Sequential extension of proliferative lifespan in human fibroblasts induced by over-expression of CDK4 or 6 and loss of p53 function.
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 approximately 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 p21(Waf1) levels but with up to a 19-fold increase in p16(Ink4a). Surprisingly however, this could not explain arrest, since expression of mutant CDK4 and/or CDK6, incapable of binding p16(Ink4a), did not confer any greater lifespan extension than the wild-type CDKs. Subsequent abrogation of p53 function by a second vector, encoding HPVE6, downregulated p21(Waf1) 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 p21(Waf1) or p16(Ink4a); and (ii) provide an in vitro model of clonal evolution through successive dysfunction of Rb and p53 pathways in a relevant human cell context.
Morris
Mark
M
Cancer Research Campaign Laboratories, Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Hepburn
Peter
P
Wynford-Thomas
David
D
D017209:Apoptosis; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002472:Cell Transformation, Viral / Q000235:genetics; D016922:Cellular Senescence / Q000235:genetics*; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000502:physiology*; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D016161:Genes, Retinoblastoma; D019942:Genes, p16; D016158:Genes, p53; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D008954:Models, Biological; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000502:physiology; D050725:Papillomavirus E7 Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000502:physiology*
C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; C059731:oncogene protein E7, Human papillomavirus type 16; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases