One of the most common biochemical correlates of alkylating agent resistance is an elevation of intracellular levels of the ubiquitous nonprotein thiol, glutathione(GSH). The overall objective of the proposed studies is to evaluate possible mechanism(s) by which GSH levels are increased in drug resistant tumor cells. It is our hypothesis that increased GSH in resistant tumor cells is attributable to drug-induced alterations in the activity of key enzymes or substrates involved in biosynthesis of this important tripeptide. Consequently we plan to focus our initial investigations on changes in what we consider to be three prime candidates: GCS, the rate limiting enzyme in GSH synthesis; gamma-glutamyl transpeptidase (GGT), the initial step in a GSH salvage pathway; and intracellular cysteine, a key amino acid precursor. We have spent the past 12 months developing the requisite assays and cloning and sequencing the cDNA for gamma-glutamylcysteine synthetase (GCS) from human kidney and believe we are now in an unique position to initiate this type of investigation. Specifically, we will compare the activity of these enzymes in select pairs of sensitive and alkylating agent resistant cell lines; correlate differences in enzyme activity with the expression of GCS and GGT genes; determine whether transfection of GCS or GGT cDNAs can alter GSH levels and response to alkylating agents; and examine the effect of buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis being evaluated clinically as a potential modifier of tumor GSH levels, on the regulation and activity of these important regulators of GSH biosynthesis. These evaluations will include assessment of these parameters in lymphocytes from patients participating in our Phase I clinical trial of BSO. This comprehensive investigation of molecular and biochemical mechanisms responsible for the elevation of GSH and their response to GSH modulating agents could contribute significantly to understanding the molecular and cellular pharmacology of this important thiol in the response of cells to anti-neoplastic agents.
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