Regulation of the cell cycle by cyclin-dependent kinases

Clinical Sciences

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Abstract

The cell cycle is regulated by the activities of cyclin-dependent kinases (Cdks). In mammals, 11 different Cdks have been identified whereas in yeast only one major Cdk drives the cell cycle. Of the 11 mammalian Cdks, Cdk4 and Cdk6 promote entry and progression through G1, Cdk2 functions in entry and progression through S-phase, Cdc2 (Cdk1) regulates M-phase, and the other Cdks function in transcription or have unknown functions. The activities of Cdks are regulated by protein-protein interactions (with cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, subcellular localization, and multiple phosphorylations. Several functions for Cdk2 have been suggested, including entry into S-phase by Rb phosphorylation, initiation of DNA replication, exit from S-phase, and progression through G2 phase. We have generated Cdk2 knockout mice and found that they are viable but sterile. Our results demonstrate that Cdk2 is essential for meiosis but not mitosis in vivo.- Aim 1:Functions of Cdk2 during tumorigenesis.- Aim 2:Genetic and biochemical analysis of Cdk2 pathways.- Aim 3:Analysis of the Cdc2 locus in the mouseAim 1:Functions of Cdk2 during tumorigenesis.In tumors, the regulation of the cell cycle is altered to allow for increased proliferation. We are now studying the loss of Cdk2 during tumorigenesis. Four different models will be employed: (1) chemically induced skin tumor model, (2) chemically induced liver tumor model, (3) gamma irradiation model, and (4) a loss of p53 induced model. For the skin tumor experiments (1), mice at 7 to 8 weeks of age were shaved and up to 400 nmoles of 9,10,-Dimethyl-1,2-Benzanthracene [DMBA] were painted on the skin. Starting 10 to 20 days later twice weekly doses of up to 10 nmoles of 12-O-tetradecanoylphorbol-13-acetate [TPA] was painted on the shaved areas. Papillomas appeared between 7 to 10 weeks after the first dose of TPA. After 20 weeks, the number of papillomas per mouse averaged between 10 and 15, which were collected along with normal skin samples after the mice were euthanized. All skin samples will be analyzed for genetic mutations in the Ras gene by PCR and at the same time protein levels of cell cycle regulators will be investigated by Western blots or immunohistochemistry. We have done a pilot experiment and are currently repeating it with a larger cohort of mice. Our results indicate that Cdk2-/- mice develop less papillomas than wild type mice and therefore are less susceptible to skin tumors. In order to study if effect of Cdk2-/- is specific to skin tumors, we will investigate other tumor models in the Cdk2-/- background. We will use a liver tumor model, gamma irradiation, and a p53 model. We have generated double mutant mice lacking Cdk2 and p53 and these mice are viable. So far, we have only few animals and we will start the analysis of these mice soon.All these experiments aim to investigate the efficiency of cancer therapy by inhibiting Cdk2. This is a very important link between our experiments and therapeutical intervention.Aim 2:Genetic and biochemical analysis of Cdk2 pathways.Cdk2-/- mice did not display a sever phenotype. Therefore, we have to define the pathways in which Cdk2 is involved in more details. We are approaching these questions by mouse genetics and biochemistry. Double knockout mice of Cdk2 and Cdk4 or p27 are being generated at this moment. So far we have determined that Cdk2-/-p27-/- double mutants are viable and that these mice display all the p27-/- as well as Cdk2-/- phenotypes. Therefore, Cdk2 did not rescue the p27-/- phenotypes and there must be other targets of p27. We have identified Cdc2 are an important target of p27 and have shown that Cdc2 can regulate S phase in the absence of Cdk2. Cdc2 achieves this by interacting with cyclin E & A and forming an active kinase. These important results have been published in Nature Cell Biology.