Posttranscriptional Regulation ofMRP/GS-XPump and γ-Glutamylcysteine Synthetase Expression by 1-(4-Amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl)-3-nitrosourea and by Cycloheximide in Human Glioma Cells

doi:10.1006/bbrc.1997.7423

Biochemical and Biophysical Research Communications

Volume 239, Issue 1, 9 October 1997, Pages 51-56

https://doi.org/10.1006/bbrc.1997.7423 Get rights and content

Abstract

Treatment of human glioma A172 cells with 1-(4amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethy-3-nitrosourea (ACNU) for 2 to 4 hr resulted in a 2- to 3-fold increase in steady-state levels of multidrug resistance-associated protein (MRP) and γ-glutamylcysteine synthetase (γ-GCS) mRNA. Nuclear run-on assays revealed a less than 0.5-fold increase in transcription rates of these genes under the same treatment conditions, suggesting that posttranscriptional regulation plays an important role for the increased mRNA levels. In the absence of ACNU, rates ofMRPandγ-GCSmRNA degradation were similar in A172 cells as determined by incubating cells with the RNase inhibitor, Actinomycin D. ACNU treatments resulted in increasedMRPmRNA stability. Induction ofMRPandγ-GCSmRNA by ACNU apparently did not requirede novoprotein synthesis as determined by the use of protein synthesis inhibitor cycloheximide (CHX). However, CHX alone could induce accumulation ofγ-GCSmRNA, also by posttranscriptional mechanism. Taken together, these results demonstrate that (i) posttranscriptional regulation is primarily involved in the induction ofMRPandγ-GCSexpression by ACNU and CHX in human glioma cells; and (ii) despite the fact that these two genes have been reported to be frequently co-expressed, their responses to the treatments of RNA and protein synthesis inhibitors are not the same.

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Trivalent arsenite (As³⁺) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and the cytotoxic effects of As³⁺. Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As³⁺ coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation, and activity. As³⁺ increased the rate of transcription of both the Gclm and the Gclc genes and induced the posttranscriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As³⁺-induced Gclc expression and attenuated induction of Gclm. As³⁺ induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and posttranscriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As³⁺ and highlight the potential importance of the GSH antioxidant defense system in regulating As³⁺-induced responses in hepatocytes.
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Glutathione (GSH) is the primary nonprotein thiol in the cell. It has many important roles in cell function, including regulating redox-dependent signal transduction pathways. The content of GSH within the cell varies with stress. In many cases, a process involving GSH synthesis results in adaptation to subsequent stressors. Sustained increases in GSH content are controlled primarily through induction of two genes, Gclc and Gclm, leading to the synthesis of the rate-limiting enzyme for GSH synthesis, glutamate cysteine ligase. Each of these genes in humans has a number of putative enhancer elements in their promoters. Overall, the most important element in both Gclc and Gclm expression is the electrophile response element. We review the evidence that has led to this conclusion and the implications for the redox-dependent regulation of this critical intracellular antioxidant.
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2002, Free Radical Biology and Medicine
Citation Excerpt :
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Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells.
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2002, Biochemical Pharmacology
Citation Excerpt :
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Regular ArticlePosttranscriptional Regulation ofMRP/GS-XPump and γ-Glutamylcysteine Synthetase Expression by 1-(4-Amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl)-3-nitrosourea and by Cycloheximide in Human Glioma Cells

Abstract

Regular Article
Posttranscriptional Regulation ofMRP/GS-XPump and γ-Glutamylcysteine Synthetase Expression by 1-(4-Amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl)-3-nitrosourea and by Cycloheximide in Human Glioma Cells