Abstract 5361: DNA damage by platinum-based drugs induces p21-dependent recruitment of PCNA as a homotrimer into the inhibited G1-phase CDK complex
Authors: He Guangan , Kuang Jian , Koomen John , Kobayashi Ryuji , Khokhar Abdul R. , Siddik Zahid H. 
Published: June 2012

DNA damage results in activation of cell cycle checkpoints and cell cycle arrest as a result of inhibition of cyclin-dependent kinases (CDK). In G1 phase, checkpoint activation is often associated with inhibition of CDK4/cyclin D1 and CDK2/cyclin E activities, usually by p21 in a p53-dependent manner. Our investigations in A2780 ovarian tumor cells with cisplatin and 1R,2R-diaminocyclohexane-(trans-diacetato)(dichloro)-platinum(IV) (DAP), a platinum analog that circumvents cisplatin resistance, demonstrated that both agents activated the p53-p21 pathway, which inhibited G1-phase CDK4 and CDK2 kinases, beginning at ∼18 h after drug treatment. However, biochemical analysis of the inhibited complex demonstrated an increase in size by ∼100-150 kDa, which was not consistent with binding of p21 alone and the anticipated stoichiometry of 1 for CDK:p21. In order to account for the p21-dependent increase in the size of the CDK complex, we undertook large-scale immunoprecipitation with antibodies to cyclin D (for the CDK4 complex) or cyclin E (for the CDK2 complex) following DAP treatment, and analyzed the inhibited complex by mass spectrometry for additional recruited proteins. This analysis revealed only one additional protein, namely PCNA, in the inhibited CDK2 and CDK4 complex. As confirmation of p21-dependence, immunodepletion of p21 in boiled cell lysates from DAP-treated cells abolished the binding of PCNA to the CDK complex in a cell-free binding assay. Similar p27 depletion did not prevent the binding of PCNA to the complex. However, PCNA binding with p21 at a stocihiometry of 1 was insufficient to account for the increase in molecular size of the inhibited CDK complex, and it was likely that PCNA was present in its trimeric state. This was confirmed by employing mutant PCNA (Y114A) which could not form the homotrimer and, consequently, was unable to bind to p21 and become recruited into the CDK complex. Moreover, inhibiting the binding of PCNA to p21 did not prevent p21 from inhibiting CDK2, and this indicated that PCNA is not vital for p21-dependent inhibition of kinase activity. On the other hand, knockdown of PCNA by siRNA in A2780 cells potentiated the cytotoxicity of DAP, whereas p21 knockout in HCT-116(p21-/-) cells decreased the sensitivity to this agent. Taken together, our data demonstrate that p21-dependent recruitment of PCNA as a homotrimer substantially increases the molecular size of the CDK complex, but whereas PCNA recruitment does not contribute to p21-dependent inhibition of the kinase, it may attenuate the effect of p21 on drug-induced cytotoxicity. (Supported by NCI RO1 CA127263).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2011-5361