R. Timothy Mulcahy

Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element

The rate-limiting step in the de novo synthesis of the cellular protectant glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS; also known as glutamine-L-cysteine ligase, GLCL), a heterodimer consisting of catalytic (GCS(h)) and regulatory (GCS(l)) subunits. Regulation of expression of the human gamma-glutamylcysteine synthetase regulatory subunit gene in response to beta-NF is mediated by an Electrophile Responsive Element (EpRE) [Moinova, H., and Mulcahy, R. T. (1998) J. Biol. Chem. 273, 14683-14689]. Oligonucleotide probes corresponding to wild-type and mutant EpRE sequences were used in gel-shift and super-shift analyses to identify proteins binding. Four protein:DNA complexes (a-d) with distinct mobilities were detected when the wild-type EpRE probe was incubated with nuclear extracts from control or beta-NF-treated HepG2 cells. Following beta-NF treatment, there was an increase in the intensity of a single band, band b. This band was eliminated in gel shifts employing mutant EpRE probes which abolish beta-NF inducibility, demonstrating a correlation between band b and transactivation. Super-shift analysis identified JunD, Nrf1, and Nrf2 in the EpRE-binding complexes. Antibodies to Nrf2 completely super-shifted the band b protein:DNA complex. These studies demonstrate that Nrf2 proteins recognize and bind the GCS(l) EpRE sequence to affect transactivation of the gene.

Regulation of γ-glutamylcysteine synthetase subunit gene expression: Insights into transcriptional control of antioxidant defenses

γ-Glutamylcysteine synthetase (GCS; also referred to as glutamate-cysteine ligase, GLCL) catalyzes the rate-limiting reaction in glutathione (GSH) biosynthesis. The GCS holoenzyme is composed of a catalytic and regulatory subunit, each encoded by a unique gene. In addition to some conditions which specifically upregulate the catalytic subunit gene, expression of both genes is increased in response to many Phase II enzyme inducers including oxidants, heavy metals, phenolic antioxidants and GSH-conjugating agents. Electrophile Response Elements (EpREs), located in 5′-flanking sequences of both the GCSh and GCSl subunit genes, are hypothesized to at least partially mediate gene induction following xenobiotic exposure. Recent experiments indicate that the bZip transcription factor Nrf2 participates in EpRE-mediated GCS subunit gene activation in combination with other bZip proteins. An AP-1-like binding sequence and an NF-κB site have also been implicated in regulation of the catalytic subunit gene following exposure to certain pro-oxidants. Potential signaling mechanisms mediating GCS gene induction by the diverse families of Phase II enzyme inducers include thiol modification of critical regulatory sensor protein(s) and the generation of the reactive oxygen species. This review summarizes recent progress in defining the molecular mechanisms operative in transcriptional control of the genes encoding the two GCS subunits, identifying areas of agreement and controversy. The mechanisms involved in GCS regulation might also be relevant to the transcriptional control of other components of the antioxidant defense battery.

Expression of Multidrug Resistance Protein/GS-X Pump and γ-Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress

Expression of the MRP1 gene encoding the GS-X pump and of the γ-GCSh gene encoding the heavy (catalytic) subunit of the γ-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like γ-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such astert-butylhydroquinone, 2,3-dimethoxy-1,4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated withtert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably γ-GCSh-transfected cell lines down-regulated endogenous MRP1 and γ-GCShexpression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of theMRP1 and γ-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation ofMRP1 and γ-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and γ-GCShexpression.

The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for γ-glutamylcysteine synthetase and γ-glutamyl transpeptidase

Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1–3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving γ‐glutamylcysteine synthetase and γ‐glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP‐independent and we hypothesize that it makes an important contribution to the anti‐atherosclerotic and antioxidant properties of nitric oxide.

DT-diaphorase in activation and detoxification of quinones

A role for DTD in the bioreductive activation of mitomycin C was supported by indirect evidence utilizing enzyme inhibitors in cellular systems. Using a cell-free system, we have confirmed that DTD can bioactivate mitomycin C using both purified rat and human DTD. Metabolism and bioactivation of mitomycin C by DTD is pH-dependent. At pH 7.8 alkylation of DTD leading to enzyme inhibition and DTD crosslinking occurs whereas at pH values of 7.4 and below metabolite formation, preservation of catalytic activity of DTD and sequence-selective DNA crosslinking occurs. Bioactivation of mitomycin C by DTD and the cytotoxicity of this drug in DTD-rich cell lines is oxygen-independent. Mitomycin C induces greater DNA crosslinking, even after chemical reduction, at lower pH values. This suggests that if mitomycin C is used in tumors with elevated DTD activity, greater therapeutic activity may be obtained by lowering intratumoral pH. Human NSCLC has elevated DTD activity relative to SCLC and normal lung and may be a target for the development of drugs which can be efficiently bioactivated by DTD. Because of the pH-dependent inactivation of DTD by mitomycin C, however, other drugs which are efficiently metabolized and bioactivated by DTD may be better candidates for the therapy of tumors high in DTD such as NSCLC.

Cloning and nucleotide sequence of a full-length cDNA for human liver γ-glutamylcysteine synthetase

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.

H2O2-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1

The gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, “redox signaling” resulting in kinase signaling pathways is distinct from “oxidative stress,” and is mediated by discrete, localized redox circuitry.

Up-regulation of γ-glutamylcysteine synthetase activity in melphalan-resistant human multiple myeloma cells expressing increased glutathione levels

Levels of intracellular glutathione (GSH) and the GSH-related enzymes γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyltranspeptidase (γ-GT) were measured in the melphalan-resistant human multiple myeloma cell line 8226/LR-5 and were compared to those measured in the drug-sensitive 8226/S and doxorubicin-resistant 8226/Dox40 cell lines. Both GSH and γ-GCS activity, the rate-limiting step in the de novo synthesis of GSH, were elevated by a factor of approximately 2 in the melphalanresistant 8226/LR-5 cells relative to the other two lines. γ-GT activity was not elevated significantly in the /LR-5 cells. Northern analysis with a probe specific for the large subunit of human liver γ-GCS identified two bands (3.2 and 4.0 kb), both of which were increased by a factor of 2–3 in the 8226/LR-5 line. Levels of γ-GCS mRNA expression were comparable in the /S and /Dox40 cell lines. Levels of γ-GT mRNA were similar in the /S and /LR-5 lines but were reduced in the /Dox40 cells. These data suggest that the increased GSH levels associated with resistance to melphalan in the 8226/LR-5 myeloma cells is attributable to up-regulation of γ-GCS. This observation is consistent with recent demonstrations of up-regulation of γ-GCS in melphalan-resistant prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells, suggesting that increased expression of γ-GCS may be an important mediator of GSH-associated resistance mechanisms.

The Survival of Thyroid Cells: In Vivo Irradiation and in Situ Repair

The survival of rat thyroid cells irradiated in vivo and removed immediately for transplantation survival assay was compared with results obtained previously for thyroid cells irradiated in vitro and with the survival of thyroid cells irradiated and left in situ for 24 hr before transplantation survival assay. The Do for thyroid cells irradiated in vivo and removed immediately for assay is 195 rad; N is 4 and Dq is 270 rad. These parameters are not significantly different from those obtained when thyroid cells are irradiated in vitro. When these parameters are compared to those of thyroid cells irradiated and left in situ for 24 hr, the latter have a greater N and Dq, but there is no significant difference in Do. These data provide further evidence for a form of repair of potentially lethal damage which is dependent on the tissue environment (in situ repair, ISR) as previously described for ir

4-Hydroxytamoxifen, an active metabolite of tamoxifen, does not alter the radiation sensitivity of MCF-7 breast carcinoma cells irradiated in vitro.

SummaryThe effect of 4-hydroxytamoxifen (4OH-TAM), the potent anti-estrogenic metabolite of tamoxifen, on the radiosensitivity of MCF-7 cells irradiatedin vitro was determined. Radiation dose response curves were generated for MCF-7 cells maintained and irradiated in phenol red-free medium containing 10−10 M estradiol (E2) with or without 10−7 M 4OH-TAM. Immediately after irradiation cells were transferred to medium containing 10−10 M E2 supplemented with bovine serum to stimulate colony formation. Estradiol-stimulated cell proliferation was inhibited by 10−7 M 4OH-TAM, but radiation sensitivity was not significantly altered (p > 0.3). Continued incubation in the absence of E2 for an additional 24 hours after irradiation likewise failed to alter the radiosensitivity of 4OH-TAM-treated MCF-7 cells. These studies indicate that growth-inhibitory concentrations of the anti-estrogen 4OH-TAM do not modify thein vitro radiation sensitivity of this line of human breast carcinoma cells.