Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione.
Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.
Miller
A C
AC
Radiation Biochemistry Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5603.
Gafner
J
J
Clark
E P
EP
Samid
D
D
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D019328:Buthionine Sulfoximine; D002453:Cell Cycle; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D012323:RNA Processing, Post-Transcriptional; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D003513:Cycloheximide; D015689:Oncogene Protein p21(ras); D005978:Glutathione