Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity

gamma-Glutamylcysteine synthetase (GCS) catalyses the first step of glutath ione (GSH) biosynthesis and is considered to be thr: rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the c atalytic subunit of GCS. These mutant cells contained a wild-type steady-st ate level of GSH and, after depletion, synthesized GSH at the same rare as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in muta nt cells by 80% but did not increase that of wildtype cells, and, in GSH-de pleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated th at GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cispla tin or nitrogen mustard.