Proliferative capacity and DNA content of aging human diploid cells in culture: A cytophotometric and autoradiographic analysis

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in Gl. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (MI) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed MI mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active MI mechanism.Normal diploid cells exhibit a finite proliferative capacity in culture (13-16, 25, 32, 43), characterized by a decreasing mitogen responsiveness (33) and an eventual arrest in GI. The frequency with which cells from different species escape cellular senescence and give rise to immortal cell lines varies extensively (23). Whereas mouse cells immortalize with a high probability (6,19,24,29) (2, 3,18,26,34,36,39,41). The role of T antigen in the maintenance of immortality of human cells has not yet been determined. Because the mechanisms regulating senescence in rodent and human cells are different, this study was undertaken to examine this question. We report that the continued expression of T antigen is required for the proliferation of T-antigen-immortalized human fibroblasts and discuss a model suggesting why it is misleading to say that T antigen is necessary for the maintenance of immortality. A similar observation for a temperature-sensitive T antigen is presented in the accompanying paper (40). METHODSCells and media. IMR-90 normal human fibroblasts (ATCC 186) were maintained in a 4:1 mixture of Dulbecco modified Eagle medium-medium 199 supplemented with 10% defined supplemented bovine calf serum (Hyclone Laboratories, Logan, Utah). When necessary, serum was depleted of steroids by being stirred overnight at 4°C with 5 mg of sterile activated charcoal (Norit A) per 100 ml of serum (10). After removal of the charcoal by spinning at 25,000 x g for 60 min, the serum was decanted and stored at -10°C. Plasmids (MCDB 202 [11]) supplemented with 400 ,ug of G418 per ml and 10-6 M dexamethasone and placed in modular incubators (gassed with 1% oxygen-5% carbon dioxide-94% nitrogen to increase ...

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“…Since Ml is postulated to be the mechanism responsible for normal cellular senescence, and senescent cells are arrested in G1 (9,42,46,50) …”

Section: Resultsmentioning

confidence: 99%

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

1989

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“…Senescent or presenescent ®broblasts have typically been characterized to arrest in G1 (Yanishevsky et al, 1974). Therefore, we analysed EJp21 cells for changes in cell cycle progression after induction of p21…”

Section: Ectopic Expression Of P21mentioning

confidence: 99%

p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53

et al. 1999

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We have shown previously that wild type p53 can rapidly induce replicative senescence in EJ human bladder carcinoma cells lacking functional p53. A major eector of p53 functions is p21, a potent cyclindependent kinase inhibitor. p21Waf1/Cip1/Sdi1 has been shown to be involved in both p53 dependent and independent control of cell proliferation, dierentiation and death. To directly investigate the eects of p21Waf1/Cip1/Sid1 in the p53 response observed in EJ tumor cells, we established p21Waf1/Cip1/Sdi1 inducible lines using the tetracyclineregulatable vector system. p21Waf1/Cip1/Sdi1 induction caused irreversible cell cycle arrest in both G1 and G2/M, and diminished Cdk2 kinase activity. In addition, p21Waf1/Cip1/ Sdi1 induction led to morphological alterations characteristic of cells undergoing replicative senescence with morphological, biochemical and ultrastructural markers of the senescent phenotype. Furthermore, sustained p21Waf1/Cip1/Sdi1 induction sensitized EJ cells to apoptotic cell death induced by mitomycin C, a cross-linking DNA damaging agent. These ®ndings support the function of p21Waf1/Cip1/Sdi1 as an inducer of replicative senescence and a major mediator of this phenomenon in response to p53. Moreover, our results imply that therapeutic intervention in human cancers might be aimed at sustained elevation of p21Waf1/Cip1/Sdi1 expression.

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“…Normal senescent cells are blocked primarily in a G 1 -like phase (30,89) but can be induced to enter a single round of replicative DNA synthesis in response to SV40 T antigen (29,39,80). Flow-cytometric analysis was employed to determine the cell cycle distribution of PBA HDF, as well as to examine whether they are responsive to mitogenic stimulation by SV40 T antigen.…”

Section: Fig 1 T Antigen Versus Rbmentioning

confidence: 99%

Human Fibroblast Commitment to a Senescence-Like State in Response to Histone Deacetylase Inhibitors Is Cell Cycle Dependent

Ogryzko

Hirai

Russanova

et al. 1996

Molecular and Cellular Biology

244

153

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Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G 1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.Cellular senescence in human diploid fibroblasts (HDF) and other human cell types has been extensively studied (17,26,35,53,73,85) yet remains incompletely understood. On the basis of current knowledge, it is not unreasonable to suppose that multiple mechanisms contribute to the senescence phenotype and that the relative contributions of such mechanisms may vary for different cell types and conditions of cell propagation. At least two senescence-associated changes, increased oxidative stress (74, 75) and telomere shortening (33,34,55,87), have been intensively studied and are widely viewed as important components of in vitro aging. Less well studied is a third aspect of senescence, namely, its apparent relatedness to differentiation (4,16,26,48,57). Although these two processes are not necessarily synonymous (60, 88), in many respects senescence resembles a partial or aberrant form of terminal differentiation, with cells appearing to acquire a phenotype that is suboptimal for tissue function and maintenance. HDF proliferative potential is dependent primarily on the number of rounds of DNA synthesis completed rather than the cumulative time in culture (17,19,27); thus, to the extent that senescence is akin to terminal differentiation, it most closely resembles model systems (e.g., murine erythroleukemia or promyelocytic leukemia cells) where a requirement for passage through the cell cycle has been demonstrated (7,45,46).Our attention to the differentiative aspects of senescence was first prompted by experiments designed to compare cell cycle arrest mechanisms in senescent and quiescent cells. Transient expression of simian virus 40 (SV40) T antigen is strongly mitogenic, i.e., is dominant over cell cycle arrest mechanisms, in both senescent cells and serum-deprived quiescent fibroblasts (29,39,80). We and others observed that in senescent HDF, but not quiescent cells, the domain in T antigen which mediates binding to retinoblastoma (Rb) family proteins is required for efficient stimulation of DNA synthesis (8a, 65). This...

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Proliferative capacity and DNA content of aging human diploid cells in culture: A cytophotometric and autoradiographic analysis

Cited by 132 publications

References 13 publications

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53

Human Fibroblast Commitment to a Senescence-Like State in Response to Histone Deacetylase Inhibitors Is Cell Cycle Dependent

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