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Kinetics of the proliferation of human fibroblasts during their lifespan in vitro
Mechanisms of Ageing and Development, 6 ( 1977) 341 - 343 © Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
341
KINETICS OF THE PROLIFERATION OF HUMAN FIBROBLASTS DURING THEIR LIFESPAN IN VITRO
A. MACIEIRA-COELHO Department of Cell Pathology, Institute of Cancerology and Immunogenetics (INSERM U50), 94800 Ville]uif (France) (Received November 8, 1976) There has been some confusion in the past in the interpretation of data concerning the kinetics of the proliferation of human fibroblasts in vitro [1]. The discussion has centred mainly around the presence of an irreversibly arrested fraction of cells that would increase in size at each subcultivation. This idea originated from the experiments of Merz and Ross [2] who showed that the number of isolated cells capable of originating colonies declined at each passage. However, if a cell cannot divide when isolated it may do so when surrounded by other cells and so the experiments of Merz and Ross cannot predict the cell behaviour at the level of the population. We describe here a model that takes into consideration all the data previously published on the kinetics of the proliferation of these fibroblast populations that may be helpful in understanding cell division during in vitro aging. Experiments performed with autoradiography have shown that during serial subcultivation of human embryonic lung fibroblasts there is a decrease in the number of cells synthesizing DNA during a 24 hour period [3, 4], a decreased number of cells entering the division cycle between subcultivation and resting stage [3], a lower saturation density [3, 5, 6], a prolonged doubling time [3, 5, 6], and a prolonged mean generation time [6]. Furthermore, there is an increased heterogeneity in the initiation of the cell cycle so that the cells become more erratic [6],while in young cultures between subcultivation and resting phase the number of cells entering the S period increases progressively to a maximum and then decreases to very low levels, in old cultures there is a burst of cells entering S, then the percentage goes down, and up again before it decreases at confluency [6]. This erratic behaviour has been confirmed by direct examination of the cells with time lapse cinematography [7]. This heterogeneity in the initiation of the division cycle can lead us to consider as non-dividers cells that still have the potential to enter the cell cycle but in a more unpredictable way as has also recently been suggested [8]. Furthermore, it seems that if there are non-dividing cells in late passage cultures, the percentage must be very small since after prolonged labelling with tritiated thymidine during two following passages the number of labelled cells came down to 8% [9]. So if there is production of non-dividing cells they must be lost. The absence of a large compartment of non-dividing cells is also suggested from experiments where the large slow-dividing cells were isolated from a mixed population and when replicated produced another population identical to the original one made of large and small cells [ 10].
342 ~ur~i~ de~
Early passage
i
Daysoffer subcultivotion
saturation density
Intermediate/.
.,~l ~
~
],
Daysafter subcultivotion saturation density
E passage
Daysoffer subcultivotic~ Fig. 1. Schematic representation of the kinetics of cell proliferation during the lifespan of human embryonic lung fibroblasts in vitro. For explanation, see text.
Primary changes in the S period have not been detected and it seems that the changes leading to the growth decline are located in the periods preceding DNA synthesis and mitosis rather than in these periods themselves [ I 1]. All the data reported above can be summarized in the model illustrated in Fig. 1. It represents three different periods of the cell population life span. On the left side of the Figure each circle and ellipse represents the generation time of a cell. In early passages most cells in the population are postulated to have short division cycles. Some cells already exist with long generation times (ellipses) which are extended mainly at the expense of G1 and G2. There is no gross prolongation of the S period and mitosis. Since the saturation density is high (vertical hatched line) most cells are able to complete their cycle before this density is reached. At this stage the growth curve of the population (right hand side of the Figure) has a short lag period, a phase of logarithmic growth with a steep slope, and high saturation density reached within 3-4 days. Towards the middle of the population life span (intermediate passage), there are still cells with short generation times, but most cells have division cycles between the two extremes. Since the saturation density is lower, an increased number of cells will not have time to complete their cycle. Thus, during a prolonged labelling with tritiated thymidine (3H-TdR) an increased number of cells will appear as unlabelled. At this stage the slope of the logarithmic portion of the growth curve will be less steep and the cells will reach a lower saturation density, although still within 3-4 days.
343 Towards the end o f the life span (late passage) most cells have very long generation times, and since the saturation density is very low, a still greater number o f cells do not have time to complete the cycle between subcultivation and confluency. Thus, even more cells will remain unlabelled after growing in the presence of 3H-TdR. The growth curve will be characterized by a long lag phase, a period of short logarithmic growth with a shallow slope, and a low saturation density reached only after 7 - 1 0 days. In Fig. 1 the prolongation of G1 and G2 is identical but it is possible that one is predominant. It should be emphasized that when one speaks about prolongation of G1, or G2, it applies only to cells that are moving through the cycle. Since the cells that are arrested due for instance to the effects of saturation density, seem to be all in G1, a distinction should and can be made when measuring the prolongation of the G 1 and G2 periods, otherwise cells that are arrested will be taken as prolonged in G 1. Methods that allow the analysis of cells in motion along the cycle should be distinguished from those measuring only instants [12, 13]. In summary, one can confirmed by direct methods fibroblasts, the spectrum of division to a normal cell cycle
say [7], cell with
that as has already been suggested before [6] and during the decline of the division potential of human activity seems to range from complete inhibition of a progressive increase of cells with long cycles.
REFERENCES 1 P. I. Good, Aging in mammalian cell populations: a review, Mech. Ageing Develop., 4 (1975) 339-348. 2 G. S. Merz, Jr. and J. D. Ross, Viability of human diploid cells as a function of in vitro age, J. Cell Physiol., 74 (1969) 219-223. 3 A. Macieira-Coelho, J. Ponten and L. Philipson, Inhibition of the division cycle in confluent cultures of human fibroblasts in vitro, Exp. Cell Res., 43 (1966) 20-29. 4 V. J. Cristofalo and B. B. Sharp, Cellular senescence and DNA synthesis. Thymidine incorporation as a measure of population age in human diploid cells, Exp. Cell Res., 76 (1973) 419-427. 5 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 583-621. 6 A. Macieira-Coelho, J. Ponten and L. Philipson, The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro, Exp. Cell Res., 42 (1966) 673-684. 7 P. M. Absher, R. G. Absher and W. D. Barnes, Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age, Exp. Cell Res., 88 (1974) 95-104. 8 G. L. Grove and V. J. Cristofalo, The transition probability model and the regulation of proliferation of human diploid cell cultures during aging, Cell Tissue Kinet., 9 (1976) 395-399. 9 A. Macieira-Coelho, Are non-dividing cells present in ageing cell cultures?, Nature, 248 (1974) 421-422. 10 Y. Mitsui and E. L. Schneider, Characterization of fraetionated human diploid fibroblast populations, Exp. Cell Res., in press. 11 A. Macieira-Coelho and J. Pont6n, Analogy in growth between late passage human embryonic and early passage human adult fibroblasts, J. Cell Biol., 43 (1969) 374-377. 12 A. Macieira-Coelho and L. Berumen, The cell cycle during growth inhibition of human embryonic fibroblasts in vitro, Proc. Soc. Exp. Biol. Med., 144 (1973) 43. 13 A. Macieira-Coelho, Cell cycle analysis in mammalian cells, in P. F. Kruse, Jr. and M. K. Patterson, Jr. (eds.), Tissue Culture: Methods and Application, Academic Press, New York, 1973, p. 412.
341
KINETICS OF THE PROLIFERATION OF HUMAN FIBROBLASTS DURING THEIR LIFESPAN IN VITRO
A. MACIEIRA-COELHO Department of Cell Pathology, Institute of Cancerology and Immunogenetics (INSERM U50), 94800 Ville]uif (France) (Received November 8, 1976) There has been some confusion in the past in the interpretation of data concerning the kinetics of the proliferation of human fibroblasts in vitro [1]. The discussion has centred mainly around the presence of an irreversibly arrested fraction of cells that would increase in size at each subcultivation. This idea originated from the experiments of Merz and Ross [2] who showed that the number of isolated cells capable of originating colonies declined at each passage. However, if a cell cannot divide when isolated it may do so when surrounded by other cells and so the experiments of Merz and Ross cannot predict the cell behaviour at the level of the population. We describe here a model that takes into consideration all the data previously published on the kinetics of the proliferation of these fibroblast populations that may be helpful in understanding cell division during in vitro aging. Experiments performed with autoradiography have shown that during serial subcultivation of human embryonic lung fibroblasts there is a decrease in the number of cells synthesizing DNA during a 24 hour period [3, 4], a decreased number of cells entering the division cycle between subcultivation and resting stage [3], a lower saturation density [3, 5, 6], a prolonged doubling time [3, 5, 6], and a prolonged mean generation time [6]. Furthermore, there is an increased heterogeneity in the initiation of the cell cycle so that the cells become more erratic [6],while in young cultures between subcultivation and resting phase the number of cells entering the S period increases progressively to a maximum and then decreases to very low levels, in old cultures there is a burst of cells entering S, then the percentage goes down, and up again before it decreases at confluency [6]. This erratic behaviour has been confirmed by direct examination of the cells with time lapse cinematography [7]. This heterogeneity in the initiation of the division cycle can lead us to consider as non-dividers cells that still have the potential to enter the cell cycle but in a more unpredictable way as has also recently been suggested [8]. Furthermore, it seems that if there are non-dividing cells in late passage cultures, the percentage must be very small since after prolonged labelling with tritiated thymidine during two following passages the number of labelled cells came down to 8% [9]. So if there is production of non-dividing cells they must be lost. The absence of a large compartment of non-dividing cells is also suggested from experiments where the large slow-dividing cells were isolated from a mixed population and when replicated produced another population identical to the original one made of large and small cells [ 10].
342 ~ur~i~ de~
Early passage
i
Daysoffer subcultivotion
saturation density
Intermediate/.
.,~l ~
~
],
Daysafter subcultivotion saturation density
E passage
Daysoffer subcultivotic~ Fig. 1. Schematic representation of the kinetics of cell proliferation during the lifespan of human embryonic lung fibroblasts in vitro. For explanation, see text.
Primary changes in the S period have not been detected and it seems that the changes leading to the growth decline are located in the periods preceding DNA synthesis and mitosis rather than in these periods themselves [ I 1]. All the data reported above can be summarized in the model illustrated in Fig. 1. It represents three different periods of the cell population life span. On the left side of the Figure each circle and ellipse represents the generation time of a cell. In early passages most cells in the population are postulated to have short division cycles. Some cells already exist with long generation times (ellipses) which are extended mainly at the expense of G1 and G2. There is no gross prolongation of the S period and mitosis. Since the saturation density is high (vertical hatched line) most cells are able to complete their cycle before this density is reached. At this stage the growth curve of the population (right hand side of the Figure) has a short lag period, a phase of logarithmic growth with a steep slope, and high saturation density reached within 3-4 days. Towards the middle of the population life span (intermediate passage), there are still cells with short generation times, but most cells have division cycles between the two extremes. Since the saturation density is lower, an increased number of cells will not have time to complete their cycle. Thus, during a prolonged labelling with tritiated thymidine (3H-TdR) an increased number of cells will appear as unlabelled. At this stage the slope of the logarithmic portion of the growth curve will be less steep and the cells will reach a lower saturation density, although still within 3-4 days.
343 Towards the end o f the life span (late passage) most cells have very long generation times, and since the saturation density is very low, a still greater number o f cells do not have time to complete the cycle between subcultivation and confluency. Thus, even more cells will remain unlabelled after growing in the presence of 3H-TdR. The growth curve will be characterized by a long lag phase, a period of short logarithmic growth with a shallow slope, and a low saturation density reached only after 7 - 1 0 days. In Fig. 1 the prolongation of G1 and G2 is identical but it is possible that one is predominant. It should be emphasized that when one speaks about prolongation of G1, or G2, it applies only to cells that are moving through the cycle. Since the cells that are arrested due for instance to the effects of saturation density, seem to be all in G1, a distinction should and can be made when measuring the prolongation of the G 1 and G2 periods, otherwise cells that are arrested will be taken as prolonged in G 1. Methods that allow the analysis of cells in motion along the cycle should be distinguished from those measuring only instants [12, 13]. In summary, one can confirmed by direct methods fibroblasts, the spectrum of division to a normal cell cycle
say [7], cell with
that as has already been suggested before [6] and during the decline of the division potential of human activity seems to range from complete inhibition of a progressive increase of cells with long cycles.
REFERENCES 1 P. I. Good, Aging in mammalian cell populations: a review, Mech. Ageing Develop., 4 (1975) 339-348. 2 G. S. Merz, Jr. and J. D. Ross, Viability of human diploid cells as a function of in vitro age, J. Cell Physiol., 74 (1969) 219-223. 3 A. Macieira-Coelho, J. Ponten and L. Philipson, Inhibition of the division cycle in confluent cultures of human fibroblasts in vitro, Exp. Cell Res., 43 (1966) 20-29. 4 V. J. Cristofalo and B. B. Sharp, Cellular senescence and DNA synthesis. Thymidine incorporation as a measure of population age in human diploid cells, Exp. Cell Res., 76 (1973) 419-427. 5 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 583-621. 6 A. Macieira-Coelho, J. Ponten and L. Philipson, The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro, Exp. Cell Res., 42 (1966) 673-684. 7 P. M. Absher, R. G. Absher and W. D. Barnes, Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age, Exp. Cell Res., 88 (1974) 95-104. 8 G. L. Grove and V. J. Cristofalo, The transition probability model and the regulation of proliferation of human diploid cell cultures during aging, Cell Tissue Kinet., 9 (1976) 395-399. 9 A. Macieira-Coelho, Are non-dividing cells present in ageing cell cultures?, Nature, 248 (1974) 421-422. 10 Y. Mitsui and E. L. Schneider, Characterization of fraetionated human diploid fibroblast populations, Exp. Cell Res., in press. 11 A. Macieira-Coelho and J. Pont6n, Analogy in growth between late passage human embryonic and early passage human adult fibroblasts, J. Cell Biol., 43 (1969) 374-377. 12 A. Macieira-Coelho and L. Berumen, The cell cycle during growth inhibition of human embryonic fibroblasts in vitro, Proc. Soc. Exp. Biol. Med., 144 (1973) 43. 13 A. Macieira-Coelho, Cell cycle analysis in mammalian cells, in P. F. Kruse, Jr. and M. K. Patterson, Jr. (eds.), Tissue Culture: Methods and Application, Academic Press, New York, 1973, p. 412.