Novel roles for cyclin -dependent kinases in checkpoint pathway function and prevention of DNA damage

Cyclin-dependent kinases (Cdks) promote cellular proliferation, are often deregulated in human cancers, and are targets of ongoing cancer chemotherapy trials. To better understand Cdk functions in cell cycle regulation, we have investigated the consequences of Cdk inhibition in human cells. We demonstrate that Cdk activity is required to maintain function of the Chk1 pathway, a key component of the response to DNA damage and stalled replication. Chk1 expression was markedly reduced in primary fibroblasts and U2OS osteogenic sarcoma cells by treatment with small molecule Cdk inhibitors or induction of a dominant negative mutant of Cdk2. The findings of decreased Chk1 activity and accumulation of Cdc25A, a protein targeted for degradation by Chk1, confirmed that Chk1 function was impaired. Strikingly, Cdk inhibition triggered a DNA damage response, characterized by the accumulation of activated forms of ATM and Chk2 as well as nuclear foci containing phosphorylated substrates of ATM/ATR, including historic H2AX (γH2AX). Moreover, both Cdk and Chk1 inhibitors enhanced the cytotoxicity of etoposide, a DNA-damaging agent. Time course experiments demonstrated that the bulk of ATM activation, mediated by Cdk inhibitors, followed Chk1 down-regulation. Ectopic expression of Chk1 significantly diminished the induction of γH2AX foci by Cdk inhibitors, indicating that Chk1 down-regulation was necessary to elicit the full phenotype. Conversely, Chk1 RNAi combined with partial inhibition of DNA replication was sufficient to evoke a DNA damage response, while Chk1 down-regulation alone was not, suggesting that replication defects also contributed to the induction of DNA damage. Accordingly, Cdk inhibition disrupted the normal dynamics of the replication complex, as evidenced by increased mid S phase chromatin association of MCM2 and MCM7, members of the putative replicative helicase. These results define a pathway through which Cdk inhibition can mediate DNA damage and potentially enhance the efficacy of extant cancer chemotherapies