Sequential extension of proliferative lifespan in human fibroblasts induced by over-expression of CDK4 or 6 and loss of p53 function

Abstract

Replicative senescence is thought to be a signi®cant barrier to human tumorigenesis, which in human ®bro-blasts, and many other cell types, can be overcome experimentally by combined loss of function of p53 and Rb`pathways'. To avoid the confounding pleiotropic e ects 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 ®broblast 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 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.

Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells

“…Wynford-Thomas (Morris et al, 2002) has recently reported that a Cdk4 mutant, R24C, was unable to cooperate with hTERT to immortalize normal human keratinocytes, and in addition resulted in the production of so much functional p53 that inactivating it was also required before the cells became immortal (Morris et al, 2002). This apparent contradiction of our current results can be explained by the different properties of wild-type and mutant Cdk4s.…”

A CDKN2A Mutation in Familial Melanoma that Abrogates Binding of p16INK4a to CDK4 but not CDK6

“…If both pRb and p53 are inactivated, the cells proliferate until they reach the M2 phase of senescence where continued cell proliferation is offset by cell death (13,14). If only one of the tumor suppressor pathways is disabled, then HDFs senesce at a stage that is operationally between M1 and M2, referred to as M INT or M1.5 (18)(19)(20). In line with these ideas, we have previously shown that p16…”

Enhanced malignant tumorigenesis in Cdk4 transgenic mice

“…While the CDK4(R24C) mutated form appears exclusively in some cases of sporadic and familial melanomas (Ohta et al, 1994;Wo¨lfel et al, 1995;Zuo, 1996;Soufir et al, 1998), amplification and/or overexpression of CDK4 have been found in a wide spectrum of human tumors (He et al, 1995;Holland et al, 1998;Kanoe et al, 1998;Nikitakis et al, 2002). Surprisingly, Morris et al (2002) have shown that the CDK4(R24C) produced no additional growth advantage compared with CDK4 overexpressing forms in human fibroblasts; indeed CDK4(R24C) was less effective than wild-type CDK4 (Morris et al, 2002). These results support the observation that CDK4(R24C) mutation is restricted to melanoma development where escape of the p16 Ink4a inhibitory effect appear to be indispensable, while overexpression of CDK4 may be a more general mechanism for an acquired growth advantage (He et al, 1994;Schmit et al, 1994;Sonoda et al, 1995;Kanoe et al, 1998;An et al, 1999;Dei Tos et al, 2000;Lam et al, 2000).…”