Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence.

Orderly cell cycle progression of mammalian cells determines their ability to undergo regulated proliferation, differentiation, senescence, and apoptosis (43, 44). Perturbations in the expression and activities of proteins that regulate these cellular phenomena determine the emergence of a cancerous state. Two important tumor suppressor pathways, which depend on the expression and activity of two key cell cycle regulatory proteins, pRb and p53, serve as critical branch points that govern controlled cell growth (14, 17, 33, 43, 44, 49). In concordance with this, it has been observed that a majority of human tumors harbor mutations that debilitate the surveillance function of the pRb and/or p53 pathways (43, 49). Moreover, patients with inactivating mutations in both pathways have a much worse prognosis compared to patients with inactivation in just one of the two pathways. While the consequences of inactivation of p53 on cancer development has been exhaustively studied using mice that are nullizygous at the p53 loci, it has been difficult to examine the effects of simultaneous inactivation of the retinoblastoma (Rb) family proteins on cancer progression, as homozygous inactivation of both Rb alleles leads to early embryonic lethality (5, 9, 19, 27). This requirement of the Rb family proteins during embryogenesis and early development has precluded the examination of their role during adult development and on cancer predisposition by using conventional gene disruption techniques (5, 6, 19, 25–28). Since homozygous inactivation of Rb leads to early embryonic lethality, studies focused on examination of the role of Rb family proteins in development and cancer have been restricted to characterization of mouse models that either (i) harbor heterozygous inactivation of Rb family genes, (ii) harbor a heterozygous inactivation of Rb family genes in addition to disruption of genes that code for other key cell cycle regulators, such as members of the cyclin kinase inhibitor (CKI) family, or (iii) have inactivation of the Rb family proteins via expression of viral proteins, such as papillomavirus protein E7 or the simian virus 40 large T-antigen protein (14, 17). However, the unequivocal importance of the retinoblastoma proteins in regulating cell cycle progression and cell proliferation and its underlying effects on immortalization and tumorigenesis has been elucidated by studies that describe the disruption of the retinoblastoma family gene loci in fibroblasts (7, 39). Loss of expression of pRb, p107, and p130 proteins in fibroblasts resulted in increased cell proliferation, shortening of the cell cycle, and immortalization. However, these studies do not allow an extrapolation of the effects of simultaneous inactivation of the retinoblastoma proteins on adult development and cancer predisposition. Cdk4, along with Cdk6, is the chief catalytic subunit of the regulatory cyclin D family of proteins that govern G1-to-S phase progression of mammalian cells via phosphorylation and inactivation of retinoblastoma family proteins (43, 44). Members of the INK4 family of proteins, chiefly p16Ink4a, are specific inhibitors of the cyclin D/Cdk4 complexes (43, 44). Mutations in CDK4 and its key kinase inhibitor p16INK4A have been implicated in the genesis and progression of familial human melanoma (20, 31). The importance of the CDK4 locus in human cancer was further emphasized upon identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4A, predisposing humans to hereditary melanoma (51, 53). This observation suggests that a mutant CDK4 gene can function as a dominant oncogene that is resistant to normal physiological inhibition by p16INK4A. To determine the consequence of the R24C germ line mutation on mouse development and cancer susceptibility, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated “knock-in” technology (37). The presence of the Cdk4R24C mutation induced hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130, and mouse embryo fibroblasts (MEFs) derived from Cdk4R24C/R24C mice escape from replicative senescence and become insensitive to contact-induced growth arrest. Moreover, the presence of the Cdk4R24C mutation resulted in transformation in vitro and spontaneous and chemical carcinogen-induced tumorigenesis in vivo, suggesting that the Cdk4R24C mutation can serve as the primary event in the progression towards a fully transformed phenotype.