This proposal focuses on the checkpoint that monitors DNA damage in G2 and prevents mitosis from occurring until DNA repair is completed. There are distinct checkpoint pathways functioning at the G1/S and G2/M transitions that delay entry into S-phase or mitosis in the presence of DNA damage. Mutations in G1/S check point genes, such as p53, are commonplace in tumors, promoting genomic instability, tumor evolution and abolish apoptotic responses leading to chemo resistance. Mutations in G2 check point genes are extremely infrequent, suggesting they are important for tumor cell viability. The long-term goal of this study is to fully dissect the biology of G2 checkpoint responses, and on the basis of this, to devise and assess targeted anti-cancer therapies, especially for the treatment of tumors bearing mutations in p53. Central to this proposal is a series of experiments that aim to investigate regulation of a key component of this checkpoint, the Chk1 protein kinase. The G2 checkpoints and the cell cycle regulators that they regulate are highly conserved in evolution. Therefore, the experiments described here will be carried out in both human cells, mice, and in the fission yeast Schizosaccharomyces pombe, the organism that has long been used as a paradigm of G2 cell cycle control. To understand mechanisms of Chk1 function and regulation, the genetics of the yeast system will be utilized in a series of screens based on a large collection of both loss- and gain-of- function Chk1 alleles. These experiments will also involve the cloning of genes already identified to alter Chk1 function and the biochemical dissection of checkpoint signaling that builds on this genetics. The mechanism(s) by which checkpoint arrested cells re-enter the cell cycle will also be investigated. In parallel, findings will be recapitulated and expanded upon in human cells in culture, taking biochemical approaches to study wildtype and mutant Chk1, and homologs of proteins identified as modulating Chk1 function in S. pombe. Finally, we will build on data already obtained in cell lines to inhibit Chk1 signaling in tumor cells in the mouse to investigate the utility of G2 checkpoint inhibition as a targeted anti-cancer therapy. The study will yield data that will be important in terms of both the basic biology of the cell cycle and stability of the genome and also in the testing of new approaches and new targets for cancer therapy. ? ?
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