Bioreductive chemotherapy is based on reductive activation of drugs by enzymes including cytosolic NAD (P) H: quinone oxidoreductases (NQO1 and NQO2) and microsomal NADPH: cytochrome P450 reductase, identification of tumors rich in those enzymes, and differences in oxygen and pH between normal and tumor tissues. Recently, we have identified and cloned the cytosolic glucose regulatory protein (GRP58) that also plays a significant role in activation of drugs. GRP58 catalyzed activation of mitomycin C that led to DNA cross-linking. GRP58 was found overexpressed in many tumors including breast, uterus, lung, and stomach. However, the mechanism of the role of GRP58 in mitomycin C-induced DNA cross-linking remains unknown. A GRP58-null mouse has not been generated, and the in vivo role of GRP58 remains largely unknown. Recent studies have shown that a significant percentage of human individuals with chemotherapy-induced secondary leukemia carry both mutant NQO1 P187S alleles and are deficient in NQO 1 protein and activity. However, it is unknown if the absence of NQO1 contributed to chemotherapy induced leukemia. NQO 1-null mice deficient in NQO 1 protein and activity were generated in our laboratory. The three specific aims of this competing renewal are as follows. 1). Determine the mechanism of the role of GRP58 in mitomycin C-induced DNA cross-linking. This will be achieved by testing the hypothesis that two thioredoxin domains in GRP58 in combination with thioredoxin reductase and/or phosphorylation of GRP58 and/or glycosylation of GRP58 play a significant role in activation of mitomycin C or transport of mitomycin C to the nucleus leading to DNA cross-linking. We plan to use site-directed mutagenesis, transfection, and other assays to achieve goals of this aim. 2). Generate GRP58-null mouse deficient in GRP58 and study the in vivo role of GRP58 in mouse development and mitomycin C toxicity. Embryonic stem cells and homologous recombination will be used to generate GRP58-null mice that will be exposed to mitomycin C and other anti-tumor drugs and analyzed for their sensitivity to DNA and membrane damage. 3). Investigate the in vivo role of NQO 1 in chemotherapy related leukemia; NQO 1-null mice will be exposed to alkylating agents or TOPO II inhibitors alone or with radiation, and the mice analyzed for CBCs, bone marrow, lymph nodes, spleen, and thymus for symptoms of myeloid and lymphoid leukemia and chromosomal aberrations. These studies are expected to provide a mechanism for the role of GRP58 in mitomycin C-induced DNA cross-linking, the in vivo role of GRP58 in mouse development and toxicity of mitomycin C and other drugs, and the in vivo role of NQO 1 in chemotherapy related leukemia.
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