Genes regulating glutathione concentrations in X-ray-transformed rat embryo fibroblasts: changes in γ-glutamylcysteine synthetase and γ-glutamyltranspeptidase expression

Abstract

The concentration of glutathione (GSH) and the expressions of gamma-glutamylcysteine synthetase and gamma-glutamyltranspeptidase (GGT) were assessed in rat embryo fibroblasts (REF) displaying various stages of X-ray-induced transformation. A secondary culture of REF cells was irradiated, and a normal-immortalized cell line (X-REF-23) was isolated. Chronic exposure of X-REF-23 cells to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) yielded cells (X-REF-23-TP) capable of benign tumor formation in nude mice. These cells exhibited GSH concentrations and gamma-glutamylcysteine synthetase heavy subunit mRNA levels that were approximately 50% less than those measured in X-REF-23 cells. Neither X-REF-23 nor X-REF-23-TP cells exhibited detectable GGT mRNA or activity. Administration of 3 Gy of X-rays followed by chronic TPA treatment yielded cells (X-REF-23-TPX) capable of malignant tumor formation in nude mice. These cells expressed GGT mRNA and Concanavalin-A minus GGT activity. One TPX clone (X-REF-23-TPX.1) was chosen for further characterization. Northern blotting of X-REF-23-TPX.1 cells indicated that gamma-glutamylcysteine synthetase heavy subunit mRNA levels were similar to those of X-REF-TP cells. X-REF-23-TPX.1 cells contained nearly the same amount of GSH as X-REF-23 cells. However, the ability of diethylmaleate (DEM) to deplete GSH was diminished in X-REF-23-TPX.1 cells compared with X-REF-23 cells. Furthermore, exposure of X-REF-23-TPX.1 cells to DEM stimulated GSH resynthesis such that the GSH concentration exceeded control values during exposure. The resynthesis of GSH during a DEM exposure was found to be dependent upon the expression of GGT, as demonstrated by inhibition with AT-125. These experiments indicate that ionizing radiation can lead to elevated constitutive expression of GGT in transformed REF cells and that expression of GGT activity was responsible for the increased rate of GSH repletion observed in X-REF-23-TPX.1 cells.

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

“…For example, the regulation of the key genes involved in glutathione synthesis has been shown to involve the stress-responsive transcription factors Nrf2 [22, 54, 97], Nrf1 [98, 99], AP-1, and NFkB [38, 100, 101]. The comprehensive gene lists presented here will facilitate future studies to identify the role of key transcription factors in modulating GSH concentrations and E h during embryonic development.…”

Nuclear Factor-κB modulates cellular glutathione and prevents oxidative stress in cancer cells

“…GSH de novo synthesis, carried out by the consecutive action of two ATP-dependent enzymes, γ-glutamate-cysteine ligase (γ-GCL) and GSH synthase (GS), primarily determines cellular GSH level [8]. γ-GCL is the rate-limiting enzyme, consisting of a heterodimer composed of a catalytic (γ-GCLC) and a regulatory subunit (γ-GCLM) [9]. We find that the IKKβ-NF-κB pathway is responsible for maintaining basal GCLC/GCLM expression, which in turn regulates GSH biosynthesis.…”

Assignment of the human gene (GLCLR) that encodes the regulatory subunit of γ-glutamylcysteine synthetase to chromosome 1p21

“…In human neoplastic diseases, it appears to modulate the therapeutic effectiveness of chemotherapeutic drugs (Mulcahy etal., 1994) and ionizing radiation (Vincenzini et al, 1993). Its expression is altered during radiation-induced neoplastic trans formation in vitro (Sierra-Rivera et al, 1994) and in chemically induced tumors in vivo (Picket et al, 1984). Since y-GCS repre sents a rate-limiting step in GSH synthesis, a better under standing of y-GCS regulation at the molecular level will provide insight into the many functions of GSH, especially the role it plays in human diseases.…”