DNA damaging agents in the form of radiation and chemotherapeutic drugs are commonly used for treatment of human cancers. Understanding the cellular response to DNA damage is crucial for improving the therapeutic effects of these treatment modalities. It is established that the treatment-induced DNA damage does not kill cancer cells directly. Instead, they initially activate one or multiple cell cycle checkpoints and subsequently induce programmed cell death. Accumulating evidence indicates that the G2 phase checkpoint response is antagonistic to the cell killing effect of DNA damaging agents and that abrogation of the G2 phase checkpoint may improve the outcome of chemotherapy. Cisplatin is a widely used DNA damaging agent in chemotherapy. One of the major problems in cisplatin-based chemotherapy is the acquired. drug resistance. DACH-acetato-Pt (DAP) is a novel cisplatin analog with great clinical potential to overcome cisplatin resistance in cancer cells. For a more effective use of this novel drug in future chemotherapy, PI?s group have been undertaking studies to elucidate the cellular responses this new agent elicits and to identify the mechanisms crucial for its unique antineoplastic activity. In their previous studies, they compared effects of DAP and cisplatin on the cell cycle checkpoints. These studies clearly demonstrated that DAP and cisplatin have dramatically different effects on the G2 phase checkpoint. While cisplatin predominantly inhibits G2 phase progression, DAP has little effect on it. This difference raises the possibility that silencing the G2 phase checkpoint response may contribute to the unique antineoplastic activity of DAP. They recently discovered that DAP is a highly potent inducer (-100 fold increases) of the universal Cdk inhibitor p2lWafl/Cipl (p21) via a p53-dependent mechanism. However, while the induced p21 binds both Gi phase and G2 phase Cdk complexes, it only inhibits G I phase Cdk activities. This suggests that DAP may activate an as yet unidentified mechanism that prevents the induced p21 from inhibiting 02 phase Cdk activities. In their effort to uncover this novel mechanism, they discovered that DAP appeared to induce dephosphorylation of Cdc2 kinase at sites that are distinct from those already known to regulate Cdc2 kinase activity. In contrast, they found that cisplatin appeared to induce phosphorylation of these sites along with its inhibition of the Cdc2 kinase activity. These novel preliminary findings lead them to hypothesize (i) that phosphorylation of Cdc2 kinase at the uncharacterized sites is a prerequisite for the induced p21 to inhibit Cdc2 kinase activity; (ii) that DAP-induced dephosphorylation of Cdc2 kinase at these sites prevents the DNA damage-induced G2 phase checkpoint response; and (iii) that cisplatin-induced phosphorylation of Cdc2 kinase at these sites promotes the cisplatin-induced G2 phase checkpoint response. To test these hypotheses, three specific aims are proposed. First, the uncharacterized sites in Cdc2 that are hypophosphorylated in DAP-treated cells and hyperphosphorylated in cisplatin-treated cells will be identified through mass spectrometry and confirmed through site directed mutagenesis. Second, the kinase activity that phosphorylates Cdc2 at these sites will be identified from crude cell lysates by biochemical fractionations and its effect on the Cdc2 kinase activity from DAP treated cells will be determined. Third, the novel Cdc2 phosphorylation sites will be mutated to nonphosphorylatable amino acid residues and the effect of the mutants on cisplatin-induced G2 phase checkpoint response will be examined by using transfected cells. The proposed studies will advance our understanding of the molecular pharmacology of DAP and cisplatin. In addition, they may fill a gap in our understanding of the molecular pathways involved in DNA damage-induced G2 phase checkpoint response, and provide new strategic approaches to abrogate G2 phase checkpoint response.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Experimental Therapeutics Subcommittee 1 (ET)
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Forry, Suzanne L
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University of Texas MD Anderson Cancer Center
Other Domestic Higher Education
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