Abstract
Replicative senescence is thought to be a significant barrier to human tumorigenesis, which in human fibroblasts, and many other cell types, can be overcome experimentally by combined loss of function of p53 and Rb ‘pathways’. To avoid the confounding pleiotropic effects of HPVE7 frequently used in such studies, here we have employed retroviral vectors over-expressing CDK4 or CDK6 as a more representative model of naturally-occurring mutations targeting the Rb pathway. We show that these can extend fibroblast lifespan by ∼10 population doublings, ending in a viable senescence-like state which contrasts with the apoptotic end-stage seen with E7. Compared with ‘normal’ senescence, this growth arrest was, in most cases, not accompanied by any further increase in p21Waf1 levels but with up to a 19-fold increase in p16Ink4a. Surprisingly however, this could not explain arrest, since expression of mutant CDK4 and/or CDK6, incapable of binding p16Ink4a, did not confer any greater lifespan extension than the wild-type CDKs. Subsequent abrogation of p53 function by a second vector, encoding HPVE6, downregulated p21Waf1 and conferred a second lifespan extension, ending in a crisis-like state, consistent with full escape from senescence. These data: (i) point to a back-up ‘senescence’ mechanism distinct from induction of p21Waf1 or p16Ink4a; and (ii) provide an in vitro model of clonal evolution through successive dysfunction of Rb and p53 pathways in a relevant human cell context.
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Acknowledgements
We are grateful to the Medical Research Council for grant support, to Jane Bond, Fiona Wyllie and Julia Skinner for technical advice, and to Theresa King and Julia Parmar for manuscript preparation. We also thank Dr Jiri Bartek and Dr Manuel Serrano for gifts of antibodies and plasmids and Dr Gordon Peters for his very helpful advice throughout the work
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Morris, M., Hepburn, P. & Wynford-Thomas, D. Sequential extension of proliferative lifespan in human fibroblasts induced by over-expression of CDK4 or 6 and loss of p53 function. Oncogene 21, 4277–4288 (2002). https://doi.org/10.1038/sj.onc.1205492
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DOI: https://doi.org/10.1038/sj.onc.1205492
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