ROLE OF METALLOTHIONEIN IN CANCER AND DRUG RESISTANCE

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This study will elucidate the characteristics of an important trans- activating factor in the control of metallothionein (MT) gene expression, and determine the potential role of this factor in cellular resistance to cadmium toxicity and the acquired resistance of tumor cells to potent anticancer drugs such as cisplatin. Our laboratory has delineated a 26 bp sequence in the promoter region of the mouse MT-I gene, designated MRE-c', that is required for basal transcription. A novel protein factor, MBF-2, that interacts with this sequence and controls MT gene expression has been identified and purified to near-homogeneity. cDNA clones representing this protein have been isolated. The four major specific aims of this proposal are as follows: (l) Complete the characterization of the MBF2 protein including tissue distribution and any post-translational modifications of the purified MBF-2 protein, confirmation that the cDNAs represent the full-length MBF-2 mRNA, and the complete sequence analysis of the cDNA. (2) Investigate whether those cells and tissues that naturally express higher basal levels of MT have more MBF-2, or perhaps more active MBF-2, than others. These experiments will utilize antibodies to MBF-2 and a reporter gene construct to compare MBF-2 function in cisplatin-sensitive and resistant cell lines. (3) Investigate whether increased levels of MBF- 2 expression lead to higher basal levels of MT gene transcription in cultured cells and if so, whether an increased resistance to cisplatin and cadmium is conferred. Expression of the MBF-2 cDNA will be driven from a strong ubiquitous promoter in these experiments. (4) Use transgenic mice to test directly the importance and function of MRE-c' and MBF-2. Mice which overexpress this basal transcription factor could ultimately prove useful as a model system for testing physiological aspects of resistance to other anti-cancer drugs and toxic compounds that might be influenced by MT expression levels. The long-term objectives of this research are to characterize the regulatory factors required for expression of the MT genes, and explore the role of MT in cadmium-induced toxicity/tumorigenesis and acquired cellular resistance to metal- containing anticancer drugs.