ALTERATION OF TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL EXPRESSION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE BY DIETHYL MALEATE

Gamma-glutamylcysteine synthetase (gamma-GCS), also known as glutamate -cysteine ligase (EC 6.3.2.2), is the rate-limiting enzyme in the synt hesis of glutathione (GSH). The gene GLCLC encodes the catalytic subun it while GLCLR encodes the regulatory subunit. Although it has been sh own that GLCLC can respond to a variety of stresses by increased trans cription, it is not known whether a similar response occurs for GLCLR. Nor is it known whether post-transcriptional regulation of either gen e product is altered during stress. The present investigation was unde rtaken to explore transcriptional and post-transcriptional regulation of GLCLC and GLCLR gene products when HepG2 cells were challenged with the radiation sensitizer diethyl maleate (DEM). Expression of steady- state GLCLC and GLCLR mRNA was enhanced 5-20-fold after DEM challenge. Nuclear run-off assays were performed on unstressed and stressed cell s to determine whether the increased expression of GLCLC and GLCLR mRN A was due to altered transcriptional activity of these genes. The DEM treatment increased the transcription rates of both genes 2-5-fold. In unstressed HepG2 cells, the half-life of GLCLC mRNA transcripts was a pproximately 4 h. In contrast, the half-life of GLCLR transcripts was approximately 8 h. In cells treated with DEM, the half-lives of all tr anscripts were increased, indicating that message stabilization contri buted to the increased expression of gene products. Finally, a PEST al gorithm has identified a PEST (proline, glutamate, serine, threonine) motif within the catalytic subunit of gamma-GCS, suggesting that this subunit might exhibit conditional proteolytic regulation. These result s imply that regulation of the products of the GLCLC and GLCLR genes m ay be altered at multiple levels during exposure to stress. (C) 1997 b y Radiation Research Society.