METHOTREXATE UPTAKE AND METABOLISM IN HEPATIC CELLS

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The polyglutamate derivatives of antifolates have been established as being a major determinant in the therapeutic efficiency of this class of antitumo agents. The formation of these derivatives has been characterized in a number of transformed cell lines and with isolated folypolyglutamate synthetase. In contrast, the process of cleavage of these y-glutamyl derivatives has not yet been evaluated in sufficient depth or detail. The focus of this study is the enzyme that catalyzes this process, y-glutamyl hydrolase (GH). Studies in the previous grant period have established a number of features about this enzyme: 1) it is a glycoprotein (Mr-55 kDa) with a peptide Mr of 33kDa, 2) it is primarily a secreted enzyme (in all tumor cells tested) whose primary cellular location is the lysosome, 3) an intracellular elevation in its activity is a mechanism of acquired drug resistance, 4) its elevation has a more profound effect in diminishing cellular antifolate polyglutamates than folyl polyglutamates, and 5) a cDNA for GH from rat hepatoma has been cloned and characterized and the deduced amino acid sequence established. A detailed evaluation of GH will be made in order to establish its role in the pharmacology of the antifolat including 1) further studies on GH gene and factors that alter its expressi , 2) the use of site directed mutagenesis and detailed physical and biochemical analysis to understand the structure of the glycoprotein and it mechanism of action, 3) determining the mechanism of alteration in enzyme activity in acquired drug resistance and in response to insulin and other perturbations in tissue culture conditions, 4) evaluating the cytolocalizat n of GH by microscopy using polyclonal antibodies and investigating its cellular trafficking, and 5) using molecular methodology to enhance or inhibit GH levels in cells and evaluate the outcome in terms of the effect antifolate activity and polyglutamylation, GH function and folate growth dependence. These investigations will be conducted to determine if GH can be exploited as a target or if the modification of its function can be util ed to enhance the therapeutic selectivity of the antifolates. Lastly, the secretion of GH will be examined in detail in order to understand its biological and pharmacological significance and to develop the tools to determine its potential as a diagnostic marker.