Nucleoside analogs have been clearly demonstrated to be effective in the treatment of a variety of hematologic malignancies and several have promise for the treatment of solid tumors and retroviral infections. In order to be efficacious, these compounds must be metabolized by enzymes located in cellular targets to the corresponding nucleoside triphosphate. Although many correlative studies have been performed in an attempt to define the relationships between the expression of these enzymes and the sensitivity or resistance of malignant cells to these agents, definitive data on cause and effect relationships among these parameters are lacking. This proposal seeks to elucidate at a basic genetic level factors controlling the expression of several enzymes that are critical to the activation of a number of clinically useful nucleoside analogs. To this end, we will first define factors involved in the regulation of expression of 2'-deoxycytidine kinase (dCK), the enzyme that catalyzes the initial, rate-limiting phosphorylation of many nucleosides. Structural elements of the gene that could modulate its expression at the transcriptional level will be examined using DNase I hypersensitive site analysis and transient transfection assays. In addition, the possible role of post- transcriptional regulation will be evaluated. Secondly, the two 5'- nucleotidases that catalyze nucleoside monophosphate phosphorolysis will be studied. The 5' regions Of the ecto-5'-nucleotidase gene that appear to mediate tissue-specific gene expression will be analyzed and the ability of nucleoside monophosphate analogs to serve as substrates or inhibitors of this enzyme will be documented. The cytoplasmic or soluble 5'- nucleotidase that has been implicated in the phosphorolysis of purine nucleoside analogs, as well as in the inverse phosphorylation of several dideoxynucleosides useful in AIDS treatment, will be purified and the corresponding cDNA cloned. The expression of this enzyme will be measured in a variety of leukemic cells to determine whether it varies in a tissue- or cell-type specific fashion. In addition, each of these enzymes will be overexpressed in leukemic cell lines using both constitutive and inducible promoters to learn whether increased dCK expression as an independent variable augments sensitivity and whether increased 5'-NT activity induces resistance to nucleoside analogs. Finally, sequential alterations in the expression of these enzymes at the mRNA and activity levels will be defined in the leukemic cells of patients undergoing treatment with 2- chlorodeoxyadenosine or fludarabine. We will thus be able to determine whether structural gene alterations or changes in gene expression occur during treatment. These studies will allow us to define the role of these nucleoside and nucleotide metabolizing enzymes in mediating nucleoside analog sensitivity and resistance. In addition, the elucidation of the endogenous factors regulating the expression of these genes could allow us to develop new approaches to selectively manipulating their expression by either pharmacologic or gene transfer methodologies.
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