s s a t i o n -AccumuI a t i o n o f s i d e p r o d u c t s o r d e s t r u c t i o n o f c e l l u l a r c o n s t i t u e n t s L o s s o f a b i I i t y t o -r e p l a c e p a r t s E r r o r s i n c a t a l y s i s U n c a t a l y s e d r e a c t i o n s •Loss o f a b i I i t y — t o . d i i f f e r e n t i a t e i n t o ; r e p Iacement e e l I s — P h y s i c a I — — i n t e r a c t i o n Geometry o f — — — — — c e l l u l a r r e l a t i o n s h i p s •Amount o r k i n d o f • -MorpHiogenesi s - C e l I movement (Random) - C e l I l o s s -Cel I s i z e " C y t o p l a s m i c v i s c o s i t y — C h e m i c a I i n t e r a c t i o n e x t r a c e l l u l a r s u b s t a n c e s C r o s s - f e e d i n g • C o m p e t i t i o n f o r n u t r i e n t s •Loss o f immune t o l e r a n c e C r o s s - t o x i t y r-Loss o f r e a d o u t dev i c e due to-J-Loss o f code -Loss o f cond i t i o n s f o r r e a d - o u t . P h y s i c a l s u p p o r t • D i f f u s i o n b a r r i e r s (Membranes) * M o d i f i e d a f t e r S t r e h l e r , B. L. T i m e , C e l l s , and A g i n g . N. Y., Academic P r e s s (1970)1 TABLE 3: AGING OF MAMMALIAN CELLS I n f o r m a t i o n l o s s |Yes| (j^  Loss i n t r IC JL Yes (NcJ-frjlnability t o cope w i t h a n t i h o m e o s t a t i c s t r e s s e s ) i t r a c e I I u I a r | G a t h e r i n g o f new h y p o t h e s e s r Yes I I n f o r m a t i o n l 0 s s | Loss i s i n c e l I communityl L o s s i s randomj Yes I Loss i s programmed L o s s o c c u r s i n n u c l e u s o c   I Yes | (hfc)-L o s s i s c Loss e x t r a c e I I u I a r I T i s s u e l e v e l C r o s s - I i n k i ng i n c o l l a g e n Orqan l e v e l I -{System l e v e l ) D i e t a r y hypophysectomy M e t a b o l i c r a t e >-Loss o c c u r s i n c y t o p l a s m hromosomal {Yes| (No)—-t Loss i s extrachromosomaI j A f f e c t s DNA s t r u c t u r e I Yes I 1§ RNA t r a n s c r i p t i o n P r o t e i n ~ Lysosoma1 t r a n s l a t i o n s t a b i 1 i t y S y n t h e t a s e s ( e g . m u t a g e n i c DNA ipol ymerases) O t h e r o r g a n e l l e s ( e g . membranes, m i t o c h o n d r i a ) M o l e c u l a r ) Yes I > Macromo I ecu I a"fj M u t a t i o n j Yes C r o s s - 1 i n k i ng i n l o n g - t e r m mo I ecu Ies E p i g e n e t i c maski ng * M o d i f i e d a f t e r C o m f o r t , A. B a s i c R e s e a r c h i n G e r o n t o l o g y , G e r o n t o l o g i a , 16:48-64 (1970) 6. As mentioned p r e v i o u s l y , t h e r e a p p e a r s t o be an i m p a i r e d immune r e s p o n s e as w e l l as a c u m u l a t i v e i n c i d e n c e of spontaneous tumors i n mice w i t h age ( 8 , 9 , 1 5 3 ) . P e r h a p s , t h e absence o f , o r d e f i c i e n c y i n , h o s t immunity t o v i r a l a n t i g e n s c o u l d a l l o w n a t u r a l l y - o c c u r r i n g v i r u s e s and t h e i r s y n e r g i s t i c c o u n t e r p a r t s t o t r a n s f o r m c e l l s t o a c a n c e r o u s c o n d i t i o n . The second mechanism i n v o l v e s s o m a t i c m u t a t i o n s c o n s t i t u t i n g t h e e s s e n t i a l c h a n g e ( s ) t o a c a n c e r c e l l i f t h e normal r e g u l a t o r y d e v i c e ( s ) c o n t r o l l i n g c e l l d i v i s i o n i s u p s e t ( 5 1 ) . C o n s i s t e n t w i t h t h i s h y p o t h e s i s i s t h e o b s e r v a t i o n o f an i n c r e a s e d i n c i d e n c e o f c a n c e r w i t h age ( 7 ) . These s o m a t i c m u t a t i o n s , however, m i g h t u l t i m a t e l y be caused by v i r u s e s ( 1 3 4 , 1 3 6 ) , i o n i z i n g r a d i a t i o n ( 4 8 , 8 1 , 1 6 7 ) , u l t r a v i o l e t l i g h t - ( 4 9 , 1 5 7 ) , and c h e m i c a l mutagens ( 9 1 , 1 3 5 ) . T h i s t h e o r y o f s o m a t i c m u t a t i o n may be d e s c r i b e d a s c a n c e r caused by a l o s s o f an e s s e n t i a l g e n e ( s ) f u n c t i o n , i n c o n t r a s t t o t h e f i r s t mechanism of v i r a l c a r c i n o g e n e s i s , w h i c h i n v o l v e s t h e a d d i t i o n o f new g e n e t i c m a t e r i a l . I t m i g h t be mentioned t h a t a t h i r d mechanism as a cause f o r c a n c e r i n v o l v e s i r r e v e r s i b l e c y t o d i f f e r e n t i a t i o n ( 1 9 , 9 3 , 1 0 6 ) , b u t e v i d e n c e i n f a v o u r o f t h i s h y p o t h e s i s i s s c a n t y a t t h i s t i m e . In f a c t , i t has been proposed t h a t a g i n g i t s e l f may be caused by c e l l u l a r o v e r d i f f e r e n t i a t i o n ( 1 4 1 , 1 6 3 ) . R e c e n t a t t e m p t s by b i o l o g i s t s a t s t u d y i n g a g i n g have f o c u s s e d on t h e s e a r e a s : 1. The i n v e s t i g a t i o n o f s u b s t a n c e s such as r a d i o - p r o t e c t i v e a g e n t s , e x e m p l i f i e d by a n t i - o x i d a n t s , (68,69) and of p r o c e d u r e s , s u c h as c a l o r i e r e s t r i c t i o n (I 16),which can p r o l o n g t h e mammalian s p a n . 2. The i n v e s t i g a t i o n o f autoimmune e f f e c t s i n a g i n g mammals (1 6 0 , 1 6 1 , 1 6 2 ) . 3. The n a t u r e o f t h e d i f f e r e n c e s between a n i m a l s w h i c h d e t e r m i n e r e l a t i v e l i f e spans ( 2 7 ) . 4. The i n v e s t i g a t i o n o f d e v e l o p m e n t a l a g e n t s such as a n t i m e t a b o l i t e s , a n t i d i f f e r e n t i a n t s , and hormones (50,57,170) w h i c h r e t a r d t h e ag i ng program. 5. The s t u d y of t h e p r o g r e s s i v e s t i f f e n i n g of c o l l a g e n w i t h age ( 1 0 , 4 5 , 6 2 , 8 0 , 1 5 8 ) . 6. The s t u d y of lysosomal membrane s t a b i l i t y i n r e l a t i o n t o a g i n g ( 4 ) . 7. The e x a m i n a t i o n o f t h e p o s s i b i l i t y o f a d e c l i n e i n t h e f u n c t i o n a l e f f i c i e n c y of n o n - p r o l i f e r a t i n g , h i g h l y - s p e c i a l i z e d c e l l s such as a g i n g neurons and a g i n g m u s c l e f i b e r s ( 4 6 , 4 7 , 1 5 9 ) . 8. The i n v e s t i g a t i o n of t r a n s c r i p t i o n enzymes, measured by a d e c r e a s e i n t h e i r a c t i v i t y w i t h age ( 1 3 , 1 7 1 ) , o r o f ' h y d r o I y t i c enzymes, • measured by an i n c r e a s e i n t h e i r a c t i v i t y w i t h age ( 8 2 , 1 7 1 ) . 9. The s t u d y of a d e t e r i o r a t i o n of t h e g e n e t i c program of a c e l l , p r o d u c i n g an a c c u m u l a t i o n o f c o p y i n g e r r o r s ( 3 , 3 7 , 1 6 4 ) . 10. The s t u d y of c l o n a l s e n e s c e n c e and t h e n a t u r e of c o n t i n u o u s c e l l l i n e s w i t h s p e c i a l r e f e r e n c e t o t h e " H a y f l i c k phenomenon", which i s t h e a p p a r e n t l i m i t a t i o n on t h e d i v i s i o n of s o m a t i c c e l l s i n v i t r o ( 7 3 ) . The i n v e s t i g a t i o n a t hand f o c u s s e d m a i n l y on t h e l a s t two a f o r e m e n t i o n e d a r e a s o f a g i n g r e s e a r c h . C u r r e n t r e s e a r c h t o p o s t pone o r p r e v e n t a g i n g must f o c u s on a g e n t s which p r o l o n g t h e l i f e - s p a n of t h e o r g a n i s m ( T a b l e 4 ) ; n o n - s p e c i f i c l i f e s h o r t e n i n g i s easy t o p r o d u c e . TABLE 4: SUBSTANCES WHICH MIGHT MODIFY THE RATE OF AGING* i AGENT THEORETICAL BASIS RESULTS A n t i o x i d a n t s Rad i o p r o t e c t a n t s P r o t e i n s y n t h e s i s i n h i b i t o r s Lysosome s t a b i l i z e r s Immunosuppressants A n t i - c r o s s I i n k i n g a g e n t s Hormonal a g e n t s - A n a b o l i c s - S o m a t o t r o p h i n - P r e d n i so I one - 1 7 - K e t o s t e r o i ds A n t i m e t a b o l i c d r u g s Scavenge f r e e r a d i c a l s ; p r e v e n t a t t a c k on DNA o r some o t h e r s y s t e m . ' A s s u m p t i o n t h a t a g i n g \ i s s i m i l a r i n n a t u r e t o r a d j a t i o n damage. Br e a k " v i c i o u s c i r c l e " i f f a u l t y s y n t h e s i s i n v o l v e d . P r e v e n t e s c a p e of enzymes ( i n c l u d i n g lysosome DNAs'e). A b o l i s h any a g i n g e f f e c t s due t o immune d i v e r g e n c e . A g i n g due t o c r o s s - l i n k s i n lo n g - t e r m m o l e c u l e s . i i M o d i f y c h e m i c a l a l l o m e t r y , r e t a r d s e n e s c e n t program P r e v e n t d e c l i n e i n p r o t e i n s t o r a g e and m u s c u l a r s t r e n g t h , M a i n t a i n "young" p a t t e r n o f p r o t e i n s y n t h e s i s . S l o w i n g o f a g i n g p r ogram; a n t i a u t o i m m u n e ? D e c l i n e most c l o s e l y p a r a l l e l s human s e n e s c e n c e . D e l a y s program; s i m u l a t e c a l o r i e r e s t r i c t i o n ; induice " a c t i v e d i a p a u s e " . " P r o l o n g l i f e " ; may o r may n o t s h i f t s p e c i f i c age ( 6 8 , 6 9 ) . E q u i v o c a l ; n o t f u l l y t e s t e d I i f e l o n g . A z o t h i o p r i n e p r o l o n g s mouse I i f e s i i g h t l y ( 1 6 0 ) . Under t e s t ( 8 7 , 8 8 ) ; BAPN, p e n i c i I I ami ne ( 8 3 ) . E f f e c t i v e c l i n i c a l l y . C a r c i n o g e n i c ; f a i l s t o p r o l o n g r a t I i f e ( 5 0 ) . Doubles l i f e s p a n i n s h o r t - l i v e d s t r a i n ( 1 4 ) . Pr o p o s e d ( S t r e h l e r ) but n o t t e s t e d . * M o d i f i e d a f t e r C o m f o r t , A. B a s i c R e s e a r c h i n G e r o n t o l o g y , G e r o n t o l o g i a , 16: 48-64 ( 970) PROBLEM The aim o f t h e p r e s e n t s t u d y was t o examine t h e phenomenon o f a g i n g i n v i t r o . Emphasis was p l a c e d on t h e b i o l o g i c a l mechanism(s) u n d e r l y i n g t h e a g i n g p r o c e s s , w h i c h may i n v o k e a change a t t h e l e v e l o f t h e DNA and t h e c e l l n u c l e u s . I t was hoped t h a t t h i s t h e s i s m i g h t c l a r i f y some o f t h e p r o c e s s e s o f a g i n g a t t h e c e l l u l a r l e v e l . C e l l s o f r a t , mouse, h a m s t e r , and human k i d n e y were c h o s e n f o r t h i s s t u d y . The m o r p h o l o g i c a l c h a n g e s , m i t o t i c r a t e and D N A - r e p I i c a t i o n s y n t h e s i s , D N A - r e p a i r ( u n s c h e d u l e d D N A - s y n t h e s i s ) c a p a c i t y , D N A - r e p a i r d u r a t i o n , t h e e f f e c t o f an e x t e r n a l a g e n t ( v i r u s ) , and t h e f r e q u e n c i e s and t y p e s o f chromosome a b e r r a t i o n s a f t e r v i r a l e x p o s u r e were a l l s e l e c t e d as c r i t e r i a t o be s t u d i e d i n aged c e l l s . The number o f p o p u l a t i o n d o u b l i n g s t h a t i t t a k e s a e e l I t o age i n v i t r o was a l s o i n v e s t i g a t e d . EXPERIMENTAL 10. A. MATERIALS AND METHODS (1) C e l I s The f o l l o w i n g c e l l s t r a i n s were u s e d : (a) P r i m a r y c u l t u r e s of human e m b r y o n i c k i d n e y c e l l s (HEK); (b) P r i m a r y c u l t u r e s o f e m b r y o n i c hooded r a t e e l I s ; ( c ) P r i m a r y c u l t u r e s o f e m b r y o n i c S w i s s mouse c e l l s ; and (d) P r i m a r y c u l t u r e s o f e m b r y o n i c S y r i a n hamster c e l l s . (2) V i r u s Human a d e n o v i r u s t y p e 12 ( p r o t o t y p e s t r a i n H u i e ) , o r i g i n a l l y o b t a i n e d from R. Huebner o f t h e N a t i o n a l I n s t i t u t e o f H e a l t h , B e t h e s d a , Md., was u s e d . The v i r u s was grown i n s u s p e n s i o n c u l t u r e s o f KB c e l l s . E x a m i n a t i o n o f t h e v i r u s s t o c k i n t h e e l e c t r o n m i c r o s c o p e showed t h a t a l l p r e p a r a t i o n s were f r e e o f a d e n o - a s s o c i a t e d v i r u s ( A A V ) . The v i r u s h a r v e s t was t i t r a t e d on p r i m a r y human e m b r y o n i c k i d n e y c e l l s m a i n t a i n e d i n medium 199 w i t h 2% c a l f s erum. (3) G e n e r a l C e l l C u l t u r e T e c h n i q u e A l l c e l l c u l t u r e s were grown a t 37*C 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 (MEM) w i t h Hanks' s a l t s , s upplemented w i t h 10$ f e t a l b o v i n e ( c a l f ) serum, p e n i c i l l i n (100 u n i t s / m l ) , s t r e p t o m y c i n (100 m i c r o g m / m l ) , \% K a n a m y c i n , and \% F u n g i z o n e . Medium was r o u t i n e l y changed t w i c e a week. MEM w i t h o u t a r g i n i n e (ADM) was used t o m a i n t a i n c e l l s a t Gj f o r p r o l o n g e d p e r i o d s ( 5 6 ) . C e l l c u l t u r e s , w h i c h were b l o c k e d i n t h i s way, were i d e a l f o r s t u d y i n g D N A - r e p a i r s y n t h e s i s because t h e y showed no D N A - s y n t h e s i s due t o D N A - r e p I i c a t i o n a t S-phase ( 5 6 , 1 3 5 ) . 11. E m b r y o n i c c e l l s were seeded on L e i g h t o n t u b e c o v e r s l i p s , k e p t i n MEM u n t i l t h e c e l l s were i n t h e i r l o g a r i t h m i c phase of g r o w t h , t h e n t h e y were t h o r o u g h l y and i n d i v i d u a l l y r i n s e d i n s e r u m - f r e e ADM, and t r a n s f e r r e d i n t o new L e i g h t o n t u b e s c o n t a i n i n g ADM w i t h 5% f e t a l b o v i n e serum. S t o c k c e l l s were grown i n 32 o z . p r e s c r i p t i o n b o t t l e s ( w i t h 4 ml NaHCC>3/800 ml medium) and passaged t o 60x15 mm.diam. p l a s t i c d i s h e s : F a l c o n P l a s t i c s , O x n a r d , C a l i f o r n i a ( w i t h 16 ml NaHC03/800 ml medium) o r L e i g h t o n t u b e s ( w i t h 4 ml NaHC03/800 ml medium). About 2 x 10^ young c e l l s were seeded p e r L e i g h t o n t u b e w i t h 2 ml o f medium; t o o b t a i n a c o m p a r a b l e s e e d i n g e f f i c i e n c y , 4 x 10^ o l d c e l l s had t o be seeded per L e i g h t o n t u b e . Embryos Put I n t o C u l t u r e Whole u t e r i , c o n t a i n i n g 15 t o 18 d a y - o l d e m b r y o s , were removed from p r e g n a n t r a t s , m ice o r h a m s t e r s i n a s t e r i l e h ood. The p a r e n t was a s p h y x i a t e d i n a c l o s e d c o n t a i n e r by d r y i c e (CO2) fumes. Each u t e r u s was washed i n a t e n - f o l d normal a n t i b i o t i c c o n c e n t r a t i o n (3.1 gm p e n i c i l l i n : 1.0 gm s t r e p t o m y c i n : 12.5 c c . p h o s p h a t e - b u f f e r e d s a l i n e , w i t h o u t , c a l c i u m and magnesium : 400 ml MEM w i t h o u t s e r u m ) . Each embryo was chopped up i n a p l a s t i c p e t r i d i s h i n 2 t o 3 ml serum-f r e e MEM c o n t a i n i n g 0.25$ t r y p s i n and t h e c e l l s were g e n t l y s e p a r a t e d w i t h a 1 c c s y r i n g e . The c e l l s u s p e n s i o n was l e f t f o r 30 - 45 m i n u t e s , s y r i n g e d a g a i n , and t h e n c e n t r i f u g e d a t 1000 rpm f o r 7 m i n u t e s . The p e l l e t was r e s u s p e n d e d i n 10 ml s e r u m - f r e e MEM and t h e n c e n t r i f u g e d a g a i n a t 1000 rpm f o r 7 m i n u t e s . The s u p e r n a t a n t was poured o f f and 10 ml f r e s h MEM w i t h \0% f e t a l b o v i n e serum and t h e a n t i b i o t i c c o n c e n t r a t i o n d e s c r i b e d under MATERIALS AND METHODS: #3 was added. The c e l l p e l l e t was d i s p e r s e d e v e n l y t h r o u g h o u t t h e medium by p i p e t t i n g . The c o n c e n t r a t i o n o f c e l l s p e r 10 ml MEM was c a l c u l a t e d w i t h a hemacytometer (AO I n s t r u m e n t Co., B u f f a l o , N.Y.) and t h e j r e q u i r e d number of c e l l s was seeded i n t o a s t o c k 32 o z . p r e s c r i p t i o n b o t t l e . O n l y t h e k i d n e y s from an i n d u c e d - a b o r t e d human embryo ( o b t a i n e d from D r . P o l a n d , Department o f P e d i a t r i c s , Vancouver G e n e r a l H o s p i t a l ) were chopped u p , t r y p s i n i z e d , and put i n t o c u l t u r e f o l l o w i n g t h e a f o r e -m e n t i o ned p r o c e d u r e f o r t h e o t h e r c e l l s t r a i n s . (5) A u t o r a d i o q r a p h y ( a ) L a b e l l i n g : T r i t i a t e d t h y m i d i n e (3H-TdR) was used t o f o l l o w D N A - r e p a i r s y n t h e s i s o r u n s c h e d u l e d D N A - s y n t h e s i s (28,29,49) a s w e l l as D N A - r e p I i c a t i o n s y n t h e s i s ( 8 9 ) . I t i s known t h a t t h y m i d i n e i s a s p e c i f i c p r e c u r s o r i n DNA; 3H-TdR l a b e l l i n g combined w i t h a u t o r a d i o g r a p h y can be used t o l o c a l i z e s i t e s of D N A - s y n t h e s i s i n a c e l l . The c e l l s were i n c u b a t e d i n t h e p r e s e n c e o f -TdR f o r 4 t o 2 hours and p r e p a r e d f o r a u t o -r a d i o g r a p h y ( s e e ( b ) ) . An amount o f 3H-TdR (Schwarz B i o r e s e a r c h , O r a n g e b u r g , N.Y.) a t a s p e c i f i c a c t i v i t y o f 17.9 c u r i e s / m i I I imole was added t o t h e c u l t u r e medium t o g i v e a f i n a l c o n c e n t r a t i o n o f 10 m i c r o c u r i e s / m I MEM o r ADM. (b) P r e p a r a t i o n of C e l l s f o r A u t o r a d i o g r a p h y : F o l l o w i n g i n c u b a t i o n w i t h t h e l a b e l l e d n u c l e o s i d e , t h e medium was d e c a n t e d and t h e L e i g h t o n t u b e c o v e r s l i p s w i t h t h e a t t a c h e d c e l l s were washed t w i c e w i t h Hanks' b a l a n c e d s a l t s o l u t i o n . The c u l t u r e s were 13. t h e n p u t i n t o \% sodium c i t r a t e f o r 10 m i n u t e s , a f t e r w h i c h t h e c e l l s were f i x e d i n a c e t i c - a l c o h o l (1 p a r t g l a c i a l a c e t i c a c i d : 3 p a r t s a b s o l u t e a l c o h o l ) t h r o u g h 4 washes o f 10 m i n u t e s e a c h , t o remove e x c e s s ( u n i n c o r p o r a t e d ) ^ H-TdR. The c o v e r s l i p s w i t h t h e c e l l s were t h e n a i r - d r i e d . F i n a l l y , t h e c o v e r s l i p s were mounted on m i c r o s c o p e s l i d e s ( c e l l s i d e up) w i t h Parawax ( A m e r i c a n Can C o . Neenah, W i s c o n s i n ) and t h e l a t t e r a l l o w e d t o c o o l b e f o r e p r o c e e d i n g . ( c ) S t a i n i n g : The c e l l s were s t a i n e d i n 2% a c e t o - o r c e i n f o r 20 t o 25 m i n u t e s , p l a c e d i n 20% a l c o h o l (3 washes of 10 m i n u t e s e a c h ) , and t h e n p u t i n t o 10$ a l c o h o l (2 washes o f 10 m i n u t e s e a c h ) . F i n a l l y , t h e c u l t u r e s were r i n s e d i n d i s t i l l e d w a t e r (3 t o 5 washes o f 10 m i n u t e s e a c h ) . The s l i d e s were a i r - d r i e d . (d) P r e p a r a t i o n o f A u t o r a d i o g r a p h s ( 1 3 5 ) : The s l i d e s w i t h mounted c o v e r s l i p s c o n t a i n i n g f i x e d and s t a i n e d ceI I s w e r e; d i p p e d d i r e c t l y i n t o t h e a u t o r a d i o g r a p h i c e m u l s i o n ( K o d a k : N u c l e a r T r a c k E m u l s i o n ) w h i c h had p r e v i o u s l y been m e l t e d a t 45° C and d i l u t e d w i t h d i s t i l l e d w a t e r (.1 p a r t e m u l s i o n : 1 p a r t w a t e r ) . E x c e s s e m u l s i o n was t h e n d r a i n e d o f f and t h e s l i d e s a l l o w e d t o d r y on e a r a c k . The s l i d e s were exposed a t 4 C i n l i g h t - t i g h t boxes c o n t a i n i n g t h e d e h y d r a t i n g a g e n t , D r i e r i t e . An e x p o s u r e t i m e of 2 weeks was u s e d . The a u t o r a d i o g r a p h s were d e v e l o p e d f o r 2 m i n u t e s i n Kodak D-19, f i x e d f o r 10 m i n u t e s a t 20°C i n Kodak R a p i d F i x , t h e n washed f o r { t o 1 hour a t 20 C i n r u n n i n g w a t e r . 14. A f t e r a i r - d r y i n g , t h e c e l l s were d e h y d r a t e d as f o l l o w s : 95$ e t h a n o l (90 s e c o n d s ) ; 100$ b u t a n o l (90 s e c o n d s ) ; 50$ b u t a n o l / 50$ x y l o l (90 s e c o n d s ) ; 100$ x y l o l (2 washes o f 5 m i n u t e s e a c h ) . The c o v e r s l i p s were mounted p e r m a n e n t l y w i t h DPX (BDH Reagent) o r Permount. (e) P h o t o g r a p h y : A u t o r a d i o g r a p h s were p h o t o g r a p h e d on Kodak High C o n t r a s t Copy F i l m . (6) P h a s e - C o n t r a s t M i c r o s c o p y o f L i v i n g C e l l C u l t u r e s (a) P r e p a r a t i o n o f C e l l C u l t u r e s : A drop o f p h y s i o l o g i c a l i s o s m o t i c g l y c e r i n was p u t on a g l a s s s l i d e . A L e i g h t o n t u b e c o v e r s l i p was p l a c e d ( c e l l s i d e down) on t o p o f t h e g l y c e r i n d r o p , w h i c h was a l l o w e d t o s p r e a d o u t e v e n l y . The c o v e r s l i p was t h e n s e a l e d w i t h p a r a f f i n . Each c u l t u r e examined by p h a s e - c o n t r a s t m i c r o s c o p y (400X m a g n i f i c a t i o n ) was d i s c a r d e d a f t e r 10 m i n u t e s t o e l i m i n a t e m o r p h o l o g i c a l a r t i f a c t s . (b) P h o t o g r a p h y : The c e l l s were p h o t o g r a p h e d on Kodak H i g h C o n t r a s t Copy F i l m . The n e g a t i v e s were t h e n f i x e d f o r 6 m i n u t e s w i t h Kodak F i x e r - 5 and p r i n t e d on Kodabromide #3 and #4 p a p e r . (7) V i r a l I n f e c t i o n o f C e l l C u l t u r e s The same p r o c e d u r e was used f o r t h e i n f e c t i o n o f young and o l d mouse c e l l c u l t u r e s . C u l t u r e s o f r a p i d l y - d i v i d i n g young c e l l s , w h i c h had n o t y e t a t t a i n e d c o n f l u e n t m o n o l a y e r s , a s w e l l a s o l d c e l l s , were s e l e c t e d and i n o c u l a t e d w i t h 0.2 m l . o f human a d e n o v i r u s 12 ( T i t e r = 5 x 1 07 TCD5 0 f o r 2 x 1 05 c e l l s / 0 . 2 ml) p e r L e i g h t o n t u b e . A l l c u l t u r e growth was a r r e s t e d by ADM p r i o r t o and a f t e r a d d i t i o n o f t h e e v i r u s , w h i c h was p e r m i t t e d t o a d s o r b f o r 4 h o u r s a t 37 C. d u r i n g w h i c h t i m e t h e c u l t u r e was g e n t l y shaken w i t h v i r u s i n o c u l a t e e v e r y 15 15. m i n u t e s o r s o . Unadsorbed v i r u s was washed f r e e by r i n s i n g t h e c e l l s 3 t i m e s w i t h f r e s h medium. The i n f e c t e d c u l t u r e s were s u b s e q u e n t l y i n c u b a t e d a t 37 C. (8) E x p o s u r e o f C e l I s t o 4 NQO A s t a n d a r d 10"3M s o l u t i o n o f 4NQ0 was p r e p a r e d by d i s s o l v i n g 1.9 mg. 4NQ0 (DaUchi P u r e C h e m i c a l Co., Tokyo) i n 0.4 ml o f 100$ e t h a n o l , w h i c h was t h e n added t o 9.6 ml o f c o m p l e t e c u l t u r e medium ( e i t h e r ADM o r MEM, de p e n d i n g on t h e e x p e r i m e n t ) . O t h e r c o n c e n t r a t i o n s were made by a p p r o p r i a t e d i l u t i o n w i t h c u l t u r e m e d i a . The c e l l c u l t u r e s were exposed t o 4NQ0 f o r 1£ h o u r s , f o l l o w e d by l a b e l l i n g w i t h 3H-TdR f o r H h o u r s , a s i n t h e e x p o s u r e o f young and o l d c e l l s t o d i f f e r e n t m o l a r i t i e s o f 4NQ0. A l t e r n a t i v e l y , a f t e r 4NQ0 p r e t r e a t m e n t f o r 1£ h o u r s , young and o l d c e l l s were exposed t o s e v e r a l p u l s e s o f 3H-Td,R (each H hours d u r a t i o n ) , a s i n t h e d u r a t i o n and c a p a c i t y o f D N A - r e p a i r . (9) C y t o l o g i c P r e p a r a t i o n s C o l c h i c i n e was used t o d e t e r m i n e t h e m i t o t i c r a t e and t h e chromosome a b e r r a t i o n s i n young and o l d c e l l s , s i n c e i t i s a metaphase-a r r e s t i n g a g e n t a t e x t r e m e l y low c o n c e n t r a t i o n s ( 5 8 , 7 9 , 9 9 ) . C o l c h i c i n e - t r e a t e d o l d c e l l s were used i n e v e r y e x p e r i m e n t t o e n s u r e t h a t 100$ o f t h e s e c e l l s were n o n - p r o l i f e r a t i n g . The f o l l o w i n g was t h e p r o c e d u r e f o r t r e a t m e n t o f any o f t h e a f o r e m e n t i o n e d mammalian c e l l s t r a i n s w i t h c o l c h i c i n e : (a) About 0.6 ml o f 0.01$ c o l c h i c i n e / m l ADM o r MEM was a p p l i e d t o t h e c e l l s f o r 4 t o 6 h o u r s . S i n c e t h e number o f metaphase p l a t e s i n c r e a s e s w i t h t h e t i m e o f c o l c h i c i n e t r e a t m e n t , a l l c e l l s were exposed t o t h i s c h e m i c a l f o r 6 h o u r s . 16. (b) C e l l s were t h e n p u t i n t o 1$ sodium c i t r a t e (10 m i n u t e s ) . ( c ) F i x a t i o n was c a r r i e d o u t w i t h 75$ a l c o h o l : 25% a c e t i c a c i d (10 mi n u t e s ) . (d) C e l l s were s t a i n e d f o r 10 m i n u t e s w i t h o r c e i n . (e) M o u n t i n g : C e l l s were put i n t o 50% e t h a n o l (30 s e c o n d s ) ; t r a n s f e r r e d t o 100$ e t h a n o l (30 s e c o n d s ) ; 1 b u t a n o l / 1 x y l o l (30 s e c o n d s ) ; 100$ x y l o l (30 s e c o n d s ) . C e l l s were l e f t i n a n o t h e r b a t h o f 100$ x y l o l f o r a minimum o f 3 m i n u t e s . The c o v e r s l i p was f i n a l l y mounted ( c e l l s i d e down) w i t h DPX o r Permount. About 3000 c e l l s on v a r i o u s a r e a s of each c o v e r s l i p c u l t u r e were s c r e e n e d f o r e i t h e r t h e i n c i d e n c e o f metaphase p l a t e s o r t h e f r e q u e n c y o f S phase n u c l e i . The f r e q u e n c i e s and t y p e s o f chromosome a b e r r a t i o n s and t h e p e r c e n t o f c e l l s a t metaphase were d e t e r m i n e d on t h e same c o v e r s l i p s . About 150 metaphase p l a t e s f o r each sample were a n a l y s e d f o r c h r o m a t i d b r e a k s , exchanges and f r a g m e n t a t i o n ( 1 3 5 ) . Chromosome a b e r r a t i o n s appear i n waves (128) a n d , f o r t h i s r e a s o n , p e r i o d i c a l samples o f v i r a l l y -i n f e c t e d c u l t u r e s were t a k e n up t o 44 h o u r s p o s t - i n f e c t i o n t o e n s u r e c a t c h i n g t h e maximum number o f a n o m a l i e s . As w e l l , a l l c e l l s were i n f e c t e d a t t h e same s t a g e o f t h e c e l l c y c l e t h r o u g h ADM a r r e s t a t G] s i n c e t h e t y p e of chromosome a b e r r a t i o n i s dependent on t h e t i m e o f v i r u s a c t i o n ( 1 2 8 ) . 17. B. RESULTS ( I ) M o r p h o l o g i c a l Changes Undergone by A g i n g C e l l s (a) C o n t r o l C u l t u r e s : The c o n t r o l s i n a l l e x p e r i m e n t s were p r i m a r y e m b r y o n i c ("young") r a t , mouse, h a m s t e r , and human k i d n e y c e l l s , w hich were a l l o w e d t o age i n v i t r o , a c c o r d i n g t o t h e p r o c e d u r e d e s c r i b e d i n ; ( c ) . A l l young c e l l s examined by phase c o n t r a s t m i c r o s c o p y e x h i b i t e d a wide a r r a y of s t r u c t u r a l d i s p a r i t y ( F i g u r e s 1,4,6,9); most o f t h e c e l l s were f i b r o b l a s t i c i n a p p e a r a n c e , b e i n g s t e l l a t e - s h a p e d o r e l o n g a t e . As w e l l , t h e y o u n g e s t c e l l s were a l l mononucIeated. (b) P r o g r e s s i v e Changes i n P r i m a r y C e l l P o p u l a t i o n s w i t h Age: As t h e c e l l s a g e d , s e v e r a l m o r p h o l o g i c a l changes o c c u r r e d . The o v e r a l l s i z e of t h e c e l l g e n e r a l l y i n c r e a s e d . There was a l s o a t r a n s f o r m a t i o n t o a p o l y g o n a l , f l a t , e p i t h e l i a l -l i k e s h a p e . A p p r o x i m a t e l y 17$ o f t h e aged c e l l s were m u l t i -n u c l e a t e d , w i t h b i n u c l e a t i o n b e i n g t h e most common n u c l e a r c o n d i t i o n ( F i g u r e s 3 , 1 6 ) . I n v a r i a b l y , an a c c u m u l a t i o n of m e t a c h r o m a t i c g r a n u l e s , r e f e r r e d t o as age pigments o r " I i p o -f u s c i n g r a n u l e s " , was e v i d e n t around t h e n u c l e u s . ( F i g u r e s 2 , 8 , 1 0 , 1 3 ) . Ragged edges o f t h e c e l l membrane were v i s i b l e i n many o l d c e l l s ( F i g u r e s 7 , 8 , 1 6 ) . The n u c l e i of aged c e l l s s t a i n e d more l i g h t l y w i t h o r c e i n t h a n t h o s e of younger c e l l s . ( c ) O b t a i n i n g Old C e l l s i n V i t r o : P r i m a r y c u l t u r e s of each c e l l s t r a i n were r e p e a t e d l y s u b c u l t u r e d upon monolayer f o r m a t i o n u n t i l a l l o r t h e m a j o r i t y o f c e l l s assumed a l a r g e , f l a t , p o l y g o n a l shape ( F i g u r e s 2,5,16) and were n o n - p r o l i f e r a t i n g ( t e s t e d by t r e a t m e n t w i t h 0.01$ c o l c h i c i n e f o r 4 t o 6 hours/ml medium). I t was found t h a t i f t h e p r i m a r y c u l t u r e s o f t h e c e l l s t r a i n s were not s u b c u l t u r e d w i t h i n a c o u p l e o f days a f t e r m o nolayer f o r m a t i o n , t h e y would n o t seed upon s u b s e q u e n t p a s s a g i n g . Each m o n o l a y e r was t r y p s i n i z e d w i t h 0.125$ t r y p s i n i n o s e r u m - f r e e MEM f o r 5 m i n u t e s a t 37 C. The c e l l s were t h e n c e n t r i f u g e d a t 1000 rpm f o r 7 m i n u t e s , r e s u s p e n d e d i n e i t h e r ADM o r MEM w i t h no s e r um, and a g a i n c e n t r i f u g e d a t 1000 rpm f o r 7 m i n u t e s . A f t e r t h e s u p e r n a t a n t had been poured o f f , f r e s h ADM o r MEM w i t h e i t h e r 5$ o r 10$ f e t a l b o v i n e s e rum, r e s p e c t i v e l y , and t h e a n t i b i o t i c s d e s c r i b e d under MATERIALS AND METHODS: #3 were a d d e d . O l d o r aged c e l l s c o u l d o n l y be k e p t i n ADM f o r 2 t o 3 days because t h e y were p r o n e t o l i f t i n g o f f t h e c u l t u r e s u r f a c e i f a l l o w e d t o r e m a i n i n t h i s medium any l o n g e r . F i g u r e 1. Young r a t c e l l s ( f i r s t p a s s a g e ) showing f a i r l y r e g u l a r o r i e n t a t i o n t o one a n o t h e r . Phase c o n t r a s t . X1400. F i g u r e 2. A poIygonaI Iy-shaped aged r a t c e l l i n v i t r o . Phase c o n t r a s t . X1400. F i g u r e 3 . A b i n u c l e a t e d aged r a t c e l l i n v i t r o . A s m a l l n u c l e a r e v a g i n a t i o n i s e v i d e n t ( a r r o w ) . Phase c o n t r a s t . X1400. 19. F i g u r e 4. A young s t e l l a t e - s h a p e d mouse c e l l . Phase c o n t r a s t . X1400. F i g u r e 5. O l d mouse c e l l s i n v i t r o . Phase c o n t r a s t . X1400. F i g u r e 6. Young S y r i a n hamster c e l l s i l l u s t r a t i n g p a r a l l e l o r i e n t a t i o n t o one a n o t h e r . Phase c o n t r a s t . X1400. F i g u r e 7. O l d S y r i a n hamster c e l l s w i t h ragged edges o f t h e c e l l membranes. Note t h e many n u c l e o l i ( a r r o w s ) i n one o f t h e c e l l s . Phase c o n t r a s t . X1400. F i g u r e 8. A b n o r m a l l y - s h a p e d n u c l e u s o f an aged S y r i a n hamster c e l l . The dense a r e a s u r r o u n d i n g t h e n u c l e u s i s age pigment ( A P ) . Phase c o n t r a s t . X I 4 0 0 . F i g u r e 9. E l o n g a t e young human k i d n e y e e l Is " ( f i r s t p a s s a g e ) . L= Lysosomes. Phase c o n t r a s t . X1400. F i g u r e 10. L a r g e r aged human k i d n e y c e l l ( f i f t h p a s s a g e ) . Phase c o n t r a s t . X1400. 22. 23. M i t o t i c R a t e and D N A - R e p I i c a t i o n S y n t h e s i s i n O l d C e l l s Young and o l d r a t , mouse, hamster and human c e l l c u l t u r e s were examined f o r t h e i r m i t o t i c r a t e s and t h e i r D N A - r e p I i c a t i o n s y n t h e s i z i n g r a t e s . H e a v i l y as w e l l as more l i g h t l y - l a b e l l e d e a r l y and l a t e S phase n u c l e i were c o u n t e d t o d e t e r m i n e t h e p e r c e n t o f D N A - s y n t h e s i z i n g c e l l s i n t h e young and o l d c u l t u r e s ( F i g u r e s 11,12,13; T a b l e 5 ) . The r a t i o o f t h e p e r c e n t o f metaphase t o i n t e r p h a s e n u c l e i gave t h e f r e q u e n c y o f metaphase p l a t e s , w h i c h were used as i n d i c a t o r s o f t h e r a t e s o f c e l l d i v i s i o n ( T a b l e 6 ) . F i g u r e 11. S-phase n u c l e i i n young r a t c e l l s ( a r r o w s ) . Note t h e p r o p h a s e i n t h e upper c e n t e r . A u t o r a d i o g r a p h . X1400. F i g u r e 12. S-phase n u c l e u s i n aged r a t c e l l ( a r r o w ) . A u t o r a d i o g r a p h . X1400. F i g u r e 13. S-phase n u c l e u s i n aged mouse c e l l ( a r r o w ) . N ote t h e dense a r e a , p r o b a b l y age pigment ( A P ) , around t h e n u c l e u s . A u t o r a d i o g r a p h . X1400. TABLE 5: DNA-SYNTHESIS IN YOUNG AND OLD CELLS DNA-SYNTHESIS($ S-Phases) DNA-SYNTHESIS(% S- P h a s e s ) CELL STRAIN YOUNG CELLS OLD CELLS Rat 14.1 9.3 Mouse 13.0 8.8 Hamster 13.7 8.9 Human Ki d n e y 16.2 10.6 TABLE 6: MITOTIC RATE IN YOUNG AND OLD CELLS MITOTIC RATE($) MITOTIC RATE(.%) CELL STRAIN YOUNG CELLS OLD CELLS Rat 2.0 0 Mouse 3.2 0 Hamster 3.7 0 Human K i d n e y 4.3 0 F i g u r e 14. N u c l e u s w h i c h a p p e a r s t o be i n t h e p r o c e s s o f s p l i t t i n g ( a r r o w ) from an aged mouse c e l l . A c e t o - o r c e i n s t a i n i n g . X 1 4 0 0 . F i g u r e 15. N u c l e u s w h i c h a p p e a r s t o be i n t h e p r o c e s s o f s p l i t t i n g ( a r r o w ) f r o m an aged mouse c e l l . A c e t o - o r c e i n s t a i n i n g . X I 4 0 0 . F i g u r e 16. B i n u c l e a t e d c o n d i t i o n o f an aged mouse c e l l . A c e t o - o r c e i n s t a i n i n g . X I 4 0 0 . 26. 27. C e l l G e n e r a t i o n Time f o r A g i n g i n V i t r o The t e c h n i q u e f o r o b t a i n i n g o l d c e l l s was r e f i n e d t o d e t e r m i n e t h e number o f c e l l g e n e r a t i o n s f o r t h e c e l l s t o become a g e d , (a) C l o n e - F o r m i n g C a p a c i t y o f Each P a s s a g e : S i n c e a l l o f t h e e m b r y o n i c c e l l s w h i c h a r e seeded do n o t d i v i d e ( F i g u r e 1 7 ) , i t was n e c e s s a r y t o f i n d o u t what p e r c e n t a g e o f t h e s e c e l l s a r e m i t o t i c a l l y - a c t i v e . T h i s was done by d e t e r m i n i n g t h e c l o n e -f o r m i n g c a p a c i t y t h r o u g h each passage o f t h e i n i t i a l p r i m a r y e m b r y o n i c c u l t u r e s , t h e n f i r s t s u b c u l t u r e , and so o n , u n t i l t h e c e l l s had aged ( i . e . , l o s s o f p r o l i f e r a t i o n ) . About 1 x 1 03 c e l l s / 5 c m p e t r i d i s h were seeded p e r pa s s a g e and t h e number o f c l o n e s c o u n t e d a f t e r 7 t o 10 d a y s . These c l o n e s , e x p r e s s e d as a p e r c e n t a g e , gave t h e c l o n e - f o r m i n g c a p a c i t y o f a p a r t i c u l a r p a s s a g e , and were an i n d e x o f t h e c e l l s w h i c h were d i v i d i n g . The number o f c e l l s i n i t i a l l y s e e d e d / p e t r i d i s h / p a s s a g e a s w e l l as t h e p e r c e n t a g e o f t h o s e c e l l s w h i c h were p r o l i f e r a t i n g was known. T h e r e f o r e , % o f p r o l i f e r a t i n g c e l l s ( c l o n e - f o r m i n g c a p a c i t y ) Number o f e e l Is seeded = Number o f c e l l s t h a t a r e s t i l l young (by t h e c r i t e r i o n o f c e l l d i v i s i o n ) . F i n a l l y , Number o f young c e l l s  Number o f c e l l s w h i c h form a c o n t a c t - i n h i b i t e d m o n o l a y e r = Number o f p o p u l a t i o n d o u b l i n g s ( i n a g e o m e t r i c p r o g r e s s i o n ) / p a s s a g e . U l t i m a t e l y , t h e numbers o f p o p u l a t i o n d o u b l i n g s / p a s s a g e were added up t o g i v e t h e t o t a l number o f c e l l g e n e r a t i o n s f o r a e e l I t o age i n v i t r o . S t a i n i n g t h e C l o n e s : The medium was decanted from t h e p e t r i d i s h and t h e c e l l s were f i x e d i n a c e t i c - a l c o h o l f o r 10 m i n u t e s . The f i x a t i v e was d e c a n t e d . The c e l l s were r i n s e d t w i c e i n each o f 70$ a l c o h o l and d i s t i l l e d w a t e r and were s t a i n e d w i t h 2% t o l u i d i n e b l u e f o r 10 m i n u t e s . The same t o l u i d i n e b l u e s o l u t i o n was not used more t h a n once because of a c e t i c a c i d c o n t a m i n a t i o n and d i l u t i o n by t h e p r e v i o u s w a t e r r i n s i n g s . The s t a i n was r i n s e d o f f w i t h d i s t i I l e d w a t e r . F i n a l l y , t h e p e t r i p l a t e s were a i r - d r i e d and t h e c l o n e s c o u n t e d . The number of p o p u l a t i o n d o u b l i n g s t h a t i t t a k e s hamster and mouse c e l l s t o l o s e t h e i r m i t o t i c a b i l i t y i s d e p i c t e d i n F i g u r e s 18 and 19, r e s p e c t i v e l y . As w e l l , t h e v a r i a t i o n i n t i m e (days i n t i s s u e c u l t u r e ) f o r t h e s e c e l l s t r a i n s t o l o s e t h e i r p r o l i f e r a t i v e c a p a c i t y i s g i v e n i n T a b l e 7. 1 2 3 4 5 PASSAGE NUMBER F i g u r e 17. COMPARISON OF SEEDING EFFICIENCY1 WITH CLONE-FORMING CAPACITY AS MOUSE CELLS ARE PASSAGED i n v i t r o . HI B S e e d i n g e f f i c i e n c y , ^ ^ C l o n e - f o r m i n g c a p a c i t y . 1000 c e l l s seeded/5 cm. p e t r i d i s h . * P r i m a r y c u I t u r e . * S e e d i n g e f f i c i e n c y i s used t o r e f e r t o c e l l s w h i c h a t t a c h t o t h e c u l t u r e s u r f a c e but do not n e c e s s a r i l y d i v i d e . 2 3 PASSAGE NUMBER F i g u r e 18. EFFECT OF PASSAGE NUMBER ON CLONE-FORMING CAPACITY OF EMBRYONIC HAMSTER CELLS i n v i t r o . f i B Data #1, Data #2. 1000 c e l l s seeded/5 cm. p e t r i d i s h . * P r i m a r y c u l t u r e . 2 3 PASSAGE NUMBER F i g u r e 19. EFFECT OF PASSAGE NUMBER ON CLONE-FORMING CAPACITY OF EMBRYONIC MOUSE CELLS i n v i t r o . B H D a t a #1, I Data #2. 1000 c e l l s seeded/5 cm. p e t r i d i s h . * P r i m a r y c u l t u r e . TABLE 7: DAYS IN CULTURE FOR VARIOUS CELL STRAINS TO LOSE PROLIFERATIVE CAPACITY CELL STRAIN NUMBER OF DAYS IN CULTURE* NUMBER OF CELL GENERATIONS FOR LOSS OF MITOTIC ACTIVITY Mouse 69; 44; 31; 30; 14 6 Hamster 90; 76; 40; 28; 18 13 * D i f f e r e n t t r i a l s o r s e t s o f d a t a . 3 3 . D u r a t i o n o f 4NQ0-Induced DNA-Repair S y n t h e s i s i n O l d C e l l s Young and o l d mouse c e l l s were c u l t u r e d i n ADM p r i o r t o and t h r o u g h o u t t h e e n t i r e e x p e r i m e n t . The d u r a t i o n o f D N A - r e p a i r was d e t e r m i n e d by p u I s e - l a b e l I i n g ( U h o u r s o f 4 x 10- 6M 4NQ0) t h e s e non-d i v i d i n g c e l l s w i t h 3H-TdR a t c o n s t a n t t i m e i n t e r v a l s o f U h o u r s d u r a t i o n a f t e r t h e i n i t i a l 4NQ0 e x p o s u r e . F o r t h e f i r s t s a m p l e , 4NQ0 and 3H-TdR were a p p l i e d s i m u l t a n e o u s l y f o r U h o u r s . T h i s e x p e r i m e n t was r e p e a t e d f o l l o w i n g t h e same p r o c e d u r e , e x c e p t f o r t h e i n d u c t i o n o f D N A - r e p a i r i n t h e mouse c e l l s w i t h 2 x 10~6M, i n s t e a d o f 4 x 10~6M, 4NQ0. The d u r a t i o n o f t h e D N A - r e p a i r s y n t h e s i s i n young and o l d mouse c e l l s a f t e r t r e a t m e n t w i t h 4 x 10*6M and 2 x 10-6M 4NQ0 i s g i v e n i n F i g u r e s 20 and 2 1 , r e s p e c t i v e l y . N u c l e i from young and o l d c e l l s exposed t o 4NQ0 i l l u s t r a t e D N A - r e p a i r i n F i g u r e s 22,23,24,25. To a v o i d t h e i n d i s c r i m i n a t e a v e r a g i n g o f a u t o r a d i o g r a p h i c g r a i n c o u n t s from n u c l e i w i t h d i p l o i d , t e t r a p l o i d , o r o c t o p l o i d DNA v a l u e s i n young and o l d c e l l s e v a l u a t e d f o r D N A - r e p a i r , a l l g r a i n c o u n t s were r e s t r i c t e d t o t h e s m a l l e s t i n t e r p h a s e n u c l e i , s i n c e t h e s e were most l i k e l y t o be d i p l o i d ( 1 3 7 ) . S m a l l , r o u n d , d a r k l y - s t a i n e d n u c l e i i n young and o l d c u l t u r e s , o b s e r v e d by S t i c h and San ( 1 3 5 ) , e x h i b i t e d a b n o r m a l l y h i g h g r a i n c o u n t s f o r D N A - r e p a i r s y n t h e s i s . 34. HOURS F i g u r e 2 0 . DURATION OF DNA-REPAIR SYNTHESIS IN AGED MOUSE CELLS, PRETREATED U HOURS WITH 4NQ0 (4x10~6M).H BNon-d i v i d i n g young mouse c e l l s a r r e s t e d by a r g i n i n e d e p r i v a t i o n , N o n - p r o l i f e r a t i n g aged mouse c e l l s . A u t o r a d i o g r a p h s . 5 10 15 H H HOURS F i g u r e 2 1 . DURATION OF DNA-REPAIR SYNTHESIS IN AGED MOUSE CELLS, PRETREATED H HOURS WITH 4NQO (2x10~5M).B 0Non-d i v i d i n g young mouse c e l l s a r r e s t e d by a r g i n i n e d e p r i v a t i o n , I J p N o n-prol i f e r a t i ng aged mouse c e l l s . A u t o r a d i o g r a p h s . F i g u r e 2 2 . N u c l e i from young mouse c e l l c u l t u r e s exposed t o 4NQ0 (4x10 M) f o r 1{ h o u r s . A c e t o - o r c e i n s t a i n i n g . A u t o r a d i o g r a p h . X I 4 0 0 . F i g u r e 2 3 . N u c l e i f r o m young r a t c e l l c u l t u r e s exposed t o 4NQ0 (4x10 M) f o r U h o u r s . A c e t o - o r c e i n s t a i n i n g . A u t o r a d i o g r a p h . X1400. F i g u r e 24. Aged mouse c e l l exposed t o 4NQ0 (4x10~6M) f o r U h o u r s . Note g r a i n s o v e r n u c l e u s . A c e t o - o r c e i n s t a i n i n g . A u t o r a d i o g r a p h . X1400. F i g u r e 2 5 . Aged r a t c e l l exposed t o 4NQ0 (4x10~6M) f o r U h o u r s . Note g r a i n s o v e r n u c l e u s . A c e t o - o r c e i n s t a i n i n g . A u t o r a d i o g r a p h s . X1400. 36. 37. (5) E f f e c t o f V a r i o u s 4NQ0 Doses on DNA-Repair i n O l d C e l l s Young and o l d r a t and mouse c e l l s were k e p t i n ADM f o r 2 t o 3 days and t h e n exposed f o r U hours t o 4NQ0 doses r a n g i n g from 5 x 1 0 ~8 t o 2 x 10~5M. T r i t i a t e d t h y m i d i n e was a p p l i e d f o r U h o u r s f o l l o w i n g t h e p r i o r t r e a t m e n t w i t h 4NQ0. The a v e r a g e number o f g r a i n s p e r n u c l e u s o f r a t and mouse c e l l s , i n d i c a t i n g t h e d e g r e e o f D N A - r e p a i r s y n t h e s i s , i s shown i n F i g u r e s 26 and 2 7 . 38. 70 -4NQ0 (M) F i g u r e 2 6 . EFFECT OF VARIOUS DOSES OF 4NQO ON DNA-REPAIR-SYNTHESIS OF AGED RAT CELLS i n v i t r o . B N o n - d i v i d i n g young r a t c e l l s a r r e s t e d by a r g i n i n e d e p r i v a t i o n , ^ ^ N o n -p r o l i f e r a t i n g aged r a t c e l l s . A u t o r a d i o g r a p h s . 39. 30 -4NQ0 (M) F i g u r e 2 7 . EFFECT OF VARIOUS DOSES OF 4NQ0 ON DNA-REPAIR SYNTHESIS OF AGED MOUSE CELLS i n v i t r o . B @ N o n - d i v i d i n g young mouse c e l l s a r r e s t e d by a r g i n i n e depr i v a t i o n , N o n - p r o l i f e r a t i n g aged mouse c e l l s . A u t o r a d i o g r a p h s . 4 0 . E f f e c t s o f A d e n o v i r u s 12 (AD 12) on D N A - S y n t h e s i s , C e l l D i v i s i o n , and Chromosome A b e r r a t i o n s i n O l d C e l l s Young ( e m b r y o n i c ) and o l d ( f i f t h p assage) mouse c e l l s were k e p t i n ADM f o r t h e d u r a t i o n o f t h e e x p e r i m e n t s t o a r r e s t c e l l d i v i s i o n . They were t h e n exposed t o 50 X i n p u t m u l t i p l i c i t y o f AD 12 f o r 4 h o u r s . I t was t h e n found t h a t a l l o f t h e c e l l s were i n f e c t e d i n a non-p r o l i f e r a t i v e s t a t e ( 1 2 8 ) . The f r e q u e n c y o f c e l l s w h i c h e n t e r e d S-phase was d e t e r m i n e d by p u l s e - l a b e l l i n g (2 h o u r s / p u l s e ) t h e c u l t u r e s w i t h 10 m i c r o c u r i e s -'H-TdR/mI ADM a t 5,8,10,16,23 and 28 h o u r s p o s t - i n f e c t i o n and c o u n t i n g t h e l a b e l l e d n u c l e i on a u t o r a d i o g r a p h y . The c o n t r o l s were n o n - i n f e c t e d , young and o l d mouse c e l l s . The e f f e c t o f AD 12 on t h e D N A - s y n t h e s i s o f t h e s e c e l l s i s shown i n F i g u r e 2 8 . The f r e q u e n c y o f d i v i d i n g c e l l s was d e t e r m i n e d by c o u n t i n g t h e metaphase p l a t e s , which were produced by e x p o s i n g t h e mouse c e l l s t o c o l c h i c i n e (6 h o u r s ) a t 14,24,34 and 44 h o u r s p o s t - i n f e c t i o n . The e x p e r i m e n t a l c o n t r o l s c o n s i s t e d o f n o n - i n f e c t e d mouse c e l l c u l t u r e s . The e f f e c t o f AD 12 on c e l l d i v i s i o n i n young and o l d mouse c e l l s i s g i v e n i n F i g u r e 2 9 . The f r e q u e n c i e s and t y p e s o f chromosome a b e r r a t i o n s i n o l d c e l l s were compared w i t h t h o s e i n young c e l l s a f t e r e x p o s u r e t o AD 12 i n bo t h c a s e s . Metaphase p l a t e s were produced a f t e r t r e a t m e n t o f t h e c e l l s w i t h c o l c h i c i n e f o r 6 h o u r s . The f i r s t two m i t o t i c d i v i s i o n s were s c r e e n e d f o r chromosome a b e r r a t i o n s ( F i g u r e s 30,31,32,33,34,35,36,37,38, 39) and t h e r e s u l t s a r e summarized i n T a b l e 8 . 10 20 30 HOURS POST-INFECT I ON F i g u r e 2 8 . EFFECT OF AD 12 ( s t r a i n H u i e ) ON DNA-SYNTHESIS IN YOUNG AND AGED MOUSE CELLS.S rBJYoung mouse c e l l s , © © Aged mouse ceI I s , C o n t r o l Aged,0 " " - t "QControl Young. C o n t r o l (Co) r e p r e s e n t s n o n - i n f e c t e d c e l l c u l t u r e s . C e l l s were p r e t r e a t e d w i t h ADM f o r 2 da y s ; 50 PFU o f v i r u s . -• 1 ffi I L_ I 1 20 40 60 HOURS POST-1NFECTI ON F i g u r e 2 9 . EFFECT OF AD 12 ( s t r a i n H u i e ) ON CELL DIVISION IN YOUNG AND AGED MOUSE CELLS. B B Young mouse c e l l s , © © Aged mouse ceI I s , 0 - 0 C o n t r o l Aged, C o n t r o l Young. C o n t r o l (Co) r e p r e s e n t s n o n - i n f e c t e d c e l l c u l t u r e s . C e l l s were p r e t r e a t e d w i t h ADM f o r 2 d a y s ; 50 PFU o f v i r u s . F i g u r e 3 0 . Metaphase p l a t e from a young n o n - i n f e c t e d mouse c e l l ( c o n t r o l ) . X5000. F i g u r e 3 1 . Metaphase p l a t e w i t h an i s o c h r o m a t i d b r e a k (arrow) f r o m a young mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 3 2 . Metaphase p l a t e w i t h d o u b l e f r a g m e n t s (*) and a s i n g l e b r e a k ( a r r o w ) f r o m a young mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 3 3 . Metaphase p l a t e w i t h a s i n g l e c h r o m a t i d b r e a k ( a r r o w ) from a young mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 34. Metaphase p l a t e w i t h a c o i l i n g d e f i c i e n c y ( " s t i c k i n e s s " ) from a young mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 3 5 . Metaphase p l a t e w i t h many s i n g l e c h r o m a t i d b r e a k s ( a r r o w s ) from an o l d mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 3 6 . Metaphase p l a t e w i t h i s o c h r o m a t i d ( a r r o w s ) and s i n g l e c h r o m a t i d (*) b r e a k s f r o m an o l d mouse c u l t u r e exposed t o AD 12. X5000. 44. F i g u r e 3 7 . Metaphase p l a t e w i t h d o u b l e f r a g m e n t s ( a r r o w ) from an o l d mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 3 8 . Metaphase p l a t e w i t h a c o i l i n g d e f i c i e n c y ( " h a z i n e s s " ) and a d o u b l e b r e a k on one c h r o m a t i d ( a r r o w ) from an o l d mouse c u l t u r e exposed t o AD 12. X5000. F i g u r e 3 9 . Metaphase p l a t e w i t h a c h r o m a t i d exchange (*) and a s i n g l e b r e a k ( a r r o w ) f r o m an o l d mouse c u l t u r e exposed t o AD 12. X5000. 45. TABLE 8: CAPACITY OF AD 12 TO INDUCE CHROMOSOME ABERRATIONS IN YOUNG AND OLD MOUSE CELLS Metaphase p l a t e s w i t h F r e q u e n c i e s o f chromosome a b e r r a t i o n s (.%)' Double C h r o m a t i d I s o c h r o m a t i d ; S i n g l e chromosome a b e r r a t i o n s (%) f r a g m e n t s exchanges breaks b r e a k s H a z i n e s s M i t o t i c r a t e (%)' Young c e l Is O l d c e l I s ' C o n t r o l 0 0 0 0 0 0 0 0 i 0 0 0 0 0.2 0 Young c e l I s O l d ceI Is . ' I n f e c t e d 62.5 86.0 25.7 24.2 0 2.7 40.0! 35.11 34.3 37.8 0 .2 7.2 7.1 C o n t r o l c u l t u r e s = No v i r u s . 2 I n f e c t e d c u l t u r e s were exposed t o AD 12 a t an i n p u t m u l t i p l i c i t y o f 50 JCD^/ c e l l f o r 4 h o u r s . 3 E x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l metaphase p l a t e s w i t h chromosome a b e r r a t i o n s :: 100 % . 4 M i t o t i c r a t e e x p r e s s e s t h e f r e q u e n c y o f c e l l s i n metaphase f o l l o w i n g 6 hours o f c o l c h i c i n e p r i o r t c s a m p l i n g a t 34 h o u r s p o s t - i n f e c t i o n . DISCUSSION 47. The r e s u l t s o f t h i s i n v e s t i g a t i o n have produced new i n s i g h t s , w h i c h B j o r k s t e n r e f e r s t o as " b a s i c t h e o r i e s " ( 1 7 ) , i n t o t h e p r i m a r y c a u s e s o f e e l l u l a r a g i n g . The s t r u c t u r a l d i s p a r i t y o f a l l t h e p r i m a r y c u l t u r e s o f mouse, r a t , hamster, and human c e l l s i s due t o t h e h e t e r o g e n e i t y o f t h e c e l l p o p u l a t i o n s . As t h e c e l l s t r a i n s a r e p a s s a g e d , a more u n i f o r m w h o r l - l i k e o r p a r a l l e l pavement i s o b t a i n e d , w i t h most e e l Is" r e s e m b l i n g f i b r o b l a s t s i n v i v o . P r o l i f e r a t i o n i n long t e r m c u l t u r e i s l e s s l i k e l y f o r f i x e d p o s t -m i t o t i c o r h i g h l y d i f f e r e n t i a t e d c e l l s ; t h u s , f i b r o b l a s t s a r e t h e c e l l t y p e most l i k e l y t o d i v i d e f o r l o n g p e r i o d s o f t i m e , r e g a r d l e s s o f t h e t i s s u e o f o r i g i n . The p o l y g o n a l e p i t h e I i a I - I i k e shape o f a l l o l d c e l l s was s i m i l a r t o t h e g i a n t sarcoma c e l l s d e s c r i b e d by Cone (34) and t h e " s p o n t a n e o u s l y " t r a n s f o r m e d g e r m - f r e e r a t c e l l s examined by Sharon and P o l l a r d ( 1 1 9 ) . P r o t o z o a n c e l l s a l s o a l t e r from a p y r i f o r m t o an o v o i d o r i r r e g u l a r form as a g i n g p r o g r e s s e s ( 1 1 1 ) . No s p e c u l a t i o n can be made on t h e s i g n i f i c a n c e o f t h i s s t r u c t u r a l t r a n s f o r m a t i o n . The i n c r e a s e d f r e q u e n c y o f ragged c e l l b o r d e r s as a g i n g p r o g r e s s e s may be e v i d e n c e f o r t h e c o r r e l a t i o n between s t r u c t u r a l changes and f u n c t i o n a l changes a t t h e c e l l u l a r l e v e l . I t has been shown t h a t t h e s t r u c t u r a l i n t e g r i t y o f t h e c e l l membrane can d i r e c t l y a f f e c t t h e f u n c t i o n o f t h e m i t o c h o n d r i a ( 9 7 ) . T h i s a g r e e s w i t h t h e d e c r e a s e i n number, t h e s t r u c t u r a l a l t e r a t i o n ( 1 1 ) , and -t h e a l t e r e d r e s p i r a t o r y r a t e o f m i t o c h o n d r i a from o l d c e l l s . A summary o f t h e c e l l u l a r a l t e r a t i o n s i n a g i n g , u s i n g both t h e l i g h t and t h e e l e c t r o n m i c r o -s c o p e s , i s g i v e n by Bakerman ( 1 1 ) . 48. The d i f f e r e n t s t a i n i n g c a p a c i t y between t h e n u c l e i of o l d and young c e l l s may i n d i c a t e c e r t a i n s t r u c t u r a l and/or f u n c t i o n a l changes i n t h e c h r o m a t i n p a t t e r n of c e l l s w i t h a g e . P u r k i n j e c e l l s from o l d i n d i v i d u a l s were o b s e r v e d t o e x h i b i t a d i f f u s e , l e s s i n t e n s e s t a i n i n g of t h e n u c l e u s compared w i t h t h o s e from young i n d i v i d u a l s ( 6 ) . A d e c r e a s e i n t h e c h r o m a t i n c o n t e n t o f c e r e b e l l a r g r a n u l e c e l l n u c l e i (21) and o l i g o d e n d r o c y t e c e l l n u c l e i ( 22) has been seen i n o l d r a b b i t s and o l d c h i n c h i l l a s . T h e r e i s a d i s t i n c t i n c r e a s e i n l i p o f u s c i n g r a n u l e s o r age pigments as c e l l s age i n v i t r o . In v i v o s t u d i e s have a l s o shown a r i s e i n age pigments i n a v a r i e t y o f a g i n g t i s s u e s , e s p e c i a I l y • n o n - d i v i d i n g n e r v e and m u s c l e c e l l s ( 6 6 ) . In f a c t , S t r e h l e r e t aj_. d emonstrated t h a t l i p o f u s c i n g r a n u l e s i n c r e a s e l i n e a r l y w i t h age i n human myocardium ( 1 4 0 ) . The i n t r a c y t o p I a s m i c a c c u m u l a t i o n of t h e i n s o l u b l e m e t a b o l i c r e s i d u e s formed from l i p i d p e r o x i d a t i o n (12) have been i m p l i c a t e d i n c r o s s - I i n k i n g r e a c t i o n s w i t h p r o t e i n ( 1 1 3 ) . The c h e m i c a l , p h y s i c a l , and e n z y m a t i c p r o p e r t i e s of t h e s e c y t o p l a s m i c i n c l u s i o n s has been d e t e r m i n e d ( 1 6 , 1 4 2 ) . Whether t h e o b s e r v e d d e g e n e r a t i v e changes d u r i n g c e l l u l a r s e n i l i t y a r e due t o a g i n g i t s e l f o r a r e one o f t h e c a u s e s l e a d i n g t o m a l f u n c t i o n o r d e a t h c a n n o t be shown. What i s e v i d e n t i s t h a t a g i n g i n v i t r o seems t o be accompanied by t h e same b a s i c changes: m o r p h o l o g i c a l t r a n s f o r m a t i o n t o a p o l y g o n a l shape; t h e a p p e a r a n c e o f a m u l t i n u c l e a t e d c o n d i t i o n ; a b e r r a n t n u c l e i ; l o s s of p r o l i f e r a t i v e c a p a c i t y ; an a c c u m u l a t i o n of age p i g m e n t s ; and t h e a ppearance of ragged c e l I b o r d e r s . T i s s u e c u l t u r e was chosen as t h e most s u i t a b l e s y s t e m f o r s t u d y i n g a g i n g i n v i t r o b ecause: ' i ) t h e r e a p p e a r s t o be a s t r o n g a n a l o g y between a g i n g i n v i t r o and a g i n g j_n v i v o ( 7 2 , 7 3 , 7 4 ) ; i i ) t h e v a r i a b l e s ( e . g . , t e m p e r a t u r e , pH, n u t r i e n t c o n c e n t r a t i o n s ) can be c o n t r o l l e d more r e a d i l y i n v i t r o ; i i i ) i t i s e a s i e r t o o b t a i n , c h a r a c t e r i z e , and examine aged c e l l s i n v i t r o ; and i v ) t h e complex i n t e r a c t i n g h o m e o s t a t i c mechanisms i n a l i v e a n i m a l , which can c o m p l i c a t e t h e c o l l e c t i o n and i n t e r p r e t a t i o n o f r e s u l t s , can be a v o i d e d . ( B i n u c l e a t i o n i s f a i r l y common i n o l d c e l l s and i s seldom o b s e r v e d i n young d i p l o i d c u l t u r e s . Any b i n u c l e a t e d c e l l s i n young c u l t u r e s m i g h t w e l l be o l d c e l l s ( i . e . , c e l l s w h i c h have c o m p l e t e d t h e i r g e n e t i c a l l y d e t e r m i n e d number o f p o p u l a t i o n d o u b l i n g s ) w h i c h have r e a c h e d t h e end of t h e i r p r o l i -f e r a t i v e c a p a c i t y . Y e t a l l c e l l s , whether young o r o l d , seem t o have t h e a b i l i t y t o s y n t h e s i z e DNA ( F i g u r e s 11,12,13; T a b l e 5 ) ; however, o l d c e l l s do have a d e c r e a s e d r a t e o f DNA s y n t h e s i s . I t i s p o s t u l a t e d t h a t t h e b i n u c l e a t e d c o n d i t i o n o f o l d c e l l s a r i s e s by D N A - r e p I i c a t i o n , f o l l o w e d by n u c l e a r d i v i s i o n w i t h o u t subsequent c y t o k i n e s i s ( F i g u r e s 14,15,16). A l s o , a few c a s e s of p o l y p l o i d y ( t e t r a p l o i d y ) were o b s e r v e d . A m i t o s i s has been seen i n v i v o i n c e r e b e l l a r P u r k i n j e c e l l s and i n h e p a t i c parenchyma from o l d e r a n i m a l s ( 5 ) . I t has been s u g g e s t e d t h a t c e l l s o n l y age i f t h e y a r e n o n - d i v i d i n g and a r e f u n c t i o n a l l y competent ( 7 4 ) . S i m i l a r l y , von Hahn (64) s u g g e s t s a c o m p a r a t i v e b i o c h e m i c a l s t u d y of DNA from p o s t - m i t o t i c o r g a n s and from t h o s e w i t h a h i g h c e l l p r o l i f e r a t i v e r a t e t o c o n f i r m t h e i d e a t h a t o n l y DNA w i t h o u t t u r n o v e r " a g e s " . The s i g n i f i c a n c e o f D N A - r e p I i c a t i o n s y n t h e s i s w i t h no m i t o s i s i n o l d c e l l s can be a t t r i b u t e d t o t h e g r e a t e r predominance o f p o l y p l o i d n u c l e i w i t h a g e . The p r o g r e s s i v e d e c r e a s e i n ' t h e number o f c e l l s 50. i n a t i s s u e w i t h age c o u l d be compensated f o r by an i n c r e a s e i n t h e number o f p o l y p l o i d n u c l e i . T h i s would a c c o u n t f o r t h e c o n s t a n t l e v e l o f DNA, i n s p i t e o f t h e d e c r e a s e d numbers o f n u c l e i ( 1 7 2 ) . R u d z i n s k a (110) and Di I l e r (39) r e f e r r e d t o a m i t o s i s as a r e s t o r a t i v e mechanism i n v o l v e d i n n u c l e a r p u r i f i c a t i o n o r r e o r g a n i z a t i o n . The d e c r e a s e i n t h e c a p a c i t y o f a d i p l o i d c e l l t o p r o l i f e r a t e as i t ages i n v i t r o , as w e l l as t h e i n c r e a s e i n t h e l e n g t h o f t h e c e l I c y c l e i n o l d c e l Is J_n_ v i v o (77,90) u n t i I u l t i m a t e l y t h e a b i l i t y f o r t h e aged c e l l t o d i v i d e i s l o s t under normal c o n d i t i o n s , can be i n t e r p r e t e d i n one o f two ways: e i t h e r c e l l u l a r a g i n g i s due t o i n h e r e n t p r o p e r t i e s o f t h e c e l l s o r o l d c e l l s have d i f f e r e n t n u t r i t i o n a l r e q u i r e m e n t s t h a n young c e l I s . C a r r e l (23,24) and o t h e r p i o n e e r i n v e s t i g a t o r s (43,44) b e l i e v e d t h a t d i p l o i d c e l l s c o u l d d i v i d e i n d e f i n i t e l y i n v i t r o w i t h o u t any k a r y o t y p i c o r m o r p h o l o g i c a l changes from t h e o r i g i n a l c e l l s t r a i n , p r o v i d e d t h e r e were no e x t r i n s i c l i m i t a t i o n s such as u n f a v o u r a b l e media c o n d i t i o n s , d r a s t i c . c h a n g e s i n t e m p e r a t u r e , e t c . The p r e s e n t s t u d y shows t h a t d i p l o i d c e l l s t r a i n s have a f i n i t e l i f e t i m e i n v i t r o . The number o f p o p u l a t i o n d o u b l i n g s f o r a c e l l t o age i n v i t r o was d e t e r m i n e d i n hamster and mouse c e l l s , a c c o r d i n g t o t h e p r e v i o u s l y d e s c r i b e d method. As t h e c e l l s were p a s s a g e d , t h e y a c q u i r e d t h e a f o r e m e n t i o n e d c h a r a c t e r i s t i c s o f s e n i l i t y and f i n a l l y c e a s e d t o p r o l i f e r a t e . The low e f f i c i e n c i e s o f t h e c l o n e - f o r m i n g c a p a c i t i e s i n p r i m a r y c u l t u r e s o f hamsl and mouse c e l l s ( F i g u r e s 18,19) can be a t t r i b u t e d t o t h e g r e a t e r predominance o f n o n - d i v i d i n g , f u l l y d i f f e r e n t i a t e d m u s c l e f i b e r s and neurons b e f o r e f i b r o -b l a s t s r a p i d l y become t h e p r e d o m i n a n t c e l l t y p e . The c u r v e s d e c l i n e a f t e r t h e second passage as most of t h e p o p u l a t i o n d o u b l i n g s have a l r e a d y o c c u r r e d and t h e c e l l s a r e a g i n g o r becoming n o n - p r o l i t e r a t i v e . I t was noted t h a t a few c u l t u r e s o f d i p l o i d mouse c e l l s were " s p o n t a n e o u s l y " t r a n s f o r m e d i n t o c o n t i n u o u s c e l l l i n e s and had abnormal a n e u p l o i d k a r y o t y p e s . T h i s o b s e r v a t i o n o f f r e q u e n t t r a n s f o r m a t i o n o f mouse c e l l s i n v i t r o has been r e p o r t e d p r e v i o u s l y ( 1 0 9 , 1 5 5 ) . Mouse c e l l s were found t o age a f t e r about 6 p o p u l a t i o n d o u b l i n g s and hamster c e l l s a f t e r about 13 p o p u l a t i o n d o u b l i n g s In each c a s e , p r o l i f e r a t i o n had ceased a f t e r t h e f i f t h passage of t h e o r i g i n a l p r i m a r y c u l t u r e s . T h i s a g r e e s w i t h t h e o b s e r v a t i o n of t h e l o n g e r l i f e span o c c u r r i n g i n S y r i a n hamsters as opposed t o S w i s s mice ( 1 1 2 ) . The l i f e s p a n o f a c e l l s t r a i n i n v i t r o does n o t seem t o depend on t h e number o f days i n c u l t u r e but r a t h e r on t h e t o t a l number o f p o p u l a t i o n d o u b l i n g s t h e c e l l c u l t u r e undergoes a f t e r c o n t a c t i n h i b i t i o n o f t h e mono-l a y e r between each passage ( R e f e r t o T a b l e 7 ) . F i n a l l y , t h e c e l l s c e a s e t o d i v i d e e n t i r e l y and w i l l n o t a t t a c h t o t h e c u l t u r e s u r f a c e i f r e p e a t e d l y s u b c u l t u r e d a t t h i s p o i n t . These o b s e r v a t i o n s agree w i t h d a t a g a t h e r e d by H a y f l i c k who f r o z e human lung f i b r o b l a s t s , c a p a b l e o f 50 p o p u l a t i o n d o u b l i n g s , a t v a r i o u s s t a g e s o f t h e i r l i f e s p a n and l a t e r r e v i v e d them; i n e v e r y c a s e , t h e c e l l s c o n t i n u e d t o d i v i d e u n t i l t h e y reached t h e i r e m p i r i c a l number o f ceI I d i v i s i o n s ( 7 3 , 7 4 ) . These d a t a would t e n d t o f a v o u r t h e i d e a o f t h e g e n e t i c t r a n s f e r e n c e o f c e l l u l a r a g i n g ( 1 1 , 2 7 , 1 6 8 ) . E v i d e n c e has been g a t h e r e d r e c e n t l y f o r D N A - r e p a i r i n mammalian c e l l s e x p o s e d t o u l t r a v i o l e t l i g h t (49,107,108) and t o c h e m i c a l mutagens (135,137) No m e n t i o n has been made o f t h e D N A - r e p a i r c a p a c i t y o f aged c e l l s . The major p a r t o f D N A - r e p a i r s y n t h e s i s i n both young and o l d mouse c e l l s i s c o m p l e t e d by 5-6 hours p o s t - t r e a t m e n t w i t h 4NQ0. A r e d u c e d , y e t s i g n i f i c a n t , u p t a k e o f "^H-TdR i n t o t h e n u c l e a r DNA of young and o l d c e l l s c o n t i n u e s f o r a p e r i o d o f up t o 17 h o u r s . T h i s a g r e e s , i n p a r t , w i t h t h e work done by S t i c h and San w i t h e m b r y o n i c hamster c e l l s ( 1 3 5 ) ; however, t h e main p o r t i o n o f t h e 52. D N A - r e p a i r s y n t h e s i s i n hamster c e l l s e x t e n d e d up t o 10 hours p o s t - t r e a t m e n t w i t h t h e same m o l a r i t y o f 4 NQO. An i n t e r e s t i n g o b s e r v a t i o n i n comparing t h e c u r v e s f o r r e p a i r s y n t h e s i s i n young and o l d mouse c e l l s ( F i g u r e 20,21) i s t h e r e d u c t i o n ( a b o u t 61%) o f t h e c a p a c i t y o f t h e D N A - r e p a i r mechanism t o r e p a i r DNA l e s i o n s i n d u c e d by 4NQ0. These l e s i o n s a r e p r o b a b l y s i n g l e -s t r a n d s c i s s i o n s w h i c h r e s u l t from t h e complexes 4 NQO forms w i t h DNA ( 1 4 4 , 1 4 9 , 1 5 0 ) . The s m a l l n u c l e i w i t h t h e h i g h degree o f 3H-TdR i n c o r p o r a t i o n i n young and o l d c u l t u r e s may r e p r e s e n t c e l l s u n d e r g o i n g p y c n o s i s ( 1 3 5 ) . The s i g n i f i c a n t l y h i g h l e v e l o f 3H-TdR i n c o r p o r a t i o n i n t h e s e n u c l e i may be i n d i c a t i v e o f e i t h e r d y i n g c e l l s o r o f c e l l s w h i c h have had t h e i r rep I i c o n s i n t h e DNA (151) a c t i v a t e d by t h e m u t a g e n i c 4 NQO and a r e , c o n s e q u e n t l y , i n a c o n t i n u a l s t a t e o f D N A - r e p I i c a t i o n r a t h e r t h a n D N A - r e p a i r s y n t h e s i s . I f t h e l a t t e r h y p o t h e s i s were t r u e , t h e s e c e l l s somehow e s c a p e t h e e f f e c t s o f a r g i n i n e d e p r i v a t i o n ; as w e l l , t h i s would be e v i d e n c e f o r t h e random a c t i o n o f 4 NQO i n i n i t i a t i n g D N A - r e p a i r s y n t h e s i s s i n c e v e r y few o f t h e s e h e a v i l y -l a b e l l e d c e l l s a r e f o u n d . V a r i o u s m u t a g e n i c a g e n t s such as r a d i a t i o n , n i t r o g e n m u s t a r d , many a l k y l a t i n g a g e n t s ( s u c h as m y l e r a n , c h l o r a m b u c i l , and t r i e t h y l e n e melamine) i n dosages o f LD50 produce a s h o r t e n e d l i f e span on a n i m a l s and an i n c r e a s e d m o r t a l i t y r a t e w i t h dosages g r e a t e r t h a n LD5Q ( 1 6 4 ) . 4 NQO has been shown t o be m u t a g e n i c ( 8 4 , 1 0 0 , 1 3 5 , 1 6 9 ) , o n c o g e n i c i n v i v o (76,101,102), and a t r a n s f o r m i n g a g e n t i n v i t r o ( 1 0 5 , 1 1 5 ) . I t a p p e a r s t h a t t h i s s y n t h e t i c c h e m i c a l does n o t have any a p p r e c i a b l e e f f e c t on t h e D N A - r e p a i r mechanism o f o l d r a t o r mouse c e l l s . S u r p r i s i n g l y , aged c e l l s seem t o respond t o v a r i o u s m o l a r i t i e s o f 4 NQO i n much t h e same way as young c e l l s ; however, t h e r e i s a s l i g h t l y g r e a t e r s e n s i t i v i t y t o 4 NQO t o x i c i t y i n aged c e l l s . Young r a t and 5 3 . mouse c e l l s s t a r t t o d i e a round 4 x 10~^M 4NQ0, w h i l e aged r a t and mouse c e l l s d i e a b o u t 2 x 10~6M 4NQ0. The c y t o t o x i c i t y o f 4NQ0 i n v i t r o has been r e c e n t l y r e p o r t e d ( 7 5 , 1 3 5 ) . The r e p a i r mechanism i n o l d c e l l s i s d o s e - d ependent,as has been d e m o n s t r a t e d f o r e m b r y o n i c S y r i a n hamster c e l l s ( 1 3 5 ) . I t i s p r o b a b l e t h a t t h i s D N A - r e p a i r o r e r r o r - c o r r e c t i n g mechanism i n mammalian c e l l s i s c a p a b l e o f r e c o g n i z i n g a v a r i e t y o f DNA l e s i o n s ( 2 5 , 6 7 , 1 3 5 ) . The t r i g g e r i n g o f DNA s y n t h e s i s , t h e s t i m u l a t i o n o f c e l l d i v i s i o n , and t h e i n d u c t i o n o f chromosome a b e r r a t i o n s i n n o n - p e r m i s s i v e s y s t e m s a r e c o n s i d e r e d as " e a r l y " v i r a l f u n c t i o n s i n n e o p l a s t i c t r a n s f o r m a t i o n . These p a r a m e t e r s were examined t o t r y t o l i n k c e l l u l a r a g i n g p r o c e s s e s w i t h n e o p l a s t i c p r o c e s s e s . I t has been d e m o n s t r a t e d r e c e n t l y t h a t young S y r i a n hamster c e l l s i n a monolayer exposed t o o n c o g e n i c p a r e n t a l AD 12 s t r a i n s and t h e i r c y t m u t ants and r e v e r t a n t s s t i m u l a t e DNA s y n t h e s i s and c e l l d i v i s i o n and p roduce chromosome a b e r r a t i o n s ( 1 2 8 ) . As w e l l , KB c e l l s i n a m o n o l a y e r i n c r e a s e t h e i r m i t o t i c r a t e when i n f e c t e d w i t h v a c c i n i a v i r u s ( 8 6 ) . I t was d e s i r a b l e t h e n t o f i n d o u t w hether n o n - p r o l i f e r a t i n g aged mouse c e l l s c o u l d be pushed i n t o DNA s y n t h e s i s and/or m i t o s i s a f t e r e x p o s u r e t o AD 12 ( p a r e n t a l s t r a i n H u i e ) i n f e c t i o n . The s t i m u l a t i o n o f c e l l u l a r DNA s y n t h e s i s i n v i r a I I y - i n f e c t e d mammalian c e l l s i s common t o many D N A - c o n t a i n i n g v i r u s e s (41,59,120,122,125, 1 4 6 ) . Young and o l d mouse c e l l s a r e s t i m u l a t e d t o i n c r e a s e t h e i r r a t e o f DNA s y n t h e s i s by 10 t o 12 h o u r s p o s t - i n f e c t i o n w i t h AD 12. The f r e q u e n c y o f S phase n u c l e i d e c l i n e s by 25 h o u r s p o s t - i n f e c t i o n . T h i s v i r a I l y - i n d u c e d c e l l u l a r D N A - r e p I i c a t i o n s y n t h e s i s c a n n o t be compared s i m p l y and d i r e c t l y w i t h normal e e l I d i v i s i o n ( 1 2 8 ) . 54. ADI2 a l s o pushed young and o l d c e l l s i n t o c e l l d i v i s i o n d e s p i t e t h e f a c t t h a t t h e c e l l s were kept i n a r e s t r i c t i v e medium f o r t h e d u r a t i o n of t h e e x p e r i m e n t . The v i r u s s t i m u l a t e d t h e s e c e l l s by 20 hours p o s t - i n f e c t i o n . T h e r e i s p r o b a b l y a s p e c t r u m o f s t r u c t u r a l and b i o c h e m i c a l changes i n v o l v e d i n a g i n g . One c a n n o t be s e l e c t i n g f o r t h e " n o t - s o - o l d " c e l l s because m i t o s i s i s s t i m u l a t e d t o t h e same deg r e e i n both young and o l d c e l l s i m p l y i n g t h e same mechanism(s) b e i n g t r i g g e r e d i n both c a s e s . If t h i s were not t h e c a s e , a l a c k o f c o r r e l a t i o n between t h e c u r v e s f o r young and o l d c e l l s ( R e f e r t o F i g u r e 29) would be e x p e c t e d s i n c e t h e " n o t - s o - o l d " ones would c e r t a i n l y be f e w e r i n number t h a n t h e t r u l y aged o r n o n - p r o l i f e r a t i n g c e l l s . ADI2 has a r e j u v e n a t i n g e f f e c t on t h e o l d c e l l s by p u s h i n g them i n t o DNA s y n t h e s i s and c e l l d i v i s i o n . I t would be i n t e r e s t i n g t o t r y t o c l o n e t h e s e " r e j u v e n a t e d " aged c e l l s t o see i f t h i s v i r a l i n d u c t i o n p e r s i s t s f o r more t h a n one m i t o t i c c y c l e o r t o see i f t r a n s f o r m a t i o n has o c c u r r e d , i n e f f e c t . A n e o p l a s t i c c o n d i t i o n m i g h t a r i s e i f t h e g e n e ( s ) a f f e c t i n g t h e r e g u l a t o r y mechanisms i n c e l l d i v i s i o n were damaged, which has an i n c r e a s e d p r o b a b i l i t y w i t h t i m e s i n c e t h e f u n c t i o n a l e f f i c i e n c y of t h e D N A - r e p a i r complex of aged c e l l s a p p e a r s t o be much reduc e d compared w i t h t h a t of a young c e l l . F u r t h e r m o r e , i t i s i m p r o b a b l e t h a t t h e n u c l e i o f n o n - p r o l i f e r a t i n g o l d c e l l s a r e any l e s s s u s c e p t i b l e t o damage t h a n t h o s e of m i t o t i c a l l y - a c t i v e young c e l l s . In f a c t , s o m a t i c c e l l s undergo m u t a t i o n s whether o r not t h e y a r e d i v i d i n g ( 3 7 ) . V a r i o u s human and s i m i a n a d e n o v i r u s e s have been shown t o in d u c e c h r o m a t i d b r e a k s i n c u l t u r e d c e l l s o f C h i n e s e and S y r i a n h a m s t e r s , r a t s , and humans ( 7 8 , 1 3 0 , 1 3 3 ) . P e r m i s s i v e and n o n - p e r m i s s i v e c e l l s exposed t o r a d i o m i m e t i c i n f e c t i o u s , c y t mutants ( 1 4 7 , 1 4 8 ) , o r t o U V - i n a c t i v a t e d ADI2 a l s o e x h i b i t e d chromosome a b e r r a t i o n s (35,36,70,71,78,94,126,127,130,132,133, 136 ) . The higher frequency of v i r a I Iy-induced chromosome a b e r r a t i o n s in o l d c e l l s (Table 8) f i t s well with the suggestion of v i r u s e s as p o s s i b l e mutagenic agents in somatic c e l l s (132,136). Of a l l metaphase p l a t e s with a b e r r a t i o n s examined in o l d c e l l s , 24.2$ and 35.1$ of the chromosome anomalies were represented by double fragments and isochromatid breaks, r e s p e c t i v e l y ; of a l l aberrant metaphase p l a t e s examined in young c e l l s , 25.7$ and 40.0$ of the chromosome anomalies were represented by double fragments and isochromatid breaks, r e s p e c t i v e l y (Figures 31,32,36,37; Table 8). M i s cellaneous chromosome a b e r r a t i o n s included s i n g l e breaks in young and o l d c e l l s (Figures 32,33,35,36,38) and chromatid exchanges and haziness in o l d c e l l s only (Figures 38, 39; Refer a l s o to Figure 3 4 ) . It i s improbable t h a t the type and frequencies of these a b e r r a t i o n s are dependent on the c e l l type employed, s i n c e s i m i l a r r e s u l t s have been reported with embryonic S y r i a n hamster c e l l s (128). The large percentage of double f r a g -ments and isochromatid breaks in young and o l d c e l l s i m p l i es t h a t the breaks occurred p r i o r to chromatid r e p l i c a t i o n and w i t h i n 10 to 12 hours post-i n f e c t i o n with the onset of the v i r a I Iy-induced DNA s y n t h e s i s . S t i c h found t h a t the m a j o r i t y of the double fragments were found p r i o r to 8 hours of post-i n f e c t i o n with AD 12 (128). There are good i n d i c a t i o n s of a c o r r e l a t i o n between induced DNA l e s i o n s and chromosome a b e r r a t i o n s d e t e c t a b l e a t the l i g h t m icroscopic level (40,135,169). The apparent decrease in the c a p a c i t y of an aged c e l l to r e p a i r i t s DNA l e s i o n s could very e a s i l y lead t o the appearance of autoimmune phenomena and the r e c o g n i t i o n of v a r i a n t c e l l s as non-self through an accumulation of somatic mutations. There i s evidence of an impaired immune response of an organism with age (152,153,154). Co i n c i d e n t with these data and s p e c u l a t i o n s would be the increased s u s c e p t i b i l i t y of aged c e l l s t o mutagens and/or 56. c a r c i n o g e n s and/or t o x i n s and/or t e r a t o g e n s , s u p p o r t e d , i n p a r t , by t h e h i g h e r i n c i d e n c e o f s p o n t a n e o u s tumours i n v i v o w i t h age (153,154) as w e l l as by t h e o b s e r v a t i o n i n v i v o o f an i n c r e a s e i n s o m a t i c m u t a t i o n s w i t h age ( 3 7 , 3 8 ) . In f a c t , a c o r r e l a t i o n e x i s t s between t h e damage in d u c e d t o DNA a t a m o l e c u l a r l e v e l and t h e s u r v i v a l o f c e l I s j_n v i t r o ( 1 3 7 ) . I t has been shown i n b a c t e r i a l s y stems t h a t an i n c r e a s e i n DNA l e s i o n s , a r e d u c t i o n i n t h e c a p a c i t y t o r e p a i r t h e s e l e s i o n s , d e f i c i e n c i e s i n r e c o m b i n a t i o n s , and t h e e n t r y o f u n r e p a i r e d DNA i n t o r e p l i c a t i o n f a v o u r an e l e v a t e d m u t a t i o n r a t e ( 1 1 8 , 1 6 6 ) . A p r o l o n g e d and i n c r e a s e d f o r m a t i o n o f mutant c e l l s would indeed l e a d t o a h e t e r o g e n e o u s c e l l p o p u l a t i o n , w h i c h a p p e a r s t o be a p r e c u r s o r c o n d i t i o n f o r n e o p l a s i a ( 1 2 7 ) . In c o n c l u s i o n , v a r i o u s c r i t e r i a f o r d i s t i n g u i s h i n g aged c e l l s f r o m young c e l l s i n v i t r o a r e summarized i n T a b l e 9. I TABLE 9: PARAMETERS DISTINGUISHING AGED CELLS FROM YOUNG CELLS i n v i t r o CHARACTERISTIC YOUNG CELL OLD CELL C e l I shape Number o f n u c l e i / c e l I M e t a c h r o m a t i c g r a n u l e s i n c y t o p l a s m M i t o s i s DNA s y n t h e s i s D u r a t i o n o f DNA r e p a i r S e n s i t i v i t y t o 4NQ0 (measured by deg r e e o f DNA r e p a i r ) 4NQ0 t o x i c i t y C l o n e - f o r m i n g c a p a c i t y F r e q u e n c y o f AD 12 - i n d u c e d chromosomal a b e r r a t i o n s AD 12 e f f e c t on DNA s y n t h e s i s AD 12 e f f e c t on c e l l d i v i s i o n A lmost i n v a r i a b l y f i b r o b l a s t i c i n appearance 1 Fewer i n number 2-5 % a r e p r o l i f e r a t i v e About 15 % o f e e l Is 6 Hours I n c r e a s e s w i t h m o l a r i t y o f 4NQ0, f o l l o w e d by a b r u p t d e c l i n e . 4 x 1 O- 6 M As h i g h as 31 % a f t e r f i r s t p assage o f em b r y o n i c hamster c e l l s 62.5 % S t i m u l a t e d t o i n c r e a s e from 5.8 % t o 19.5 % Pushed i n t o m i t o s i s : from 0.2 % t o 7.1 % Round o r p o l y g o n a l ; e p i t h e l i a l - l i k e . M u I t i n u c l e a t i o n common. Many i n number About 9 % o f e e l Is 6 Hours I n c r e a s e s w i t h m o l a r i t y of 4NQ0, f o l l o w e d by a b r u p t d e c l i n e . 2 x l 0 "6 M 86.0 % S t i m u l a t e d t o i n c r e a s e from 0.5 % t o 14.7 Pushed i n t o m i t o s i s : from 0 % t o 7.2 % BIBLIOGRAPHY 58. (1) Adelman, R.C. R e a p p r a i s a l o f B i o l o g i c a l A g i n g . N a t u r e , 22: 1094-1096 ( 1 9 7 0 ) . (2) A l b r i g h t , J . F . and T. M a k i n o d a n . Growth and Senescence o f A n t i b o d y -Forming C e l l s . J . C e l l . P h y s i o l . , 67: 185-206 ( 1 9 6 6 ) . (3) A l e x a n d e r , P. I s t h e r e a R e l a t i o n s h i p Between A g i n g , t h e S h o r t e n i n g o f L i f e - S p a n by R a d i a t i o n and t h e I n d u c t i o n o f S o m a t i c M u t a t i o n s ? In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 6 ) . (4) A l l i s o n , A.C. The R o l e o f Lysosomes i n P a t h o l o g y . P r o c . Roy. S o c . Med., 59: 867-868 ( 1 9 6 6 ) . (5) Andrew, W. A m i t o t i c D i v i s i o n i n S e n i l e T i s s u e s as a P r o b a b l e Means o f S e l f - P r e s e r v a t i o n o f C e l l s . J . G e r o n t . , 10: 1-12 ( 1 9 5 5 ) . (6) Andrew, W. Changes i n t h e N u c l e u s w i t h A d v a n c i n g Age o f t h e O r g a n i s m , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h " . S t r e h l e r , B.L., E d . N.Y., Academic P r e s s . V.I ( 1 9 6 4 ) . (7) Andrew, W a r r e n . Tumors and A g i n g . N a t . C a n c e r I n s t . Monogr., 31 : 129-140 ( 1 9 6 9 ) . (8) A o k i , T., T e l l e r , M.N.,, and Mary-Lynn R o b i t a i l l e . A g i n g and C a n c e r i g e n e s i s . I I . E f f e c t o f Age on P h a g o c y t i c A c t i v i t y o f t h e R e t i c u l o e n d o t h e l i a l System on Tumor G r o w t h . J . N a t l . C a n c e r I n s t . , 34: 255-264 ( 1 9 6 5 ) . 59. (9) A o k i , T. and M.N. T e l l e r . A g i n g and C a n c e r i g e n e s i s . 111. E f f e c t o f Age on I s o a n t i b o d y F o r m a t i o n . C a n c e r R e s . , 26: 1648-1652 ( 1 9 6 6 ) . (10) B a i l e y , A . J . The S t a b i l i z a t i o n o f t h e I n t e r m o l e c u l a r C r o s s l i n k s o f C o l l a g e n w i t h A g i n g . G e r o n t o l o g i a , 15: 65-76 ( 1 9 6 9 ) . (11) Bakerman, S. E d . T h e o r i e s , In " A g i n g L i f e P r o c e s s e s " . Bakerman, S. E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 9 ) . (12) B a r b e r , A.A. and F. B e r n h e i m . L i p i d P e r o x i d a t i o n : I t s Measurement, O c c u r r e n c e , and S i g n i f i c a n c e i n Animal T i s s u e s , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h " , S t r e h l e r , B.L., E d . N.Y., Academic P r e s s . V.2 pp. 355-403 ( 1 9 6 7 ) . (13) B a r r o w s , C.H. Enzymes i n t h e Study o f B i o l o g i c a l A c t i v i t y , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas, pp. 169-181 ( 1 9 6 6 ) . (14) B e l l a m y , D. Longterm A c t i o n o f P r e d n i s o l o n e P h o s p h a t e on a S t r a i n o f S h o r t l i v e d M i c e . E x p . G e r o n t . , 4: 327-334 ( 1 9 6 8 ) . (15) B i g g s , P.M., C h u r c h i l l , A.E., R o o t e s , D.G., and R.C. Chubb. E t i o l o g y of Marek's D i s e a s e - An O n c o g e n i c Herpes-Type V i r u s , In " P e r s p e c t i v e s i n V i r o l o g y " . P o l l a r d , M., E d . N.Y., Academic P r e s s . V.6 p p . 211-237 ( 1 9 6 8 ) . (16) B j b V k e r u d , S. I s o l a t e d L i p o f u s c i n G r a n u l e s - A S u r v e y o f a New F i e l d , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h " . S t r e h l e r , . B.L., E d . N.Y., Academic P r e s s . V.I ( 1 9 6 4 ) . 6 0 . (17) B j o r k s t e n , J . T h e o r i e s , In " A g i n g L i f e P r o c e s s e s " . Bakerman, S., E d . ( 1 9 6 9 ) . Same as r e f e r e n c e #11. (18) B l a c k , L.M. A V i r u s Tumor D i s e a s e o f P l a n t s . Amer. J . B o t a n y , 32: 408-415 ( 1 9 4 5 ) . (19) Bui l o u g h , W.S. "The E v o l u t i o n o f D i f f e r e n t i a t i o n " . London, Academic P r e s s ( 1 9 6 7 ) . (20) B u r d e t t e , W.J. Tumors, Hormones and V i r u s e s i n D r o s o p h i l a . N a t . C a n c e r I n s t . Monogr., 31: 303-321 ( 1 9 6 9 ) . (21) Cammermeyer, J . C y t o l o g i c a l M a n i f e s t a t i o n s o f A g i n g i n R a b b i t and C h i n c h i l l a B r a i n s . J . G e r o n t . , 18: 41-54 ( 1 9 6 3 ) . (22) Cammermeyer, J . S i m i l a r i t i e s between O l i g o d e n d r o c y t e and C e r e b e l l a r G r a n u l e C e l l N u c l e i i n Mammalia and A v e s . Am. J . A n a t . , 112: 111-140 ( 1 9 6 3 ) . (23) C a r r e l , A. On t h e Permanent L i f e o f T i s s u e s O u t s i d e o f t h e O r g a n i s m . J . E x p t l . Med., 15: 516-528 ( 1 9 1 2 ) . (24) C a r r e l , A. P r e s e n t C o n d i t i o n o f a S t r a i n o f C o n n e c t i v e T i s s u e Twenty-E i g h t Months O l d . J . E x p t l . Med., 20: 1-2 ( 1 9 1 4 ) . (25) Cerda-Olmedo, E. and P.C. H a n a w a l t . R e p a i r o f DNA Damaged by N - M e t h y l -N - N i f r o - N - N i t r o s o g u a n i d i n e i n E s c h e r i c h i a c o l i . M u t a t i o n R e s . , 4: 369-371 ( 1 9 6 9 ) . (26) C h u r c h i l l , A.E., P a y n e , L.N. and R.C. Chubb. Imm u n i z a t i o n a g a i n s t Marek's D i s e a s e U s i n g L i v e A t t e n u a t e d V i r u s . N a t u r e , 221: 744-747 ( 1 9 6 9 ) . 6 1 . (27) C l a r k , A.M. G e n e t i c F a c t o r s A s s o c i a t e d w i t h A g i n g , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h " . S t r e h l e r , B.L., E d . N.Y., Academic P r e s s , p.207 ( 1 9 6 4 ) . (28) C l e a v e r , J . E . D e f e c t i v e R e p a i r R e p l i c a t i o n o f DNA i n Xeroderma Pigmentosum, N a t u r e , 218: 652-656 ( 1 9 6 8 ) . (29) C l e a v e r , J . E . R e p a i r R e p l i c a t i o n o f Mammalian C e l l DNA : E f f e c t s o f Compounds t h a t I n h i b i t DNA S y n t h e s i s o r Dark R e p a i r . R a d i a t i o n R e s . 37: 334-348 ( 1 9 7 0 ) . (30) Cohen, A. "Textbook o f M e d i c a l V i r o l o g y " . O x f o r d and E d i n b u r g h , B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s . p p . 468-469 ( 1 9 6 9 ) . (31) C o m f o r t , A. " A g i n g : The B i o l o g y o f S e n e s c e n c e " . London, R o u t l e d g e and Kegan P a u l . ( 1 9 6 4 ) . (32) C o m f o r t , A. P h y s i o l o g y , H o m e o s t a s i s , and A g i n g . G e r o n t o l o g i a , 14: 224-234 ( 1 9 6 8 ) . (33) C o m f o r t , A. B a s i c R e s e a r c h i n G e r o n t o l o g y . G e r o n t o l o g i a , 16: 48-64 ( 1 9 7 0 ) . (34) Cone, C D . O b s e r v a t i o n s of S e l f - I n d u c e d M i t o s i s and A u t o s y n c h r o n y i n Sarcoma C e l l N e t w o r k s . C a n c e r R e s . , 28: 2155-2161 ( 1 9 6 8 ) . (35) C o o p e r , J.E.K., S t i c h , H.F., and D.S. Yohn. V i r u s e s and Mammalian Chromosomes. V.111. Dose r e s p o n s e S t u d i e s w i t h Human Adeno-v i r u s e s Types 18 and 4. V i r o l o g y , 33: 533-541 ( 1 9 6 7 ) . 6 2 . (36) C o o p e r , J.E.K., Yohn, D.S., and H.F. S t i c h . V i r u s e s and Mammalian Chromosomes. X. C o m p a r a t i v e S t u d i e s o f t h e Chromosome Damage Induced by Human and S i m i a n A d e n o v i r u s e s . E x p . C e l l R e s . , 53: 225-240 ( 1 9 6 8 ) . (37) C u r t i s , H . J . The P o s s i b i l i t y o f I n c r e a s e d L o n g e v i t y by t h e C o n t r o l o f M u t a t i o n s , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 6 ) . (38) C u r t i s , H . J . B i o l o g i c a l Mechanisms U n d e r l y i n g t h e A g i n g P r o c e s s . S c i e n c e , 141: 686-694 ( 1 9 6 3 ) . (39) D i I l e r , W.F. N u c l e a r R e o r g a n i z a t i o n P r o c e s s e s i n Paramecium a u r e l i a w i t h D e s c r i p t i o n s o f Autogamy and " H e m i x i s " . J . Morph., 59: 11-67 ( 1 9 3 6 ) . (40) D u b i n i n , N.P., and V.N. S o y f e r . Chromosome Breakage and Complete G e n i e M u t a t i o n P r o d u c t i o n i n M o l e c u l a r Terms, M u t a t i o n R e s . , 8: 353-365 ( 1 9 6 9 ) . (41) D u l b e c c o , R., H a r t w e l l , L.H. and M. V o g t . I n d u c t i o n o f C e l l u l a r DNA S y n t h e s i s by Polyoma V i r u s . P r o c . N a t . A c a d . S c i . (Wash.), 53: 403-410 ( 1 9 6 5 ) . (42) D u l b e c c o , R. C e l l T r a n s f o r m a t i o n by V i r u s e s . S c i e n c e , 166: 962-968 ( 1 9 6 9 ) . (43) E b e r l i n g , A.H. The Permanent L i f e o f C o n n e c t i v e T i s s u e O u t s i d e o f t h e O r g a n i s m . J . E x p t l . Med., 17: 237-285 ( 1 9 1 3 ) . 6 3 . (44) E b e r l i n g , A.H. A Ten Y e a r O l d S t r a i n o f F i b r o b l a s t s . J . E x p t l . Med., 35: 755-759 ( 1 9 2 2 ) . (45) E l d e n , H.R. A g i n g i n C o l l a g e n , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 6 ) . (46) E r m i n i , M. The A g i n g o f S k e l e t a l M u s c l e : R e s t i t u t i o n o f P h o s p h o c r e a t i n e i n R a t s A f t e r Work. G e r o n t o l o g i a , 16: 65-71 ( 1 9 7 0 ) . (47) E r m i n i , M. The A g i n g o f S k e l e t a l M u s c l e : M y o f i b r i l l a r - ATPase -A c t i v i t i e s i n R a t s o f V a r i o u s A g e s . G e r o n t o l o g i a , 16: 72-76 ( 1 9 7 0 ) . (48) E v a n s , R.G. and A. Norman. R a d i a t i o n - S t i m u l a t e d I n c o r p o r a t i o n o f T h y m i d i n e i n t o t h e DNA o f Human L y m p h o c y t e s . N a t u r e , 217: 455-456 ( 1 9 6 8 ) . (49) E v a n s , R.G. and A. Norman. U n s c h e d u l e d I n c o r p o r a t i o n o f T h y m i d i n e i n U l t r a v i o l e t - I r r a d i a t e d Human L y m p h o c y t e s . R a d i a t i o n R e s . , 36: 287-298 ( 1 9 6 8 ) . (50) E v e r i t t , A.V. The E f f e c t o f P i t u i t a r y Growth Hormone on t h e A g i n g M ale R a t . J . G e r o n t . , 14: 415-424 ( 1 9 5 9 ) . (51) F a i l l a , G. The A g i n g P r o c e s s and S o m a t i c M u t a t i o n s , In "The B i o l o g y o f A g i n g " . S t r e h l e r , B.L., e t a j _ . , E d s . P u b l . No.6, Am. A s s o c . B i o l . S c i . , W a s h i n g t o n , p p . 170-175 ( I 9 6 0 ) . 6 4 . (52) F a l z o n e , J.A., J r . and N.W. S h o c k . P h y s i o l o g i c a l L i m i t a t i o n s and Age. P u b l i c H e a l t h Rep., 71: 1185-1193 ( 1 9 5 6 ) . (53) F a w c e t t , D.W. E l e c t r o n M i c r o s c o p i c O b s e r v a t i o n s o f I n t r a c e l l u l a r V i r u s - L i k e P a r t i c l e s A s s o c i a t e d w i t h t h e C e l l s o f t h e Lucke Renal A d e n o c a r c i n o m a . J . B i o p h y . B iochem. C y t o l . , 2: 725-742 ( 1 9 5 6 ) . (54) F e n n e r , F. "The B i o l o g y o f Animal V i r u s e s " . N.Y., Academic P r e s s , V.1 p p . 305-309 ( 1 9 6 8 ) . (55) F o o t p r i n t s o f Human C a n c e r V i r u s e s ? N a t u r e , 228: 907-908 ( 1 9 7 0 ) . (56) F r e e d , J . J . and S.A. S h a t z . Chromosome A b e r r a t i o n s i n C u l t u r e d C e l l s D e p r i v e d o f a S i n g l e E s s e n t i a l Amino A c i d . E x p . C e l l R e s . , 55: 393-409 ( 1 9 6 9 ) . F r i e d m a n , S.M. and C.L. F r i e d m a n . P r o l o n g e d T r e a t m e n t w i t h P i t u i t a r y Powder i n Aged R a t s . E x p . G e r o n t . , 1: 37-48 ( 1 9 6 4 ) . G a u l d e n , M.E. and J.G. C a r l s o n . C y t o l o g i c a l E f f e c t s o f C o l c h i c i n e on t h e G r a s s h o p p e r N e u r o b l a s t i n V i t r o w i t h S p e c i a l R e f e r e n c e t o t h e O r i g i n o f t h e S p i n d l e . E x p t l . C e l l R e s . , 2: 416-433 ( 1 9 5 1 ) . G e r s h o n , D., S a c h s , L. and E.M. W i n o c o u r . The I n d u c t i o n o f C e l l u l a r DNA S y n t h e s i s by S i m i a n V i r u s 40 i n C o n t a c t - I n h i b i t e d and i n X - l r r a d i a t e d C e l l s . P r o c . N a t l . A c a d . S c i . (Wash.), 56: 918-925 ( 1 9 6 6 ) . (57) (58) (59) 6 5 . (60) G o l d s c h m i d t , L. M o n t h l y V a r i a t i o n s i n Thermal F r a g i l i t y o f E r y t h r o c y t e s i n Man: I n f l u e n c e o f Age and S e x . N a t u r e , 201: 791-793 ( 1 9 6 4 ) . (61) Good, R.A. and J . F i n s t a d . E s s e n t i a l R e l a t i o n s h i p Between t h e Lymphoid S y s t e m , Immunity, and M a l i g n a n c y . N a t . C a n c e r I n s t . Monogr., 31: 41-58 ( 1 9 6 9 ) . (62) Grahame, R. and P . J . L . H o l t . The I n f l u e n c e o f A g i n g on t h e i n V i v o E l a s t i c i t y o f Human S k i n . G e r o n t o l o g i a , 15: 121-139 ( 1 9 6 9 ) . (63) H a b e l , K. The B i o l o g y o f V i r a l C a r c i n o g e n e s i s . C a n c e r R e s . , 28: 1825-1831 ( 1 9 6 8 ) . (64) Hahn, H.P. v o n . A g i n g i n M o l e c u l e s - DNA, In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I I I i n o i s , C h a r l e s C. Thomas ( 1 9 6 6 ) . (65) Hahn, H.P. v o n . S t r u c t u r a l and F u n c t i o n a l Changes i n N u c l e o p r o t e i n D u r i n g t h e A g i n g o f t h e C e l l . G e r o n t o l o g i a , 16: 116-128 ( 1 9 7 0 ) . (66) H a m p e r l , H. D i e F l u o r e s c e n z m i k o s k o p i e M e n s c h l i c h e r Gewebe. V i r c h o w s A r c h . P a t h o l . A n a t . u. P h y s i o l . , 292: 1-51 ( 1 9 3 4 ) . (67) H a n a w a l t , P.C. and R.H. Haynes. R e p a i r R e p l i c a t i o n o f DNA i n B a c t e r i a : I r r e l e v a n c e o f Che m i c a l N a t u r e o f Base D e f e c t . B i o c h e m . B i o p h y s . R e s . Commun., 19: 462-467 ( 1 9 6 5 ) . (68) Harman, D. P r o l o n g a t i o n o f t h e Normal L i f e s p a n and I n h i b i t i o n o f Spontaneous C a n c e r by A n t i o x i d a n t s . J . G e r o n t . , 16: 247-254 (1.961). 66. (69) Harman, D. F r e e R a d i c a l Theory o f A g i n g : E f f e c t o f F r e e R a d i c a l R e a c t i o n I n h i b i t o r s on t h e M o r t a l i t y R a te o f Male LAF^ M i c e . J . G e r o n t . , 23: 476-482 ( 1 9 6 8 ) . (70) Hausen, H. z u r . I n d u c t i o n o f S p e c i f i c Chromosomal A b e r r a t i o n s by A d e n o v i r u s Type 12 i n Human Embryonic K i d n e y C e l l s . J . V i r o l . , 1: 1174-1185 ( 1 9 6 7 ) . (71) Hausen, H. z u r . Chromosomal A b e r r a t i o n s and C l o n i n g E f f i c i e n c y i n A d e n o v i r u s Type 12 I n f e c t e d Hamster C e l l s . J . V i r o l . , 2: 915-917 ( 1 9 6 8 ) . (72) Hay, R . J . C e l l and T i s s u e C u l t u r e i n A g i n g R e s e a r c h , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h " . S t r e h l e r , B.L., E d . N.Y., Academic P r e s s , V.2 pp. 121-158 ( 1 9 6 7 ) . (73) Hayf I i c k , L. The Li mi t e d j_n_ V i t r o L i f espan o f Human D i p l o i d Ce I I S t r a i n s . E x p . C e l l R e s . , 37: 614-636 ( 1 9 6 5 ) . (74) H a y f l i c k , L. Senescence and C u l t u r e d C e l l s , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s G. Thomas ( 1 9 6 6 ) . (75) H o r i k a w a , M. N i k a i d o , 0 . , and T. S u g a h a r a . D i f f e r e n t i a l S e n s i t i v i t y t o a Chemical C a r c i n o g e n ( 4 N i t r o q u i n o I i n e - N - O x i d e ) i n Mammalian C e l I s i n V i t r o . E x p t l . C e l l R e s . , 55: 65-67 ( 1 9 6 9 ) . (76) H o s h i n o , H.Y., Kawazoe, Y., and F. Fu k u o k a . D e t e c t i o n o f P o t e n t i a l Weak C a r c i n o g e n s and P r o c a r c i n o g e n s . 1. E f f e c t o f t h e D e r i v a t i v e s o f 4 - N i t r o q u i n o l i n e - 1 - O x i d e on S u b m a n i f e s t i o n a I Dose of 4 - N i t r o q u i n o l i n e - 1 - O x i d e . Gann., 60: 523-527 ( 1 9 6 9 ) . 6 7 . (77) Howes, E.L. and S.C. H a r v e y . Age F a c t o r i n V e l o c i t y o f Growth o f F i b r o b l a s t s i n t h e H e a l i n g Wound. J . E x p . Med., 55: 577-590 ( 1 9 3 2 ) . (78) Huang, C C . I n d u c t i o n of a H i g h I n c i d e n c e o f Damage t o t h e X Chromosomes o f R a t t u s (Mastomys) n a t a l ens i s by Base A n a l o g u e s , V i r u s e s , and C a r c i n o g e n s . Chromosoma, 23: 162-179 ( 1 9 6 7 ) . (79) Inoue, S. E f f e c t o f C o l c h i c i n e on t h e M i c r o s c o p i c and S u b m i c r o s c o p i c S t r u c t u r e o f t h e M i t o t i c S p i n d l e . E x p t l . C e l l R e s . , S u p p l . 2: 305-318 ( 1 9 5 2 ) . (80) J a c k s o n , D.S. and F.S. S t e v e n . Age Changes i n t h e C r o s s - L i n k i n g o f Human C o l l a g e n . G e r o n t o l o g i a , 15: 77-84 ( 1 9 6 9 ) . (81) J o n e j a , M.G. and H.F. S t i c h . Chromosomes o f Tumor C e l l s I V . C e l l P o p u l a t i o n Changes i n Thymus, S p l e e n , and Bone Marrow D u r i n g X-Ray-Induced L e u k e m o g e n e s i s . J . N a t . C a n c e r I n s t . , 35: 421-434 ( 1 9 6 5 ) . (82) K i r k , J . E . A g i n g i n Enzyme A c t i v i t i e s o f Human A r t e r i a l T i s s u e , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas, p p . 182-192 ( 1 9 6 6 ) . (83) Kohn, R.R. and A.M. L e a s h . Longterm L a t h y r o g e n A d m i n i s t r a t i o n t o R a t s w i t h S p e c i a l R e f e r e n c e t o A g i n g . E x p . M o l e c . P a t h o l . , 7: 354-361 ( 1 9 6 7 ) . (84) Kondo, S. M u t a g e n i c i t y V e r s u s R a d i o - S e n s i t i v i t y i n E s c h e r i c h i a c o l i , 12th I n t e r n . C o n g r . G e n e t i c s , 11: 126-127 ( 1 9 6 8 ) . 6 8 . (85) K o r e n c h e v s k y , V. P h y s i o l o g i c a l and P a t h o l o g i c a l A g i n g . B o u r n e , G.H., E d . , N.Y., H a f n e r P u b . Co., I n c . ( 1 9 6 1 ) . (86) K o z i o r o w s k a , J . and K. W l o d a r s k i . E f f e c t o f Crude P r e p a r a t i o n s o f V a c c i n i a on M i t o s i s and DNA S y n t h e s i s o f KB C e l l s . J . E x p . Med. 124: 199-208 ( 1 9 6 6 ) . (87) L a B e l l a , F.S. P h a r m a c o l o g i c a l R e t a r d a t i o n o f A g i n g . G e r o n t o l o g i s t , 6: 46-50 ( 1 9 6 6 ) . (88) L a B e l l a , F.S. The E f f e c t o f C h r o n i c D i e t a r y L a t h y r o g e n on R a t S u r v i v a l . G e r o n t o l o g i s t , 8: 13 ( 1 9 6 8 ) . (89) L a m e r t o n , L.F., and R.J.M. F r y , E d s . " C e l l P r o l i f e r a t i o n " . O x f o r d , B l a c k S c i e n t i f i c P u b l i c a t i o n s ( 1 9 6 3 ) . (90) L e s h e r , S., F r y , R.J.M. and H . I . Kohn. Age and t h e G e n e r a t i o n Time o f t h e Mouse Duodenal E p i t h e l i a l C e l l s . E x p . C e l l R e s . , 24: 334-343 ( 1 9 6 1 ) . (91) L i e b , M. Enhancement o f U l t r a v i o l e t - l n d u c e d M u t a t i o n i n B a c t e r i a by C a f f e i n e . Z. V e r e r b u n g s l e h r e , 92: 416-429 ( 1 9 6 1 ) . (92) L u c k e , B. K i d n e y C a r c i n o m a i n t h e Leopard F r o g : A V i r u s Tumor. An n . N.Y. A c a d . S c i . , 54: 1093-1109 (1952)'. (93) M a r k e r t , C.L. N e o p l a s i a : A D i s e a s e o f C e l l D i f f e r e n t i a t i o n . Cancer-R e s . , 28: 1908-1914 ( 1 9 6 8 ) . 6 9 . (94) M c D o u g a l l , J.K. E f f e c t o f A d e n o v i r u s e s on t h e Chromosomes o f Normal Human C e l l s and C e l l s T r i s o m i c f o r an E Chromosome. N a t u r e , 225: 456-458 ( 1 9 7 0 ) . (95) McKeown, F. " P a t h o l o g y o f t h e Aged". London, B u t t e r w o r t h s ( 1 9 6 5 ) . (96) M e l e n d e z , L.V., Hu n t , R.D., D a n i e l , M.D., G a r c i a , F.G., and C.E.O. F r a s e r . Herpes V i r u s S a i m i r i 2 . E x p e r i m e n t a I Iy-1nduced M a l i g n a n t Lymphoma i n P r i m a t e s . L a b . Animal C a r e , 19: 378 ( 1 9 6 9 ) . (97) M e n e f e e , M.G. and V . J . E v a n s . S t r u c t u r a l D i f f e r e n c e s i n Mammalian C e l l s by Changes i n T h e i r E n v i r o n m e n t . J . N a t l . C a n c e r I n s t . , 25: 1303-1323 ( 1 9 6 0 ) . (98) M i z e l l , M., T o p l i n , I., and J . J . I s a a c s . Tumor I n d u c t i o n i n D e v e l o p i n g F r o g K i d n e y s by a Zon a l C e n t r i f u g e P u r i f i e d F r a c t i o n o f t h e F r o g Herpes-Type V i r u s . S c i e n c e , 165: 1134-1137 ( 1 9 6 9 ) . (99) M o l e - B a j e r , J . C i n e - M i c r o g r a p h i c Ana I y s i s o f C - M i t o s i s i n Endosperm. Chromosoma, 9: 332-358 ( 1 9 5 8 ) . (100) N a g a i , S. P r o d u c t i o n o f R e s p i r a t i o n - D e f i c i e n t M u t a n t s i n Y e a s t by a C a r c i n o g e n , 4 - N i t r o q u i n o I i n e - 1 - O x i d e . M u t a t i o n R e s . , 7: 333-337 ( 1 9 6 9 ) . •' " (101) N a k a h a r a , W., F u k u o k a , F., and T. S u g i m u r a . C a r c i n o g e n i c A c t i o n o f 4 - N i t r o q u i n o l i n e - N - O x i d e . Gann, 48: 129-137 ( 1 9 5 7 ) . (102) N a k a h a r a , W., F u k u o k a , F., and S. S a k a i . The R e l a t i o n Between C a r c i n o g e n i c i t y and Chemical S t r u c t u r e o f C e r t a i n Qui no I i n e D e r i v a t i v e s . Gann, 49: 33-41 ( 1 9 5 8 ) . 70. (103) N i g r e l l i , R.F., K e t c h e n , K.S., and G.D. R u g g i e r i . S t u d i e s on V i r u s D i s e a s e s o f F i s h e s . E p i z o o t i o l o g y o f E p i d e r m a l Tumors i n t h e S k i n o f F l a t f i s h e s o f t h e P a c i f i c C o a s t , w i t h S p e c i a l R e f e r e n c e t o t h e Sand S o l e ( P s e t t i c t h y s m e l a n o s t i c u s ) from N o r t h e r n H e c a t e S t r a i t , B r i t i s h C o l u m b i a . Z o o l o g i c a , 50: 115-122 ( 1 9 6 5 ) . (104) N o r r i s , A.H., Sh o c k , N.W., Landowne, M., and J.A. F a l z o n e J r . Pulmonary F u n c t i o n S t u d i e s : Age D i f f e r e n c e s i n Lung Volumes and B e l l o w F u n c t i o n . J . G e r o n t . , 11: 379-387 ( 1 9 5 6 ) . (105) O k a b a y a s h i , T.M. I d e , Y o s h i m o t o , A., and M. O t s u b o . M u t a g e n i c A c t i v i t y o f 4 - N i t r o q u i n o l i n e - 1 - O x i d e and 4-Hydroxyaminoquino I i n e - 1 - 0 x i d e on B a c t e r i a . Chem. Pharm. B u l l . , T o k y o , 13: 610-613 ( 1 9 6 5 ) . (106) P a u l , J . M a s k i n g o f Genes i n C y t o d i f f e r e n t i a t i o n and C a r c i n o g e n e s i s . Reuk, A.V.S.de, and J . K n i g h t , E d s . C i b a F o u n d a t i o n Symposium. London, J . and A. C h u r c h i l l , p p . 196-207 ( 1 9 6 7 ) . (107) Rasmussen, R.E., and R.B. P a i n t e r . R a d i a t i o n - S t i m u l a t e d D N A - S y n t h e s i s i n C u l t u r e d Mammalian C e l l s . J . C e l I B i o l . , 29: 11-19 ( 1 9 6 6 ) . (108) Regan, R.D., T r o s k o , J . E . , and W.L. C a r r i e r . E v i d e n c e f o r E x c i s i o n o f U l t r a v i o l e t - I n d u c e d P y r i m i d i n e Dimers from t h e DNA o f Human C e l I s i n V i t r o . B i o p h y s . J . , 8: 319-325 ( 1 9 6 8 ) . (109) R o t h f e l s , K.H., K u p e l w e i s e r , E.B., and R.C. P a r k e r . E f f e c t s o f X-I r r a d i a t e d F e e d e r L a y e r s on M i t o t i c A c t i v i t y and Development o f A n e u p l o i d y i n Mouse-Embryo C e l l s i n V i t r o , In "C a n a d i a n C a n c e r C o n f e r e n c e " . B egg, R.W., E d . N.Y., Academic P r e s s , V.5 ( 1 9 6 3 ) . 7 1 . (110) R u d z i n s k a , M.A. The Use o f a P r o t o z o a n f o r S t u d i e s on A g i n g . 11. The M a c r o n u c l e u s i n Young and O l d Organisms of To k o p h r y a i n f u s i o n u m : L i g h t and E l e c t r o n M i c r o s c o p e O b s e r v a t i o n s . J . G e r o n t . , 16: 326-334 ( 1 9 6 1 ) . (111) R u d z i n s k a , M.A. The Use o f a P r o t o z o a n f o r S t u d i e s on A g i n g . 1. The D i f f e r e n c e s Between Young and O l d Organisms o f T o k o p h r y a i n f u s i o n u m as R e v e a l e d by L i g h t and E l e c t r o n M i c r o s c o p y . J . G e r o n t . , 16: 213-224 ( 1 9 6 1 ) . (112) R u s s e l l , E.S. L i f e s p a n and A g i n g P a t t e r n s , In " B i o l o g y o f t h e L a b o r a t o r y Mouse". G r e e n , E.L., N.Y., M c G r a w - H i l l Book C o . ( 1 9 6 6 ) . (113) R u s s e l l , P . J . L i p i d s and P i g m e n t s , In " A g i n g L i f e P r o c e s s e s " . Bakerman, S. E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 9 ) . (114) R u s s e l l , P . J . Same as r e f e r e n c e #113. (115) S a t o , H. and T. K u r o k i . P r o c . J a p . A c a d . , 42: 1211 ( 1 9 6 6 ) . (116) S c h l e t t w e i n - G s e l I , D. N u t r i t i o n as a F a c t o r i n A g i n g , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas, pp. 280-286 ( 1 9 6 6 ) . (117) S c h w a r t z , R.S. E d . " I m m u n o l o g i c a l A s p e c t s o f N e o p l a s i a " . P r o g r e s s i n E x p t l . Tumor R e s . B a s e l , N.Y., Munchen, S. K a r g e r V.13 ( 1 9 7 0 ) . (118) S e t l o w , R.B. The P h o t o c h e m i s t r y , P h o t o b i o l o g y and R e p a i r o f P o l y n u c l e o -t i d e s . P r o g . N u c l . A c i d . R e s . , 8: 257-295 ( 1 9 6 8 ) . 7 2 . (119) S h a r o n , N. and M. P o l l a r d . Spontaneous N e o p l a s t i c T r a n s f o r m a t i o n o f Germ-Free Rat Embryo C e l l C u l t u r e . C a n c e r R e s . , 29: 1523-1526 ( 1 9 6 9 ) . (120) S h e i n i n , R. DNA S y n t h e s i s i n Rat Embryo C e l l s I n f e c t e d w i t h Polyoma-V i r u s . V i r o l o g y , 29: 167-170 ( 1 9 6 6 ) . (121) S h i k a t a , E. and K. Maramorosch. An E l e c t r o n M i c r o s c o p i c Study o f P l a n t N e o p l a s i a Induced by Wound Tumor V i r u s . J . N a t . C a n c e r I n s t . , 36: 97-116 ( 1 9 6 6 ) . (122) S h i m o j o , H. and T. Y a m a s h i t a . I n d u c t i o n o f DNA S y n t h e s i s by A d e n o v i r u s e s i n C o n t a c t - I n h i b i t e d Hamster C e l l s . V i r o l o g y , 36: 422-423 ( 1 9 6 8 ) . (123) S h o c k , N.W. Age Changes i n Some P h y s i o l o g i c a l P r o c e s s e s . G e r i a t r i c s , 12: 40-48 ( 1 9 5 7 ) . (124) Shope, R.E. I n f e c t i o u s P a p i l l o m a t o s i s o f R a b b i t s , w i t h a Note on t h e H i s t o p a t h o l o g y . J . E x p t l . Med., 58: 607-624 ( 1 9 3 3 ) . (125) S k o o g , L., N o r d e n s k j t t l d , B.A., and U. L i n d b e r g . D e o x y r i b o n u c l e o t i d e P o o l s and D e o x y r i b o n u c l e i c A c i d S y n t h e s i s i n Mouse Embryo C e l l s I n f e c t e d w i t h T h r e e C l a s s e s o f Polyoma V i r u s P a r t i c l e s . J . V i r o l . , 6: 28-32 ( 1 9 7 0 ) . (126) S t i c h , H.F. A d e n o v i r u s - l n d u c e d A n o m a l i e s o f Chromosomes and M i t o t i c A p p a r a t u s and t h e i r R o l e i n O n c o g e n e s i s . Japan J . G e n e t i c s , 44: 65-70 ( 1 9 6 9 ) . 7 3 . (127) S t i c h , H.F. The I n d u c t i o n o f G e n e t i c a l l y H e t e rogeneous C e l l P o p u l a t i o n s by. A d e n o v i r u s e s , In " G e n e t i c C o n c e p t s and N e o p l a s i a " , T w e n t y - T h i r d Annual Symposium on Fundamental C a n c e r R e s e a r c h , H o u s t o n , T e x a s , ( 1 9 6 9 ) . (128) S t i c h , H.F. O n c o g e n i c and Non-Oncogenic M u t a n t s o f A d e n o v i r u s 12: I n d u c t i o n o f Chromosome A b e r r a t i o n s and C e l l D i v i s i o n . P r o g . E x p t l . Tumor R e s . , i n P r e s s . (129) S t i c h , H.F. ( I n P r e s s ) Same as r e f e r e n c e #128. (130) S t i c h , H.F., Van H o o s i e r , G.L. and J . J . T r e n t i n . V i r u s e s and Mammalian Chromosomes. Chromosome A b e r r a t i o n s by Human A d e n o v i r u s Type 12.. E x p . C e l l R e s . 34: 400-403 ( 1 9 6 4 ) . (131) S t i c h , H.F. and D.S. Y o h n . C a p a c i t y o f A d e n v i r u s e s f o r Mammalian C e l l s . N a t u r e , 216: 1292-1294 ( 1 9 6 7 ) . (132) S t i c h , H.F. and D.S. Yohn. The M u t a g e n i c C a p a c i t y o f A d e n o v i r u s e s f o r Mammalian CeI I s . N a t u r e , 216: 1292-1294 ( 1 9 6 7 ) . (133) S t i c h , H.F., A v i l a , L.R. and D.S. Yohn. V i r u s e s and Mammalian Chromosomes I X . The C a p a c i t y o f U V - l m p a i r e d A d e n o v i r u s Type 18 t o Induce Chromosome A b e r r a t i o n s . E x p . C e l l R e s . , 53: 44-54 ( 1 9 6 8 ) . (134) S t i c h , H.F., H s u . T.C., and F. Rapp. V i r u s e s and Mammalian Chromosomes.. 1. L o c a l i z a t i o n o f Chromosome A b e r r a t i o n s A f t e r I n f e c t i o n w i t h Herpes S i m p l e x V i r u s . V i r o l o g y , 22: 439-445 ( 1 9 6 4 ) . 74. (135) S t i c h , H.F. and R.H.C. S a n . DNA R e p a i r and C h r o m a t i d A n o m a l i e s i n Mammalian C e l l s Exposed t o 4 - N i t r o q u i n o I i n e - 1 - O x i d e . M u t a t i o n R e s . , 10: 389-404 ( 1 9 7 0 ) . (136) S t i c h , H.F. and D.S. Yohn. V i r u s e s and Chromosomes. P r o g r . Med. V i r o l . , 12: 78-127 ( 1 9 7 0 ) . (137) S t i c h , H.F., S a n , R.H.C., and Y. Kawazoe. DNA R e p a i r S y n t h e s i s i n Mammalian C e l l s Exposed t o a S e r i e s o f O n c o g e n i c and Non-Oncogenic D e r i v a t i v e s o f 4 - N i t r o q u i n o l i n e - 1 - O x i d e . N a t u r e , i n P r e s s . (138) S t i c h , H.F., S a n , R.H.C, and Y. Kawazoe. ( I n P r e s s ) Same as r e f e r e n c e #137. (139) S t o c k w e l l , R.A., and C H . B a r n e t t . Changes i n P e r m e a b i l i t y o f A r t i c u l a r C a r t i l a g e w i t h Age. N a t u r e , 201: 835-836 ( 1 9 6 4 ) . (140) S t r e h l e r , B.L., M a r k , D., M i I d v a n , A.S., and M. Gee. Rate and Ma g n i t u d e o f Age Pigment A c c u m u l a t i o n i n t h e Human M y o c a r d i u m . J . G e r o n t . , 14: 430-439 ( 1 9 5 9 ) . (141) S t r e h l e r , B.L. Dynamic T h e o r i e s o f A g i n g , In " A g i n g : Some S o c i a l and B i o l o g i c a l A s p e c t s . " S h o c k , N.W., E d . P u b l . No.65, Am. A s s o c . Advancement S c . , Wash. pp. 273-303 ( 1 9 6 0 ) . (142) S t r e h l e r , B.L. On t h e H i s t o c h e m i s t r y and U I t r a s t r u c t u r e o f Age P i g m e n t , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h . " N.Y. Academic P r e s s . V . I . p p . 343-348 ( 1 9 6 4 ) . 75. (143) S t r e h l e r , B.L. "Time, C e l l s and A g i n g " N.Y. Academic P r e s s ( 1 9 7 0 ) . (144) S u g i m u r a , T., O t a k e , H., and T. M a t s u s h i m a . S i n g l e S t r a n d S c i s s i o n s o f DNA Caused by a C a r c i n o g e n , 4-HydroxyI a m i n o q u i n o l i n e - 1 - 0 x i d e . N a t u r e 218: 392 ( 1 9 6 8 ) . (145) S y v e r t o n , J . T . The P a t h o g e n e s i s o f t h e R a b b i t Papi I l o m a - t o - C a r c i n o m a Sequence. Ann. N.Y. A c a d . S c i . , 54: 1126-1140 ( 1 9 5 2 ) . (146) T a k a h a s h i , M., van H o o s i e r , G.L. and J . J . T r e n t i n . S t i m u l a t i o n o f DNA S y n t h e s i s i n Human and Hamster C e l l s by Human A d e n o v i r u s Type 12 and 5 . P r o c . S o c . E x p t l . B i o l . Med., 122: 740-746 ( 1 9 6 6 ) . (147) T a k e m o r i , N. R i g g s , J . L . , and C. A l d r i c h . G e n e t i c S t u d i e s w i t h T u m o r i g e n i c A d e n o v i r u s e s . 1. I s o l a t i o n o f C y t o c i d a l ( c y t ) Mu t a n t s o f A d e n o v i r u s Type 12. V i r o l o g y , 36: 575-586 ( 1 9 6 8 ) . (148) T a k e m o r i , N., R i g g s , J . L . and C. A l d r i c h . G e n e t i c S t u d i e s w i t h Tumori-g e n i c A d e n o v i r u s e s . 11. H e t e r o g e n e i t y o f C y t M u t a n t s o f Adeno-v i r u s Type 12. V i r o l o g y , '38: 8-15 ( 1 9 6 9 ) . (149) T a n o o k a , H., Kawazoe, Y., and M. A r a k i . R e a c t i o n Mechanism o f 4-HydroxyI a m i n o q u i n o l i n e - 1 - O x i d e and R e l a t e d Compounds i n I n a c t i v a t i o n o f t h e T r a n s f o r m i n g A c t i o n o f D e o x y r i b o n u c l e i c A c i d . Gann, 60: 537-543 ( 1 9 6 9 ) . (150) T a t a , M., T a d a , M., and T. K a k a h a s h i . I n t e r a c t i o n o f a C a r c i n o g e n , 4-HydroxyI a m i n o q u i n o l i n e - 1 - O x i d e , w i t h N u c l e i c A c i d . B i o c h . B i o p h . R e s . Comm., 29: 4 6 9 - 4 7 7 ( 1 9 6 7 ) . 76. T a y l o r , J .H. and P. M i n e r . U n i t s o f DNA R e p l i c a t i o n i n Mammalian Chromosomes. C a n c e r R e s . , 28: 1810-1814 ( 1 9 6 8 ) . T e l l e r , M.N., M i k e l l , M., and J . J . Freeman. Growth o f Human Tumor H.Ep. #3 i n N o n - C o n d i t i o n e d S w i s s M i c e . P r o c . Amer. A s s . C a n c e r R e s . , 3: 273 ( 1 9 6 1 ) . T e l l e r , M.N., S t o k e s , G., C u r l e t t , W., K u b i s e k , M.L. and D. C u r t i s . A g i n g and C a n c e r i g e n e s i s . 1. Immunity t o Tumor and S k i n G r a f t s . J . N a t . C a n c e r I n s t . 33: 649-656 ( 1 9 6 4 ) . T e l l e r , M.N., C u r l e t t , W., R o s e , B., L a r d i s , M.P., and G. S t o h r . A P o s s i b l e C o r r e l a t i o n Between O l d Age, De p r e s s e d Immune R e s p o n s e , and Spontaneous Tumor I n c i d e n c e i n M i c e . P r o c . Amer. A s s . C a n c e r R e s . , 3: 367 ( 1 9 6 2 ) . T o d a r c o , G . J . and H. G r e e n . Q u a n t i t a t i v e S t u d i e s on t h e Growth o f Mouse Embryo C e l l s i n C u l t u r e and T h e i r Development i n t o E s t a b l i s h e d L i n e s . J . C e l l B i o l . , 17: 299 ( 1 9 6 3 ) . T o d a r c o , G . J . , G r e e n , H., and M.R. S w i f t . S u s c e p t i b i l i t y o f Human D i p l o i d F i b r o b l a s t S t r a i n s t o T r a n s f o r m a t i o n by SV 40 V i r u s . S c i e n c e , 153: 1252-1254 ( 1 9 6 6 ) . T r o s k o , J . E . , C h u , E.H.Y. and W.L. C a r r i e r . The I n d u c t i o n o f Thymine Dimers i n U l t r a v i o l e t - I r r a d i a t e d Mammalian C e l l s . R a d i a t i o n R e s . 667-672 ( 1 9 6 5 ) . V e r z a r , F. The S t a g e s and Consequences o f A g i n g o f C o l l a g e n . G e r o n t o l o g i a , 15: 233-239 ( 1 9 6 9 ) . 77. V e r z a r , F. and M. E r m i n i . D e c r e a s e o f C r e a t i n e - P h o s p h a t e R e s t i t u t i o n o f M u s c l e i n O l d Age and t h e I n f l u e n c e o f G l u c o s e . G e r o n t o l o g i a 16: 223-230 ( 1 9 7 0 ) . W a l f o r d , R.L. F u r t h e r C o n s i d e r a t i o n s Towards an Immunologic Theory o f A g i n g . E x p . G e r o n t o l . , 1: 67-76 ( 1 9 6 4 ) . W a l f o r d , R.L. and G.M. T r o u p . A u t o i m m u n i t y T h e o r i e s , In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas, pp. 351-358 ( 1 9 6 6 ) . W a l f o r d , R.L. "The Immunological Theory o f A g i n g " . Copenhagen, Munksgaard ( 1 9 6 9 ) . W e i s s , P. A g i n g : A C o r o l l a r y o f Development, In " P e r s p e c t i v e s i n E x p e r i m e n t a l G e r o n t o l o g y " . S h o c k , N.W., E d . S p r i n g f l e I d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 6 ) . W e l c h , J . P . S o m a t i c M u t a t i o n s and t h e A g i n g P r o c e s s , In "Advances i n G e r o n t o l o g i c a l R e s e a r c h " . S t r e h l e r , B.L., E d . N.Y. Academic P r e s s . V.2. pp. 4-16 ( 1 9 6 7 ) . Wei l i n g s , S.R., and R.G. C h u i n a r d . E p i d e r m a l P a p i l l o m a s w i t h V i r u s -L i k e P a r t i c l e s i n F l a t h e a d S o l e , H i p p o q l o s s o i d e s e l a s s o d o n . S c i e n c e , 146: 932-934 ( 1 9 6 4 ) . W i t k i n s , E.M. U l t r a v i o l e t - I n d u c e d M u t a t i o n and D N A - R e p a i r . Ann. Rev. G e n e t i c s , 3: 525-552 ( 1 9 6 9 ) . 78. (167) W o l f f , E. and J.M. K i r r m a n . T e r a t o g e n i c E f f e c t s o f I o n i z i n g R a d i a t i o n s on t h e Embryonic Development o f t h e H i g h e r V e r t e b r a t e s . I n t e r n a t . Rev. E x p . P a t h o l . R i c h t e r , G.W. and M.A. E p s t e i n , E d s . N.Y. Academic P r e s s . V.3 ( 1 9 6 4 ) . (168) . W o l s t e n h o l m e , G.E.W. and M. O'Connor, E d s . Water and E l e c t r o l y t e M e t a b o l i s m i n R e l a t i o n t o Age and S e x . C i b a F o u n d a t i o n C o l l o q u i a on A g i n g . B o s t o n , L i t t l e , V.4 ( 1 9 5 8 ) . (169) Y o s h i d a , T.H. K u r i t a , Y. and K. M o r i w a k i . Chromosomal A b e r r a t i o n s i n Y o s h i d a Sarcoma C e l l s T r e a t e d w i t h 4 - N i t r o q u i n o l i n e - 1 - O x i d e . Gann, 56: 523-528 ( 1 9 6 5 ) . (170) Y u an, G.C. and R.S. Chang. T e s t i n g o f Compounds f o r C a p a c i t y t o P r o l o n g P o s t m i t o t i c L i f e s p a n o f C u l t u r e d Human Amnion C e l l s . J . G e r o n t . 24: 82-85 ( 1 9 6 9 ) . (171) Z o r z o l i , A. Enzymes and C e l l u l a r M e t a b o l i s m , In " A g i n g L i f e P r o c e s s e s " Bakerman, S., E d . S p r i n g f i e l d , I l l i n o i s , C h a r l e s C. Thomas ( 1 9 6 9 ) . (172) Zs-Nagy, I., von Hahn, H.P. and F. V e r z a r . A g e - R e l a t e d A l t e r a t i o n s in t h e C e l l N u c l e i and t h e DNA C o n t e n t o f R a t T a i l T endon. G e r o n t o l o g i a , 15: 258-264 ( 1 9 6 9 ) . " 

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