Transfection of complementary DNAs for the heavy and light subunits of human gamma-glutamylcysteine synthetase results in an elevation of intracellular glutathione and resistance to melphalan.

Although glutathione (GSH) has long been implicated in resistance to certain common chemotherapeutic agents, including alkylating agents, platinum analogues, and doxorubicin, evidence establishing a direct role in the resistant phenotype has been lacking. We cotransfected COS cells with the cDNAs for the two subunits of gamma-glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting step in the de novo synthesis of GSH and is itself up-regulated in some drug-resistant tumor cells. Transfection resulted in increased GCS activity and elevated GSH levels (up to 2.6-fold). Cotransfection with the two subunits greatly enhanced the synthetic efficiency of the heavy subunit. A direct correlation (P < 0.01) between intracellular GSH levels and the LD99 dose of melphalan was observed, signifying that elevation of the thiol secondary to GCS expression is sufficient to confer the resistance phenotype.