The project seeks to define the regulation of ornithine decarboxylase (DDC), a potential therapeutic target for neoplasia, at the level of gene expression. We intend to explore the role of elevated ODC gene expression in carcinogenesis and in the response of normal and neoplastic cells to drug induced polyamine depletion. We will extend our preliminary finding that an ODC-deficient cell line transfected with a human ODC gene, cloned in our lab, exhibited tumorigenic properties not present in the parental cell line. Normal human fibroblasts, NIH 3T3 cells and human colon and lung carcinoma cell lines will be transfected with the human ODC gene. Transfected cells expressing elevated ODC activity will be characterized with regard to ODC mRNA and enzyme activity levels, and polyamine concentrations. These parameters will be correlated with the cells' growth rate, ability to grow in soft agar, loss of contact inhibition, tumorigenic potential in nude mice, and response to ODC inhibition by difluoromethyl ornithine (DFMO) and bis(ethyl)spermidine (BES). Cells will also be transfected with ODC gene expression under the control of an inducible, heterologous promoter, and the same properties assayed in induced and non- induced cells. We propose to examine mechanisms of ODC gene regulation by characterizing, structurally and functionally, the promoter elements involved in constitutive and induced ODC gene transcription. Structure analysis will include nucleotide sequencing of the ODC gene 5' flanking region, mapping the transcription initiation site, and identifying sequences that interact with DNA-binding proteins. Such proteins may themselves be future chemotherapeutic targets to manipulate ODC gene expression and regulate polyamine synthesis. Promoter function will be analysed by monitoring the ability of normal and mutated ODC promoter sequences to drive transcription of a chloramphenicol acetyltransferase (CAT) reporter gene in transfected cells. Some mutated ODC promoter sequences will be re-ligated to the ODC structural gene; and the effect of the altered ODC expression on cell growth, transformation and response to ODC inhibition by DFMO and BES in cells in culture and in nude mice xenografts will be examined.
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