Abstract
Ectopic expression of telomerase blocks both telomeric attrition and senescence, suggesting that telomeric attrition is a mitotic counting mechanism that culminates in replicative senescence. By holding human fibroblast cultures confluent for up to 12 weeks at a time, we confirmed previous observations and showed that telomeric attrition requires cell division and also, that senescence occurs at a constant average telomere length, not at a constant time point. However, on resuming cell division, these long-term confluent (LTC) cultures completed 15–25 fewer mean population doublings (MPDs) than the controls prior to senescence. These lost divisions were mainly accounted for by slow cell turnover of the LTC cultures and by permanent cell cycle exit of 94% of the LTC cells, which resulted in many cell divisions being unmeasured by the MPD method. In the LTC cultures, p27KIP1 accumulated and pRb became under-phosphorylated and under-expressed. Also, coincident with permanent cell cycle exit and before 1 MPD was completed, the LTC cultures upregulated the cell cycle inhibitors p21WAF and p16INK4A but not p14ARF and developed other markers of senescence. We then tested the relationship between cell cycle re-entry and the cell cycle-inhibitory proteins following subculture of the LTC cultures. In these cultures, the downregulation of p27KIP1 and the phosphorylation of pRb preceded the complete resumption of normal proliferation rate, which was accompanied by the down-regulation of p16INK4A. Our results show that most normal human fibroblasts can accumulate p16INK4A, p21WAF and p27KIP1 and senesce by cell division-independent mechanism(s). Furthermore, this form of senescence likely requires p16INK4A and perhaps p27KIP1.
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Acknowledgements
The authors are very grateful to H Vaziri for the generous gift of the pBabest2 retroviral construct, to Karen Vousden and Gordon Peters for gifts of p14ARF antibodies, to the Cancer Research Campaign and the European Molecular Biology Organisation for financial support (K Steeghs), to Keith Vass for help with statistical analysis and to John Wyke for critical reading of the manuscript.
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Munro, J., Steeghs, K., Morrison, V. et al. Human fibroblast replicative senescence can occur in the absence of extensive cell division and short telomeres. Oncogene 20, 3541–3552 (2001). https://doi.org/10.1038/sj.onc.1204460
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DOI: https://doi.org/10.1038/sj.onc.1204460
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