GEMZAR--MECHANISMS OF CYTOTOXICITY & RADIOSENSITIZATION

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Gemcitabine is a new nucleoside analog with clinial activity in several solid tumor types, including pancreatic and non-small cell lung cancer. While gemcitabine has shown good activity as a single agent or in combination with other chemotherapeutic agents in patients with these tumor types, we have demonstrated recently that gemcitabine also enhances the sensitivity of solid tumor cells to ionizing radiation. Preclinical studies demonstrate that gemcitabine can radiosensitize human tumor cells derived from pancreatic cancer, colorectal carcinoma, head and neck cancer, breast cancer and glioblastoma. Translation of these studies to a Phase I trial in patients with unresectable head and neck cancer demonstrated that gemcitabine can sensitize tumor tissue in vivo as well, with most patients achieving a complete response to treatment. With these encouraging results, we now propose to extend these studies both in vitro and in vivo to determine the mechanism by which gemcitabine radiosensitizes tumor cells and determine whether it is distinct from the mechanism of cytotoxicity. These studies will be performed in human head and neck cancer cells, based on our encouraging preliminary Phase I results, and we will also evaluate response of human glioblastoma cells to radiosensitization with gemcitabine based radiosensitization in vitro. Preliminary data suggests that radiosensitization with gemcitabine requires a substantial decrease in dATP, due to inhibition of ribonucleotide reductase, and not affected by the amount of gemcitabine triphosphate or the amount of drug in DNA. Furthermore, new data indicates that radiosensitization is less successful in cell lines that express wild-type p53. The studies proposed here will evaluate the roles of dATP depletion and gemcitabine nucleotide in DNA in cytotoxicity versus radiosensitization. The effect of p53 will be assessed using matched wild type and mutant p53 cell lines, as well as eliminating wt p53 using the E6 protein of human papillomavirus. These studies will be performed these cell lines in vitro and in vivo in nude mice. In addition, infusion of gemcitabine in patients prior to surgical removal of tumors will allow measurement of critical proteins required for gemcitabine metabolism, phosphorylation of gemcitabine, effect on ribonucleotide reductase and p53 status. These studies will bridge the gap between in vitro and in vivo studies and help to optimize radiosensitizing therapy with gemcitabine.