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

Abstract

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 ...

Metformin‐mediated increase in DICER1 regulates microRNA expression and cellular senescence

“…5A,B,F). In IDH4 cells, senescence is induced by removal of dexamethasone (dex) (Wright et al ., 1989). Dex was removed, and cells were cultured in the presence of metformin for 7 days; metformin treatment reduced the number of SA‐β‐gal‐positive IDH4 cells (Fig.…”

“…IDH4 cells were further supplemented with 1 μg mL −1 dexamethasone (Wright et al ., 1989). WI‐38 human fetal lung diploid fibroblasts (HDFs, Coriell Cell Repositories) were grown in DMEM supplemented with 10% FBS and 1% nonessential amino acids.…”

SEN6, a locus for SV40-mediated immortalization of human cells, maps to 6q26-27

“…However, such SV40-transformed (preimmortal) cells nonetheless die after a period of extended lifespan (Neufeld et al, 1987;Ray and Kraemer, 1992). Permanent reversal of senescence requires one or more cellular mutations as well as persistent expression of large T antigen (Radna et al, 1989;Wright et al, 1989). Two of our laboratories have previously shown that preimmortal cells undergo a rearrangement involving the long arm of chromosome 6 upon immortalization.…”

Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes

“…Although these events are necessary to induce immortality (Wright et al, 1989;Radna et al, 1989) they are still insu cient and a further genetic alteration which activates telomerase (Counter et al, 1992(Counter et al, , 1994) is usually essential. Several human chromosomes have been shown to harbour antiproliferative genes which have been mapped to 1q25 (Karlsson et al, 1996), 1q42 (Karlsson et al, 1996), 2 (Uejima et al, 1995), 3p21 (Rimessi et al, 1994), 4q32-q34 (Ning et al, 1991; O Pereira-Smith ± personal communication), 6q14-q21 (Sandhu et al, 1996), 6q21-qter (Sandhu et al, 1994), 7q31-32 (Ogata et al, 1993), 11p15 (Koi et al, 1993), 17 (Casey et al, 1993), 18 (Sasaki et al, 1994) and Xp11-ter respectively.…”