- Email: [email protected]
No male-female difference in In vitro lifespan of skin fibroblasts from humans and mice
Experimental Gerontology, Vol. 18, pp. 323-324, 1983
0531-5565/83 $3.00 + .00 Copyright © 1983 Pergamon Press Ltd
Printed in the USA. All rights reserved.
NO MALE-FEMALE DIFFERENCE IN IN VITRO LIFESPAN OF SKIN FIBROBLASTS FROM HUMANS AND MICE
P. EBBESEN The Institute of Cancer Research (sponsored by the Danish Cancer Society), Radiumstationen, DK-8000 A a r h u s C, D e n m a r k
(Received 12 May 1983)
Abstract- To determine if the difference in mean survival time of males and females is related to the m e c h a n i s m restricting the number of in vitro cell doublings, we established cultures of h u m a n skin fibroblasts and murine embryo cells. The m a x i m u m n u m b e r of in vitro doublings was found to be very similar for the two sexes in both species. Furthermore, h u m a n cultures receiving repeated treatments with three alkylating carcinogens had the same in vivo survival as untreated controls.
INTRODUCTION
IN MANY m a m m a l i a n species, including man and mice, females have a longer mean survival than males (Russell, 1966; Goodnick, 1975). This difference is often assumed to derive ultimately from influence exerted by the sex chromosomes. Interspecies differences in maximum survival time parallel interspecies differences in in vitro doubling potential of fibroblasts (Hayflick, 1977), suggesting a general aging mechanism which is also operative under in vitro conditions. At present, we are studying the possibility that intersex differences also are reflected in the in vitro doubling potential. MATERIALS AND M E T H O D P u n c h biopsies were obtained from 26 healthy adult human males aged 17-56 (mean 33) and 22 females aged 17-59 (mean 33). The uppermost area of dermis was dissected out (Harper, 1979) and seeded. We used Eagle's medium with Hank's buffer and 10°70 non-inactivated fetal calf serum of a batch containing 12,000 picogram of estron sulfate, 41 picogram of estron, less than 10 picogram of estradiol and 160 picogram of testosterone. Dihydrotestosterone, A-4 androstendion and dehydroepiandrosterone sulfate were not detectable'. From each primary culture four sublines were established in 75 cm 2 Falcon bottles and kept in different incubators; one subline from each donor was carcinogen treated. Bottles were harvested at near confluency, the cells counted, and 105 cells reseeded. Plating efficiency (percent attached 24 hours after seeding) and cloning efficiency (160 cells distributed to 200 Falcon microtiter wells) were determined every tenth passage. Mouse cultures were established by seeding 103 cells from 19-day-old sex-determined embryos. All in vitro doublings of cells from a given embryo took place in one 250 cm 2 bottle. Alkylating chemical carcinogens (KaKunaga, 1977) N-methyI-N'-nitro-N-nitrosoguanidine (4I',0, methyl methanesulfonate (MS), and 4-nitro-quinoline-l-oxide (NQO) were purchased from Merck, Darmstadt, West Germany. The h u m a n cultures to be treated were given m e d i u m without serum containing 10-6, 10 -s, and 10-6s 'The testing was kindly performed by Paul Bennet, The State Serum Institute, Copenhagen. 323
324
ESBESEN
M, respectively, of carcinogen for one week. Each culture received 4N during the second passage, MS during the fifth passage and NQO during the eighth passage. Preliminary testing had shown the dose to be 1 log lower than the dose decreasing the number of cells. Statistics were carried out with Student's two-tailed t-test and Wilcoxon U-statistic. RESULTS C u l t u r e s were e s t a b l i s h e d f r o m all h u m a n biopsies. P l a t i n g efficiency when reseeding was close to 65°7o in all tests a n d c l o n i n g efficiency very low ( < 1 percent). O b t a i n e d d o u b l i n g s were c a l c u l a t e d on the a s s u m p t i o n t h a t all a t t a c h e d cells also divide. T h e m e a n d o u b l i n g value was 43 + 16 f o r the u n t r e a t e d cells f r o m m a l e d o n o r s a n d 44 + 14 for the u n t r e a t e d cells f r o m f e m a l e d o n o r s . C a r c i n o g e n - t r e a t e d cells f r o m m a l e d o n o r s reach a m e a n d o u b l i n g value o f 46 + 14 while it was 48 + 17 for the female d o n o r s . Neither t-test n o r W i l c o x o n ' s statistic s h o w e d a n y d i f f e r e n c e b e t w e e n the m e a n values at the 5°7o level. M o u s e cells h a d a p r i m a r y p l a t i n g efficiency o f a b o u t 11°70 a n d a cloning efficiency o f 3 % . Eleven B A L B / c m a l e a n d 14 f e m a l e fetus r e a c h e d 9 + 4 a n d 10 + 4 d o u b l i n g s respectively while the figures for 22 C57/B1 males a n d 10 females were 11 + 3 a n d 10 -4- 5 d o u b l i n g s . DISCUSSION F o r h u m a n s the d i f f e r e n c e in m e a n survival t i m e between males a n d females in the ind u s t r i a l i z e d c o u n t r i e s is o f the o r d e r o f five to seven percent. In l a b o r a t o r y mice the sex d i f f e r e n c e in survival t i m e varies f r o m t w e n t y - f i v e to zero p e r c e n t d e p e n d i n g on the w a y the a n i m a l s are g r o u p e d in their boxes. W h e n a b o x c o n t a i n s m o r e t h a n one m a l e the m e a n survival t i m e o f the males will be lower t h a n t h a t o f females irrespective o f w h e t h e r the m a l e s e n g a g e in p h y s i c a l fighting or not. H o w e v e r , w h e n males are kept a l o n e or in c o m p a n y with females, b o t h sexes s h o w a high m e a n survival t i m e ( E b b e s e n , 1968). F r o m the present w o r k it a p p e a r s t h a t the m a x i m u m n u m b e r o f in vitro d o u b l i n g s o f n o r m a l h u m a n a n d m u r i n e f i b r o b l a s t s d o not d i f f e r b e t w e e n the sexes to a degree d e t e c t a b l e by the m e t h o d used here. W e , t h e r e f o r e , still f a v o r the h y p o t h e s i s t h a t sex differences in survival time are largely d e p e n d e n t on i n t e r p e r s o n a l stress factors. T h a t t r e a t m e n t with a l k y l a t i n g c a r c i n o g e n i c agents does n o t influence in vitro survival suggests t h a t there m a y be no state i n t e r m e d i a r y b e t w e e n " n o r m a l " in vitro lifespan ( H a y f l i c k a n d M o o r h e a d , 1961) a n d the i m m o r t a l i z a t i o n so c h a r a c t e r i s t i c o f m a l i g n a n t cells. REFERENCES EBBESEN, P. (1968) J. Exp. Med. 127, 387. GOODNICK, C.L. (1975) J. Gerontol. 30, 257. HARPER, R.A. (1979) Science 204, 526. HAYFLICK,L. (1977) In: Handbook of the Biology of Aging. C.E. Finch and L. Hayflick (eds.). p. 167. van
Nostrand Reinhold Company, New York. HAYFLICK,L. and MOORHEAD,P.S. (1961) Exp. Cell Res. 25, 585. KAKUNAGA,T. (1977) In: Origins of Human Cancer. H.H. Hyatt, J.D. Watson and J.A. Winsten (eds.). Book C. p. 1537. Cold Spring Harbor Laboratory. RUSSELL,E.S. (1966) In: Biology of the Laboratory Mouse. E.L. Green (ed.). p. 511. McGraw-Hill, New York.
0531-5565/83 $3.00 + .00 Copyright © 1983 Pergamon Press Ltd
Printed in the USA. All rights reserved.
NO MALE-FEMALE DIFFERENCE IN IN VITRO LIFESPAN OF SKIN FIBROBLASTS FROM HUMANS AND MICE
P. EBBESEN The Institute of Cancer Research (sponsored by the Danish Cancer Society), Radiumstationen, DK-8000 A a r h u s C, D e n m a r k
(Received 12 May 1983)
Abstract- To determine if the difference in mean survival time of males and females is related to the m e c h a n i s m restricting the number of in vitro cell doublings, we established cultures of h u m a n skin fibroblasts and murine embryo cells. The m a x i m u m n u m b e r of in vitro doublings was found to be very similar for the two sexes in both species. Furthermore, h u m a n cultures receiving repeated treatments with three alkylating carcinogens had the same in vivo survival as untreated controls.
INTRODUCTION
IN MANY m a m m a l i a n species, including man and mice, females have a longer mean survival than males (Russell, 1966; Goodnick, 1975). This difference is often assumed to derive ultimately from influence exerted by the sex chromosomes. Interspecies differences in maximum survival time parallel interspecies differences in in vitro doubling potential of fibroblasts (Hayflick, 1977), suggesting a general aging mechanism which is also operative under in vitro conditions. At present, we are studying the possibility that intersex differences also are reflected in the in vitro doubling potential. MATERIALS AND M E T H O D P u n c h biopsies were obtained from 26 healthy adult human males aged 17-56 (mean 33) and 22 females aged 17-59 (mean 33). The uppermost area of dermis was dissected out (Harper, 1979) and seeded. We used Eagle's medium with Hank's buffer and 10°70 non-inactivated fetal calf serum of a batch containing 12,000 picogram of estron sulfate, 41 picogram of estron, less than 10 picogram of estradiol and 160 picogram of testosterone. Dihydrotestosterone, A-4 androstendion and dehydroepiandrosterone sulfate were not detectable'. From each primary culture four sublines were established in 75 cm 2 Falcon bottles and kept in different incubators; one subline from each donor was carcinogen treated. Bottles were harvested at near confluency, the cells counted, and 105 cells reseeded. Plating efficiency (percent attached 24 hours after seeding) and cloning efficiency (160 cells distributed to 200 Falcon microtiter wells) were determined every tenth passage. Mouse cultures were established by seeding 103 cells from 19-day-old sex-determined embryos. All in vitro doublings of cells from a given embryo took place in one 250 cm 2 bottle. Alkylating chemical carcinogens (KaKunaga, 1977) N-methyI-N'-nitro-N-nitrosoguanidine (4I',0, methyl methanesulfonate (MS), and 4-nitro-quinoline-l-oxide (NQO) were purchased from Merck, Darmstadt, West Germany. The h u m a n cultures to be treated were given m e d i u m without serum containing 10-6, 10 -s, and 10-6s 'The testing was kindly performed by Paul Bennet, The State Serum Institute, Copenhagen. 323
324
ESBESEN
M, respectively, of carcinogen for one week. Each culture received 4N during the second passage, MS during the fifth passage and NQO during the eighth passage. Preliminary testing had shown the dose to be 1 log lower than the dose decreasing the number of cells. Statistics were carried out with Student's two-tailed t-test and Wilcoxon U-statistic. RESULTS C u l t u r e s were e s t a b l i s h e d f r o m all h u m a n biopsies. P l a t i n g efficiency when reseeding was close to 65°7o in all tests a n d c l o n i n g efficiency very low ( < 1 percent). O b t a i n e d d o u b l i n g s were c a l c u l a t e d on the a s s u m p t i o n t h a t all a t t a c h e d cells also divide. T h e m e a n d o u b l i n g value was 43 + 16 f o r the u n t r e a t e d cells f r o m m a l e d o n o r s a n d 44 + 14 for the u n t r e a t e d cells f r o m f e m a l e d o n o r s . C a r c i n o g e n - t r e a t e d cells f r o m m a l e d o n o r s reach a m e a n d o u b l i n g value o f 46 + 14 while it was 48 + 17 for the female d o n o r s . Neither t-test n o r W i l c o x o n ' s statistic s h o w e d a n y d i f f e r e n c e b e t w e e n the m e a n values at the 5°7o level. M o u s e cells h a d a p r i m a r y p l a t i n g efficiency o f a b o u t 11°70 a n d a cloning efficiency o f 3 % . Eleven B A L B / c m a l e a n d 14 f e m a l e fetus r e a c h e d 9 + 4 a n d 10 + 4 d o u b l i n g s respectively while the figures for 22 C57/B1 males a n d 10 females were 11 + 3 a n d 10 -4- 5 d o u b l i n g s . DISCUSSION F o r h u m a n s the d i f f e r e n c e in m e a n survival t i m e between males a n d females in the ind u s t r i a l i z e d c o u n t r i e s is o f the o r d e r o f five to seven percent. In l a b o r a t o r y mice the sex d i f f e r e n c e in survival t i m e varies f r o m t w e n t y - f i v e to zero p e r c e n t d e p e n d i n g on the w a y the a n i m a l s are g r o u p e d in their boxes. W h e n a b o x c o n t a i n s m o r e t h a n one m a l e the m e a n survival t i m e o f the males will be lower t h a n t h a t o f females irrespective o f w h e t h e r the m a l e s e n g a g e in p h y s i c a l fighting or not. H o w e v e r , w h e n males are kept a l o n e or in c o m p a n y with females, b o t h sexes s h o w a high m e a n survival t i m e ( E b b e s e n , 1968). F r o m the present w o r k it a p p e a r s t h a t the m a x i m u m n u m b e r o f in vitro d o u b l i n g s o f n o r m a l h u m a n a n d m u r i n e f i b r o b l a s t s d o not d i f f e r b e t w e e n the sexes to a degree d e t e c t a b l e by the m e t h o d used here. W e , t h e r e f o r e , still f a v o r the h y p o t h e s i s t h a t sex differences in survival time are largely d e p e n d e n t on i n t e r p e r s o n a l stress factors. T h a t t r e a t m e n t with a l k y l a t i n g c a r c i n o g e n i c agents does n o t influence in vitro survival suggests t h a t there m a y be no state i n t e r m e d i a r y b e t w e e n " n o r m a l " in vitro lifespan ( H a y f l i c k a n d M o o r h e a d , 1961) a n d the i m m o r t a l i z a t i o n so c h a r a c t e r i s t i c o f m a l i g n a n t cells. REFERENCES EBBESEN, P. (1968) J. Exp. Med. 127, 387. GOODNICK, C.L. (1975) J. Gerontol. 30, 257. HARPER, R.A. (1979) Science 204, 526. HAYFLICK,L. (1977) In: Handbook of the Biology of Aging. C.E. Finch and L. Hayflick (eds.). p. 167. van
Nostrand Reinhold Company, New York. HAYFLICK,L. and MOORHEAD,P.S. (1961) Exp. Cell Res. 25, 585. KAKUNAGA,T. (1977) In: Origins of Human Cancer. H.H. Hyatt, J.D. Watson and J.A. Winsten (eds.). Book C. p. 1537. Cold Spring Harbor Laboratory. RUSSELL,E.S. (1966) In: Biology of the Laboratory Mouse. E.L. Green (ed.). p. 511. McGraw-Hill, New York.