@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Zoology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Atchison , Brad"@en ; dcterms:issued "2011-04-28T17:31:15Z"@en, "1971"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """The purpose of this study was to examine the phenomenon of aging at the cellular level in vitro. Some of the biological mechanisms underlying the aging process, which may invoke a change at the level of the DNA were studied. Morphological changes were analysed with phase-contrast microscopy and functional changes by the use of tritiated thymidine in combination with autoradiography as well as by cell treatment with colchicine. A method is described for obtaining "aged" or "old" cells in vitro. In the human cells (human embryonic kidney) as well as the rat, mouse, and Syrian hamster cells, morphological changes in vitro are basically the same as aging progresses. These include transformation to a polygonal, epithelial -1ike shape; binucleation; an accumulation of "age pigments" around the nucleus; the appearance of ragged edges of the cell membrane; an increase in the overall cell size; and a loss of a regular (often parallel) orientation to adjacent cells. The mitotic rate and DNA-synthesizing capacity in "young" and "aged" cells were examined using autoradiography and cell treatment with colchicine. Evidence is presented that DNA-synthesizing aged cells are non-proliferating while DNA-synthesizing young cells are mitotically active. The significance of DNA-synthesis in non-dividing "aged" cells is discussed. The number of population doublings (generation times or cell divisions) that it takes hamster and mouse cells to age in vitro was also investigated. Thirteen and six cell generation times were found to cause hamster and mouse cells to age with a loss of proliferative capacity. The effect of various molarities of 4-nitroquinoline-1 oxide (4-NQO), on DNA of aged cells, which results in an unscheduled DNA-repair synthesis, was studied using autoradiography. It appears that an aged cell responds to these concentrations in much the same way as young cells; however, there does seem to be a slightly greater sensitivity to toxic doses of 4-NQO in aged cells. Autoradiographic studies also revealed that the duration of DNA-repair is the same in both aged and young cells, but the former appear to have a decreased capacity to repair damage to the DNA of the pretreated cells caused by the 4-NQO. The significance of this apparent decrease in the DNA-repair capacity of "aged" cells is discussed. Mouse cells, aged in vitro, were exposed to human adenovirus type 12 (strain Huie). Evidence is presented that this external agent stimulated these aged cells to increase DNA-synthesis and also pushed them into mitosis (for at least one cell division). The possibility that this might be an accountable mechanism for the observation of an accumulation of mutations in aged cells is evaluated. Aged cells were examined for frequencies and types of chromosome aberrations after exposure to adenovirus type 12. Among the most common are chromatid breaks and double fragments. As well, old cells exhibited a much higher frequency of chromosome aberrations than young cells after viral exposure. A comparison of this in vitro system of cell aging with an in vivo system is presented. The application of all of the aforementioned results and observations concerning the cellular aging process to the problem of carcinogenesis and neoplasia is emphasized."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/34096?expand=metadata"@en ; skos:note "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 . 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G e r o n t o l o g i a , 15: 258-264 ( 1 9 6 9 ) . \" "@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0093286"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Zoology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Aging of mammalian cells in vitro"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/34096"@en .