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Growth promotion by preventing G0-arrest does not enhance the replicative life span of human diploid fibroblasts
Mechanisms of Ageing and Development, 15 (1981) 379-383
379
© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
G R O W T H P R O M O T I O N BY P R E V E N T I N G G o - A R R E S T DOES NOT E N H A N C E T H E R E P L I C A T I V E L I F E SPAN O F H U M A N DIPLOID FIBROBLASTS
TADAO OHNO DzVision of Pharmaceutical Sciences, National Institute Of Radiological Sciences, 4.9.1, Anagawa, Chiba-shi, 260 (Japan)
(Received July 1, 1980; in revised form September 30, 1980)
SUMMARY Platelet.derived growth factor promotes cell growth by stimulating the potential of Go-arrested cells to synthesize DNA and by preventing replicating cells from entering Go phase. Although platelet-derived growth factor markedly increased the growth rate of IMR-90 cells, it failed to enhance the replicative life span of the cells, thereby suggesting that Go phase experienced in the replicative life is not involved in determining the life span and that stimulation of growth rate can be dissociated from increasing life span.
INTRODUCTION One of the important purposes in studies on cellular aging is to determine the factors which clearly extend the limited life span of normal human diploid cells. As reported previously [1] the replicative life span of human embryonic lung fibroblasts, IMR-90, is to some extent regulated by the concentration of dialyzed serum in the culture medium. This implies that serum growth factors must play an important role in control of the cellular life span. Recent investigations indicate that a potent serum growth factor, platelet-derived growth factor (PDGF), promotes growth of different species of cultured cells [2, 3]. PDGF stimulates Go-arrested cells to become sufficiently "competent" to enter the S phase [4] and prevents replicating cells from entering Go [5]. These findings led to a study of the effect of PDGF on the life span of IMR-90 cells. The results suggest that the mechanism of determination of cellular life span which starts working before the end of replication is independent of the Go phase of the cell cycle.
380 MATERIALS AND METHODS IMR-90 cells were maintained as described previously [1] in an enriched MEM (TOM-H) containing 10% fetal bovine serum (FBS; GIBCO Lot. R370521). Growth rate of the cells was also determined as described previously [1]. Briefly, cells were seeded at a density of 1000 cells/cm2 and incubated overnight. After washing the attached cells twice with phosphate-buffered saline (PBS), TOM-H medium containing appropriate amounts of dialyzed FBS and a growth factor was added. The volume was 0.25 ml/cm 2 . Cells were cultured for 4 days. Growth rate was expressed as the increase per 4 days of the population doubling level (PDL increase) which was calculated on the cell number per cm 2 before and after the 4-day culture. To prepare the medium containing 100% dialyzed FBS, FBS was extensively dialyzed against PBS and then equilibrated with 10 volumes of TOM-H medium. This equilibrated serum was utilized as 100% dialyzed FBS for the experiment shown in Fig. 1. For the following experiments, FBS after first being extensively dialyzed against PBS was added at levels of 10% to the TOM-H medium. To minimize the PDGF activity in this dialyzed FBS, cationic proteins were adsorbed to a cation-exchange resin. A 25-ml volume of this dialyzed FBS was applied on a CM-Sephadex C-50 column (100-ml gel-bed volume) equilibrated with 80 mM NaCI-10 mM sodium phosphate (pH 7.4). The breakthrough fraction eluted with the above buffer was concentrated on a PM-10 membrane (Amicon) to its original volume. NaCI was added to a final concentration 0.14 M and it was then used as the non-cationic dialyzed FBS (NC-d-FBS) at 10% in TOM-H medium. PDGF was partially purified from disqualified human platelet concentrates (kind gifts from the Japan Red Cross) by the methods of Antoniades et al. [6, 7] up to the step of cation-exchange chromatography. A Dowex 50W-X8 column [6] and a CMSephadex C-50 column [7] were adopted for PDGF preparation used in the experiments shown in Figs. 1 and 2, respectively. The final protein concentrations used were 21.8/ag/ ml and 19.2 #g/ml for the former and the latter preparations, respectively. Addition of these PDGF preparations results in the increase of growth rates up to 31.5% and 18.1% for 39-PDL ceils and 46-PDL cells, respectively, cultured in TOM-H medium containing 10% dialyzed FBS. At every subculture, which was done once a week in the experiment shown in Fig. 2, PDGF was added after overnight incubation to avoid any inhibitory effect on cellular attachment to the surface of the culture dish. The medium was replaced 4 days after the subculture. Epidermal growth factor (EGF) was purified from male mouse submaxillary gland by the method of Savage and Cohen [8]. EGF was added at a concentration 10 ng]ml. The same preparations of PDGF, EGF, dialyzed FBS, or NC-d-FBS were used in a series of experiments to avoid batch-to-batch variations. RESULTS Growth promotion by the PDGF preparation was confirmed under variotis concentrations of dialyzed FBS, as shown in Fig. 1. A marked increase in the growth rate was
381
KI 13 .,¢
(,J
03
~
i
(o
d-FBS conc. (%)
~o
~oo
Fig. 1. Stimulation of the growth rate of IMR-90 cells by PDGF under various concentrations of dialyzed FBS (d-FBS). (e), control ceils; (O), cells treated with PDGF; (X), ceils treated with epidermal growth factor. The upper three curves are for cells at 39 PDL and the lower two curves are for ceils at 68 PDL. Each point represents the mean of duplicate cultures. The range of variation was less than 5% for any one point.
o.-OS.°===~ - .
.~'~.-I/ A /
,'":" ""
6c
,11
.,J t-~
5(
/
A
/
40
•
~
X
Weeks
;
;
1'0
Fig, 2. Effect of PDGF on the rep•cative life span of IMR-90 calls, Each point represents the mean of two culture lines which were maintained parallel but independently of each other, The experiment was started at 37.0 PDL, the mid-point of the ]fie span. The life spans of the two lines were: 72.6 and 74.6 PDL for dialyzed FBS (O); 75,0 and "/3,4 PDL for dialyzed FBS plus the PDGF (O); 71.5 and 69.3 PDL for NC-d-FBS (A) (cationic proteins were eliminated from the dialyzed FBS to minimize PDGF activity; see text); 69.0 and 69.5 PDL for NC-d-FBS plus PDGF (A).
observed in the cells at the 39th PDL, in the range 0 . 3 - 3 0 % dialyzed FBS, but this effect disappeared at 100% dialyzed FBS. This effect was superior to that of EGF at the optimal concentration. The effect of PDGF was also evident, though small, in the old 68-PDL
382 cells. No adverse effect of the PDGF preparation has been observed at any PDLs so far tested by measuring cellular growth rates. Figure 2 illustrates cumulative growth curves of IMR-90 cells cultured with or without PDGF. The ceils cultured with 10% non.cationic dialyzed FBS (NC-d-FBS), which was prepared to minimize PDGF activity in the serum, showed a 24% reduction of the growth rate and a 9% reduction of the replicative life span during the experimental period as compared with the control cells cultured with 10% dialyzed FBS. Although the reduced growth rate was recovered by addition of the PDGF, the life span did not recover to the level of the control cells. Similar results were observed in the ceils cultured with 10% dialyzed FBS plus PDGF. DISCUSSION
It is well characterized that in the course of the replicative life span of normal human diploid cells in vitro, interdivision times of individual cells are highly variable for both young and old populations [9], and that the average period of the G1 phase but not of other phases in the cell cycle is extended considerably toward the end of the life span [10]. The individual cells may enter Go in a sort of random fashion. However, the socalled "non-dividing" cells in phase III do synthesize DNA when they are incubated with [3HI thymidine for a prolonged period [11, 12]. This indicates that senescent cells have not been held in the Go phase. Moreover, when young diploid fibroblasts were arrested for months in Go by density inhibition of growth, they reached maximum doubling levels equivalent to the non-arrested control ceils after release from the arrest [13, 14]. Contrary to the case of density inhibition of growth, PDGF prevents the Go-arrest [4, 5]. Nevertheless, the results shown in Fig. 2 indicate the failure of changing life span. All these findings, therefore, strongly suggest that the Go phase experienced in the replicative life is not committed to the determination of the limited division potential of the diploid cells, though the possibility that senescent cells enter Go at the very end of their life span is not necessarily ruled out. However, it is more likely that the limitation of proliferation takes place for the daughter cells, rather than by being fLxed in Go, by transferring from an original cycling state to a different cycling state in which the cells are given lesser probability for cycling than that of the mother ceils, since this mode of proliferation is observable in rive in the hemopoietic stem cells most of which are retained in Go under normal physiological conditions [I 5]. In the previous paper, I described that there was a strict relationship between dialyzed serum concentration and cellular life span [1]. The serum concentration also regulates the growth rate of IMR-90 cells [ 1]. However, the present observations show that stimulation of the growth rate with PDGF can be dissociated from increasing life span. It is known that different serum lots give a different life span in normal human cells [16]. Therefore, there must be a factor(s) regulating life span in the serum which is probably different from factors regulating the Go phase of the cell cycle.
383 ACKNOWLEDGMENTS I t h a n k Prof. I. Y a m a n e , University o f T o h o k u , for fruitful discussion and Dr. M. Ohara, K y o t o University, for critical reading o f the manuscript.
REFERENCES 1 T. Ohno, Strict relationship between dialyzed serum concentration and cellular life span in vitro. Mech. Ageing Dev., 11 (1979) 179-183. 2 R. Ross and A. Vogel, The platelet-derived growth factor. Cell, 14 (1978) 203-210. 3 C. D, Scher, R. C. Shepard, H. N. Antoniades and C. D. Stiles, Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim. Biophys. Acta, 560 (1979) 217-241. 4 W. J, Pledger, C. D. Stiles, H. N. Antoniades and C. D. Scher, Induction of DNA synthesis in BALB/c 3T3 cells by serum components: Reevaluation of the commitment process. Proc. Natl. Acad. Sei. U.S.A., 74 (1977) 4481-4485. 5 C. D. Scher, M. E. Stone and C. D. Stiles, Platelet-dedved growth factor prevents Go growth arrest. Nature, 281 (1979) 390-392. 6 H. N. Antoniades, D. Stathakos and C. D. So,her, Isolation of a cationic polypeptide from human serum that stimulates proliferation of 3T3 cells. Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 2635 -2639. 7 H. N. Antoniades, C. D. Seller and C. D. Stiles, Purification of human platelet-derived growth factor. Proc. Natl. Aead. Sei. U.S.A., 76 (1979) 1809-1813. 8 C. R. Savage and S. Cohen, Epidermal growth factor and a new derivative: Rapid isolation procedure and biological and chemical characterization. £ Biol. Chem., 247 (1972) 7609-7611. 9 E. Bell, L. F. Marek, D. S. Levinstone, C. Merrfl, S. Sher, I. T. Young and M. Eden, Loss of division potential in vitro: Aging or differentiation? Science, 202 (1978) 1158-1163. 10 G. L. Grove and V. J. Cristofalo, Characterization of the cell cycle of cultured human diploid cells. Effects of aging and hydrocortisone. J. Cell, Physiol., 90 (1976) 415-422. 11 A. Maeieira-Coelho, Are non-dividing cells present in ageing cell culture? Nature, 248 (1974) 421-422. 12 T. Matsumura, Z. Zerrudo and L. Hayflick, Senescent human diploid cells in culture: Survival, DNA synthesis and morphology. Z Gerontol., 34 (1979) 328-334. 13 R. T. Dell'Orco, J. G. Mertens and P. F. Kruse, Doubling potential, calender time and senescence of human diploid cells in culture. Exp. CellRes., 77 (1973) 356-360. 14 K. Kaji and M. Matsuo, Doubling potential and calender time of human diploid cells in vitro. Exp. Gerontol., 14 (1979) 329-334. 15 F. Vassort, M. Winterholer, E. Frindel and M. Tubiana, Kinetic parameters of bone marrow stem cells using in vivo suicide by tritiated thymidine or by hydroxyurea. Blood, 41 (1973) 789-796. 16 E. L. Schneider, K. Braunschweiger and Y. Mitsui, The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors. Exp. Cell Res., 115 (1978) 47-52.
379
© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
G R O W T H P R O M O T I O N BY P R E V E N T I N G G o - A R R E S T DOES NOT E N H A N C E T H E R E P L I C A T I V E L I F E SPAN O F H U M A N DIPLOID FIBROBLASTS
TADAO OHNO DzVision of Pharmaceutical Sciences, National Institute Of Radiological Sciences, 4.9.1, Anagawa, Chiba-shi, 260 (Japan)
(Received July 1, 1980; in revised form September 30, 1980)
SUMMARY Platelet.derived growth factor promotes cell growth by stimulating the potential of Go-arrested cells to synthesize DNA and by preventing replicating cells from entering Go phase. Although platelet-derived growth factor markedly increased the growth rate of IMR-90 cells, it failed to enhance the replicative life span of the cells, thereby suggesting that Go phase experienced in the replicative life is not involved in determining the life span and that stimulation of growth rate can be dissociated from increasing life span.
INTRODUCTION One of the important purposes in studies on cellular aging is to determine the factors which clearly extend the limited life span of normal human diploid cells. As reported previously [1] the replicative life span of human embryonic lung fibroblasts, IMR-90, is to some extent regulated by the concentration of dialyzed serum in the culture medium. This implies that serum growth factors must play an important role in control of the cellular life span. Recent investigations indicate that a potent serum growth factor, platelet-derived growth factor (PDGF), promotes growth of different species of cultured cells [2, 3]. PDGF stimulates Go-arrested cells to become sufficiently "competent" to enter the S phase [4] and prevents replicating cells from entering Go [5]. These findings led to a study of the effect of PDGF on the life span of IMR-90 cells. The results suggest that the mechanism of determination of cellular life span which starts working before the end of replication is independent of the Go phase of the cell cycle.
380 MATERIALS AND METHODS IMR-90 cells were maintained as described previously [1] in an enriched MEM (TOM-H) containing 10% fetal bovine serum (FBS; GIBCO Lot. R370521). Growth rate of the cells was also determined as described previously [1]. Briefly, cells were seeded at a density of 1000 cells/cm2 and incubated overnight. After washing the attached cells twice with phosphate-buffered saline (PBS), TOM-H medium containing appropriate amounts of dialyzed FBS and a growth factor was added. The volume was 0.25 ml/cm 2 . Cells were cultured for 4 days. Growth rate was expressed as the increase per 4 days of the population doubling level (PDL increase) which was calculated on the cell number per cm 2 before and after the 4-day culture. To prepare the medium containing 100% dialyzed FBS, FBS was extensively dialyzed against PBS and then equilibrated with 10 volumes of TOM-H medium. This equilibrated serum was utilized as 100% dialyzed FBS for the experiment shown in Fig. 1. For the following experiments, FBS after first being extensively dialyzed against PBS was added at levels of 10% to the TOM-H medium. To minimize the PDGF activity in this dialyzed FBS, cationic proteins were adsorbed to a cation-exchange resin. A 25-ml volume of this dialyzed FBS was applied on a CM-Sephadex C-50 column (100-ml gel-bed volume) equilibrated with 80 mM NaCI-10 mM sodium phosphate (pH 7.4). The breakthrough fraction eluted with the above buffer was concentrated on a PM-10 membrane (Amicon) to its original volume. NaCI was added to a final concentration 0.14 M and it was then used as the non-cationic dialyzed FBS (NC-d-FBS) at 10% in TOM-H medium. PDGF was partially purified from disqualified human platelet concentrates (kind gifts from the Japan Red Cross) by the methods of Antoniades et al. [6, 7] up to the step of cation-exchange chromatography. A Dowex 50W-X8 column [6] and a CMSephadex C-50 column [7] were adopted for PDGF preparation used in the experiments shown in Figs. 1 and 2, respectively. The final protein concentrations used were 21.8/ag/ ml and 19.2 #g/ml for the former and the latter preparations, respectively. Addition of these PDGF preparations results in the increase of growth rates up to 31.5% and 18.1% for 39-PDL ceils and 46-PDL cells, respectively, cultured in TOM-H medium containing 10% dialyzed FBS. At every subculture, which was done once a week in the experiment shown in Fig. 2, PDGF was added after overnight incubation to avoid any inhibitory effect on cellular attachment to the surface of the culture dish. The medium was replaced 4 days after the subculture. Epidermal growth factor (EGF) was purified from male mouse submaxillary gland by the method of Savage and Cohen [8]. EGF was added at a concentration 10 ng]ml. The same preparations of PDGF, EGF, dialyzed FBS, or NC-d-FBS were used in a series of experiments to avoid batch-to-batch variations. RESULTS Growth promotion by the PDGF preparation was confirmed under variotis concentrations of dialyzed FBS, as shown in Fig. 1. A marked increase in the growth rate was
381
KI 13 .,¢
(,J
03
~
i
(o
d-FBS conc. (%)
~o
~oo
Fig. 1. Stimulation of the growth rate of IMR-90 cells by PDGF under various concentrations of dialyzed FBS (d-FBS). (e), control ceils; (O), cells treated with PDGF; (X), ceils treated with epidermal growth factor. The upper three curves are for cells at 39 PDL and the lower two curves are for ceils at 68 PDL. Each point represents the mean of duplicate cultures. The range of variation was less than 5% for any one point.
o.-OS.°===~ - .
.~'~.-I/ A /
,'":" ""
6c
,11
.,J t-~
5(
/
A
/
40
•
~
X
Weeks
;
;
1'0
Fig, 2. Effect of PDGF on the rep•cative life span of IMR-90 calls, Each point represents the mean of two culture lines which were maintained parallel but independently of each other, The experiment was started at 37.0 PDL, the mid-point of the ]fie span. The life spans of the two lines were: 72.6 and 74.6 PDL for dialyzed FBS (O); 75,0 and "/3,4 PDL for dialyzed FBS plus the PDGF (O); 71.5 and 69.3 PDL for NC-d-FBS (A) (cationic proteins were eliminated from the dialyzed FBS to minimize PDGF activity; see text); 69.0 and 69.5 PDL for NC-d-FBS plus PDGF (A).
observed in the cells at the 39th PDL, in the range 0 . 3 - 3 0 % dialyzed FBS, but this effect disappeared at 100% dialyzed FBS. This effect was superior to that of EGF at the optimal concentration. The effect of PDGF was also evident, though small, in the old 68-PDL
382 cells. No adverse effect of the PDGF preparation has been observed at any PDLs so far tested by measuring cellular growth rates. Figure 2 illustrates cumulative growth curves of IMR-90 cells cultured with or without PDGF. The ceils cultured with 10% non.cationic dialyzed FBS (NC-d-FBS), which was prepared to minimize PDGF activity in the serum, showed a 24% reduction of the growth rate and a 9% reduction of the replicative life span during the experimental period as compared with the control cells cultured with 10% dialyzed FBS. Although the reduced growth rate was recovered by addition of the PDGF, the life span did not recover to the level of the control cells. Similar results were observed in the ceils cultured with 10% dialyzed FBS plus PDGF. DISCUSSION
It is well characterized that in the course of the replicative life span of normal human diploid cells in vitro, interdivision times of individual cells are highly variable for both young and old populations [9], and that the average period of the G1 phase but not of other phases in the cell cycle is extended considerably toward the end of the life span [10]. The individual cells may enter Go in a sort of random fashion. However, the socalled "non-dividing" cells in phase III do synthesize DNA when they are incubated with [3HI thymidine for a prolonged period [11, 12]. This indicates that senescent cells have not been held in the Go phase. Moreover, when young diploid fibroblasts were arrested for months in Go by density inhibition of growth, they reached maximum doubling levels equivalent to the non-arrested control ceils after release from the arrest [13, 14]. Contrary to the case of density inhibition of growth, PDGF prevents the Go-arrest [4, 5]. Nevertheless, the results shown in Fig. 2 indicate the failure of changing life span. All these findings, therefore, strongly suggest that the Go phase experienced in the replicative life is not committed to the determination of the limited division potential of the diploid cells, though the possibility that senescent cells enter Go at the very end of their life span is not necessarily ruled out. However, it is more likely that the limitation of proliferation takes place for the daughter cells, rather than by being fLxed in Go, by transferring from an original cycling state to a different cycling state in which the cells are given lesser probability for cycling than that of the mother ceils, since this mode of proliferation is observable in rive in the hemopoietic stem cells most of which are retained in Go under normal physiological conditions [I 5]. In the previous paper, I described that there was a strict relationship between dialyzed serum concentration and cellular life span [1]. The serum concentration also regulates the growth rate of IMR-90 cells [ 1]. However, the present observations show that stimulation of the growth rate with PDGF can be dissociated from increasing life span. It is known that different serum lots give a different life span in normal human cells [16]. Therefore, there must be a factor(s) regulating life span in the serum which is probably different from factors regulating the Go phase of the cell cycle.
383 ACKNOWLEDGMENTS I t h a n k Prof. I. Y a m a n e , University o f T o h o k u , for fruitful discussion and Dr. M. Ohara, K y o t o University, for critical reading o f the manuscript.
REFERENCES 1 T. Ohno, Strict relationship between dialyzed serum concentration and cellular life span in vitro. Mech. Ageing Dev., 11 (1979) 179-183. 2 R. Ross and A. Vogel, The platelet-derived growth factor. Cell, 14 (1978) 203-210. 3 C. D, Scher, R. C. Shepard, H. N. Antoniades and C. D. Stiles, Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim. Biophys. Acta, 560 (1979) 217-241. 4 W. J, Pledger, C. D. Stiles, H. N. Antoniades and C. D. Scher, Induction of DNA synthesis in BALB/c 3T3 cells by serum components: Reevaluation of the commitment process. Proc. Natl. Acad. Sei. U.S.A., 74 (1977) 4481-4485. 5 C. D. Scher, M. E. Stone and C. D. Stiles, Platelet-dedved growth factor prevents Go growth arrest. Nature, 281 (1979) 390-392. 6 H. N. Antoniades, D. Stathakos and C. D. So,her, Isolation of a cationic polypeptide from human serum that stimulates proliferation of 3T3 cells. Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 2635 -2639. 7 H. N. Antoniades, C. D. Seller and C. D. Stiles, Purification of human platelet-derived growth factor. Proc. Natl. Aead. Sei. U.S.A., 76 (1979) 1809-1813. 8 C. R. Savage and S. Cohen, Epidermal growth factor and a new derivative: Rapid isolation procedure and biological and chemical characterization. £ Biol. Chem., 247 (1972) 7609-7611. 9 E. Bell, L. F. Marek, D. S. Levinstone, C. Merrfl, S. Sher, I. T. Young and M. Eden, Loss of division potential in vitro: Aging or differentiation? Science, 202 (1978) 1158-1163. 10 G. L. Grove and V. J. Cristofalo, Characterization of the cell cycle of cultured human diploid cells. Effects of aging and hydrocortisone. J. Cell, Physiol., 90 (1976) 415-422. 11 A. Maeieira-Coelho, Are non-dividing cells present in ageing cell culture? Nature, 248 (1974) 421-422. 12 T. Matsumura, Z. Zerrudo and L. Hayflick, Senescent human diploid cells in culture: Survival, DNA synthesis and morphology. Z Gerontol., 34 (1979) 328-334. 13 R. T. Dell'Orco, J. G. Mertens and P. F. Kruse, Doubling potential, calender time and senescence of human diploid cells in culture. Exp. CellRes., 77 (1973) 356-360. 14 K. Kaji and M. Matsuo, Doubling potential and calender time of human diploid cells in vitro. Exp. Gerontol., 14 (1979) 329-334. 15 F. Vassort, M. Winterholer, E. Frindel and M. Tubiana, Kinetic parameters of bone marrow stem cells using in vivo suicide by tritiated thymidine or by hydroxyurea. Blood, 41 (1973) 789-796. 16 E. L. Schneider, K. Braunschweiger and Y. Mitsui, The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors. Exp. Cell Res., 115 (1978) 47-52.