The broad long-term goal of this project is to elucidate the molecular basis of cell cycle checkpoint responses to Polycyclic Aryl-Hydrocarbon (PAH)-induced DNA damage. Cell cycle checkpoints are important tumor suppressive mechanisms that are frequently abrogated in human cancers. Loss of tumor-suppressive checkpoints can play a causal role in the initiation and progression of malignancies. Additionally, cell cycle checkpoints can be exploited therapeutically to treat human tumors. Therefore, a knowledge of checkpoint regulation will further our understanding of mechanisms of tumorigenesis and will impact our ability to treat human cancers.
The specific aims of this work are threefold: (1) To test the hypothesis that an ATM and/or ATR-mediated signaling pathway elicits Chkl phosphorylation and S-phase arrest in response to aryl-hydrocarbons. (2) To test the hypothesis that Chkl substrate proteins are downstream effectors of PAH-induced S-phase arrest. (3) To test the hypothesis that Radl7 and the Radl/Rad9/Husl protein complex mediate DNA damage signaling and cell cycle responses to aryl-hydrocarbons. These studies will test the mechanisms of activation of Chkl by DNA damage signals (SA 1 ). We will also identify the effectors of Chkl signaling that are responsible for PAH-induced S-phase arrest (SA 2). Furthermore, we will test the roles of the putative DNA damage-sensors Rad17, Rad1, Rad9, and Hus1 in regulation of Chkl activity and S-phase arrest (SA 3). We will use 'loss of function' strategies to perturb putative DNA damage signaling pathways in cultured cell lines. These strategies include expression of mutant dominant-negative proteins and the use of cell lines from transgenic animals. We will test the effects of the resulting perturbations on signal transduction pathways and cell cycle responses to PAHs. These experiments will identify novel tumor-suppressive checkpoint control mechanisms that protect against environmental carcinogens. The signaling pathways identified by our studies represent potential new targets for the rational design of chemotherapies to manipulate checkpoint control. Such drugs could be exploited to prevent and treat human cancers.
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