Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.
Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the 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 catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate 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 mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.
Physicochimie et pharmacologie des macromolécules biologiques, Institut Gustave Roussy, Villejuif, France.
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Vincent
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Mousset
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Jacquemin-Sablon
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004351:Drug Resistance; D004792:Enzyme Precursors / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis* / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry*; D006706:Homeostasis; D007700:Kinetics; D008168:Lung; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation
D000970:Antineoplastic Agents; D004792:Enzyme Precursors; C065566:buthionine sulfoximine ethyl ester; D008717:Methionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine