Cyclin-dependent kinase 4 (Cdk4) is an important regulator of G1/S cell cycle progression of mammalian cells in culture. Germline mutations in the 24th codon of this gene (R to C) results in a predisposition of the individuals to the development of melanoma. To understand the role of this enzyme in vivo, we have targeted the mouse cdk4 locus by homologous recombination in embryonic stem cells and generated strains of mice that either lack Cdk4 expression (Cdk4 neo/neo) or express an activated form of this enzyme (Cdk4 R24C/R24C), which cannot interact with the Cyclin Kinase Inhibitor p161NK4A. Homozygous Cdk4 (Cdk4 neo/neo) null mutant mice are viable but express defects in growth, spermatogenesis and oogenesis. In addition, these mice were found to be diabetic exhibiting defective pancreatic beta-cell development, polyuria and polydypsia. In contrast, Cdk4 R24C/R24C mice exhibit a growth advantage, and pancreatic beta-cell hyperplasia which resembled insulinomas. Moreover, these mice spontaneously develop tumors of varying cellular origin, clearly demonstrating the consequence of de-regulated Cdk4 mediated cellular pathways in the progression of various types of cancer. To gain further insight into the role of Cdk4 in cell cycle regulation and neoplasia, we propose: (1) To carry out a detailed characterization of MEFs and lymphocytes derived from cdk4 (R24C/R24C) mice to determine the effect of R24C mutation on cell cycle kinetics, ability of embryonic fibroblasts to undergo senescence, susceptibility of fibroblasts and lymphocytes to apoptotic stimuli; and susceptibility of MEFs to neoplastic transformation by oncogenes. (2) To extend experiments described in Aim 1 to whole animal model systems by carrying out experiments aimed at determining the molecular basis for the enhanced susceptibility of the Cdk4 R24C/R24C mice to the development of carcinomas following treatment with chemical carcinogens such as TPA and/or DMBA. (3) To investigate the collaboration between the Cdk4-pRb and p53 pathways, using Cdk4neo/neo and Cdk4R24C/R24C mice and mice deficient in p53. (4) To test the co-operating effects of ras activation and pRb inactivation by examining the tumor development characteristics of mice that harbor both mutations by crossing Cdk4R24C/R24C mice with mice that carry an activating germline mutation in the ras gene.
