The long-term goal of this project is to understand the functions of Chk2 in the DNA damage checkpoint and tumor suppression in breast cancer. Chk2 has recently been identified as a protein kinase involved in the DNA damage and replication checkpoint pathways. Like p53, its function as a potential tumor suppressor gene in breast cancer was indicated through studies of Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype. It is believed that Chk2 plays a critical role in maintenance of genomic stability. Loss of Chk2 functions may contribute to cancer formation or progression in breast cancer. However, the biological significance of Chk2?s role in the DNA damage checkpoint and in tumor suppression for the general population still remains to be elucidated. In this proposal, we will first generate human epithelial cells containing a Chk2 gene knockout for use as a model system. By studying the responses to genotoxic stress and transformation potential in Chk2 null cells, we can define Chk2?s function in checkpoints and in suppressing transformation in human epithelial cells. In addition, we plan to systematically identify the Chk2 substrates and its associated proteins by screening the human expression library and by mass spectrometry. The functional properties of these Chk2 substrates will be further studied by determining their specificity toward different genotoxic stresses. We will also identify the Chk2-associated proteins collected from the cells before or after DNA damage. The differential binding profiles will provide a profound insight into the signals transduced by Chk2 at checkpoints as well as DNA repair. Finally, we will extend our research from studying the molecular mechanisms of Chk2 in cultured cells to its clinical implications. More than two hundred breast cancer specimens will be analyzed for mutations of Chk2 gene or aberrant expression of Chk2 protein. The prognostic value of these Chk2 deficiencies will then be determined. We believe that the mechanisms and targets discovered in our studies will be critical for developing more effective approaches in breast cancer therapy in the future.