The fundamental goal of the cell division cycle is to produce two daughter cells that each bears complete and accurate copies of the parental genome. To this end, normal cells contain an elaborate surveillance apparatus whose function is to insure that critical cell-cycle events, such as copying of the genome (a process termed DNA replication) and the subsequent segregation of the duplicated chromosomes into the daughter cells, are performed with the appropriate timing and with high fidelity. The genome is under continuous assault by environmental toxins and the byproducts of normal cellular metabolism, which include potentially mutagenic compounds (estrogen metabolites are a relevant example) and reactive free oxygen species. When genetic damage is detected, these `checkpoint¿ mechanisms halt DNA synthesis and alert the DNA repair machinery to the presence of the DNA lesion. If the damage is irreparable, the cell is instructed to undergo irreversible growth arrest or programmed cell death. Breakdown of cell-cycle checkpoints leads to genetic instability and pathological sequelae ¿ loss of normal cell function and cancer development. Indeed, genetic instability is a universal characteristic of human cancer cells and serves as a major contributing factor to cancer progression from treatment-responsive to incurable disease. The linkage between breakdown of cell cycle checkpoints and cancer development is dramatically underscored by studies of the genes involved in heritable forms of breast cancer. Substantial public attention has been given to the BRCA1, BRCA2, ATM, and CHK2, which, when rendered dysfunctional due to mutation, increase the risk of breast cancer development at an early age. Interestingly, all of the proteins encoded by these genes are components of genome surveillance pathways in normal human cells. It is particularly striking that, while the breast cancer susceptibility proteins function throughout the body, breast cancer appears as a predominant malignancy in humans bearing defects in these proteins. These findings suggest that breast tissue is especially sensitive to mutations that impair the signaling pathways that guard genome integrity. In this project, we will test the idea that the lessons learned from breast cancer families are providing important clues concerning the etiology of the more common, sporadic form of the disease. Our hypothesis is that defects in the surveillance mechanisms that monitor the process of DNA replication in proliferating breast epithelial cells drive the progression of premalignant breast lesions to full-fledged malignant tumors. We are particularly interested in testing one possible scenario that would favor the emergence of breast cancer cells bearing defects in the DNA replication checkpoint. Developing tumors in normal breast tissue inevitably outstrip the ability of the nearby blood vessels to supply sufficient amounts of oxygen and nutrients to sustain growth of the abnormal cell mass. This situation causes the proliferating cells to pause, and we propose that this growth arrest is due in part to a checkpoint-imposed block to continued DNA replication. As the cells adapt to the oxygen-starved (hypoxic) conditions, cells that have negotiated their way past the DNA replication checkpoint acquire a selective growth advantage. This checkpoint bypass simultaneously confers genetic instability, which favors rapid evolution of more aggressive cancer cells. Our goals in this project are twofold: (1) we will define the molecular events leading to suppression of the DNA replication checkpoint in hypoxic breast cancer cells, and (2) we will test the idea that replication checkpoint defects promote the evolution of more advanced breast cancer, but concomitantly mark these cells for sensitivity to certain cancer chemotherapeutic agents.