METALLOTHIONEIN AND CYTOTOXIC DRUG RESISTANCE

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The purpose of this award is the development of a physician scientist in cancer pharmacology. The candidate has demonstrated a potential to contribute to clinical urologic oncology and is committed to an academic career. However, he lacks formal basic science training. The sponsor is a recognized cancer researcher with an established record for training physicians in a basic science environment. The proposed investigation of metallothionein (MT) and cytotoxic drug resistance seeks to train a clinician in the discipline of molecular biology, pharmacology, and experimental therapeutics and to identify new strategies to overcome tumor resistance to chemotherapy. The research proposal is designed to investigate the role of MT in conferring resistance to cis-platin (cDDP) and related anti-cancer agents. cDDP is active against a variety of human tumors and is believed to act by forming covalent cross links in chromosomal DNA. Unfortunately, the development of resistance by tumor cells limits the therapeutic usefulness of cDDP. MT, a cysteine rich protein that protects cells against heavy metal toxicity, is an important candidate to modulate cellular response to cytotoxic drugs, especially those reactive with sulfhydryl groups. We will examine the elevation of MT in drug resistant cell lines utilizing enzyme linked immunosorbent assay (ELISA) using antibodies against MT. Since human MT exists in several isomeric forms, monoclonal antibodies specific for each human isoform will be developed and ELISA will be used to examine if drug-resistant cell lines exhibit increases in any particular isoform of MT. In addition, the sensitivity to cDDP will be determined in cells with MT levels manipulated by established inducers of MT. The mechanism of over expression of MT will be studied by characterizing the MT gene copy number, mRNA level and stability of message of MT. Finally, mRNA and protein levels of MT will be determined in tissue samples from patients with urologic malignancy to evaluate if MT influences responsiveness to cDDP and whether cDDP alters human tumor expression of MT. The University of Pittsburgh is particularly well suited for the development of physician investigators in oncology because of its Pittsburgh Cancer Institute which was recently awarded a Cancer Center Support Grant for three years from the NIH.