Elevations of intracellular glutathione (GSH) levels are among the earliest and most common biochemical changes associated with inmate or acquired resistance to alkylating agents, analogs of cisplatinum and doxorubicin. Despite a long-standing association between elevated GSH and drug resistance, details of the molecular mechanisms responsible for alterations of GSH homeostasis in resistant tumor cells have remained undefined. We have demonstrated that an increase in GSH concentration in resistant cells is frequently associated with an increase in the activity of gamma- glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting reaction in the de novo synthesis of GSH. We have also established that increased GCS expression involves increased steady-state levels of mRNA for both the heavy (catalytic, GCSh) and light (regulatory, GCS1) subunits comprising the GCS holoenzyme. In the case of the heavy subunit gene (light subunit not yet examined) this over-expression is attributable to an increase in the rate of gene transcription. Further, we have cloned the human GCS catalytic and regulatory subunit genes and sequenced their 5'flanking regions. We have further demonstrated that co-transfection with cDNAs for both subunits results in GSH elevations and increased resistance to the alkylating agent, L-PAM. The long term objective of our studies is to define the molecular pathway(s) associated with the elevation of GSH commonly detected in cells resistant to alkylating agents and platinum analogs. We hypothesize that the frequent increase in GSH levels observed in resistant cells is the result of up-regulation of the rate-limiting enzyme (GCS) in the GSH synthetic pathway in response to drug exposure and involves specific cis- and trans-acting elements which mediate response to challenge with these cytotoxic agents. The specific objective of the proposed investigations is to define those cis- and trans-acting factors which mediate up-regulation of the GCSh and GCSI subunit genes in drug-resistant tumor cells which express increased levels of GSH. To this end we propose to; 1. determine whether co-transfection with GCSh and GCS1 cDNAs results in increased GSH levels and resistance to alkylators and cisplatin using an inducible expression system based on the tetracycline-resistance operon of E. coli to establish a direct relationship between increased GCS activity and drug resistance and to establish the relationship between the two subunits required for expression of resistance; 2. determine whether expression of GCS1 is transcriptionally up-regulated in drug-resistant tumor cells and complete sequencing and characterization of the promoter region of the human GCS1 and GCSh subunit genes; 3. identify cis-acting elements regulating expression of GCSh and GCS1 subunit gene expression in cells resistant to alkylating agents or cisplatinum; and 4. identify specific trans-acting factors expressed in drug-resistant cells which regulate increased transcription of GCS subunit genes. A comprehensive investigation of the molecular mechanisms responsible for the elevation in GSH will contribute significantly to understanding the molecular and cellular pharmacology of this important thiol in the response of cells to anti-neoplastic agents and its role in the evolution of drug- resistance.
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