ATR (Ataxia Telangiectasia and Rad3 related) is a member of the phosphatidylinositol-kinase (PIK) related kinase family with homology to human ATM, and the important yeast cell cycle checkpoint genes S. cerevisiae MEC1 and S. pombe Rad3. Experiments from model systems suggest that inhibition of ATR homologues may be particularly toxic to cells with defects in pathways commonly altered in cancers and knockout mice develop tumors at an increased frequency. Work that the applicant has done demonstrates that inhibition of ATR: 1) sensitizes cells to many current anticancer therapies in transformed fibroblasts; 2) is not tolerated in ATM and some cancer cell lines; and 3) ATR protein expression is altered in many cancer cell lines. In this grant application, we propose to: 1. Determine the prevalence and functional significance of ATR gene mutations in cancer. In a logical step-wise fashion, the etiology of altered ATR protein expression in human cancer cell lines will be determined. We will use RT-PCR, Northern blotting, techniques to determine methylation-dependent gene silencing and mutational screening by Denaturing HPLC (D-HPLC). These studies will be extended into primary specimens from cancers from the relevant organ sites and the functional significance of alterations determined in vitro. 2. Identify ATR-dependent genes that are transcriptionally regulated in response to DNA damage and investigate the influence on such response by the presence of other genetic alterations. Using model systems we will determine genes that are transcriptionally regulated in response to S-phase genotoxic DNA stress and determine novel members of ATR-dependent pathways of response. These studies will be extended to facilitate functional analysis of ATR alterations and to examine the functional consequence of ATR inactivation in the context of other genetic alterations. 3. Investigate whether ATR disruption is selectively toxic to cells containing wtATR but harboring alterations common in human cancers. Viral vectors will be constructed that will allow us to disrupt ATR function in many cancer cell lines to test the hypothesis that ATR disruption is more damaging to cells with defects in other DNA damage response pathways. Using xenograft models we will develop a gene therapy model of ATR disruption to test the safety and efficacy of targeting ATR for anticancer therapy.
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| Lewis, Kriste A; Mullany, Sally; Thomas, Bijoy et al. (2005) Heterozygous ATR mutations in mismatch repair-deficient cancer cells have functional significance. Cancer Res 65:7091-5 |
