Glutathione plays an important role in drug resistance of tumor cells and in their ability to resist oxidative stress. Improved salvage of glutathione can be obtained through increased activity of γ‐glutamyltransferase (GGT), which is of importance in the maintenance of cellular glutathione homeostasis. We investigated the regulation of GGT in 2 cisplatin‐resistant and 1 cisplatin‐sensitive colon carcinoma cell lines. Enzyme activity was induced in all 3 cell lines after acute exposure to cisplatin. The elevation was significantly higher in sensitive cells (3.3‐fold) than in resistant (1.6‐ to 1.7‐fold) cells. Exposure of cells to oxidative stress generated by menadione also resulted in enzyme induction but only in cisplatin‐sensitive cells. Addition of anti‐oxidants had different effects on the 2 inductions: N‐acetylcysteine blocked the induction of both cisplatin and menadione, whereas catalase and glutathione‐ester blocked only the menadione induction. Glutathione depletion alone was not sufficient to induce GGT in these cells. The data show that GGT is regulated by multiple mechanisms during anti‐tumor drug treatment and oxidative stress and that reactive oxygen species were involved in the menadione, but not cisplatin, induction of the enzyme. Int. J. Cancer 88:464–468, 2000. © 2000 Wiley‐Liss, Inc.

Injection of the rat colon carcinoma cell line CC531 into spleen of syngeneic rats results in considerable amounts of liver metastases within 14 days. We investigated whether preincubation of the cells with butyrate reduced their metastatic ability in vivo and whether this was accompanied by reduction in related properties such as secretion of metalloproteinases and their ability to withstand oxidative stress. Butyrate incubation reduced cell growth rate and initiated apoptosis in a dose- and time-related manner, but proliferation was retrieved when cultivation was continued in medium without butyrate. Splenic injection of butyrate treated, proliferating cells resulted in significantly reduced amounts of tumor mass compared to untreated cells. The butyrate treated cells were more susceptible to oxidative stress than control cells, as demonstrated by increased number of apoptotic cells and reduced cell growth after exposure to menadione. A reduction in cellular glutathione was found after prolonged incubation with butyrate. Butyrate appeared not to alter the secretion of active metalloproteinases from the cells although an apparent increase in proforms was demonstrated. Neither did butyrate alter the synthesis of metalloproteinase inhibitors. Lastly, a reduced adhesion of the tumor cells to collagen coated matrix was found after butyrate treatment. Thus, the inhibitory effects of butyrate on tumor malignancy are caused by a diversity of mechanisms.

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Gamma-glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 5S-radiolabeled GSH. The enzyme was also induced by interferon-gamma and tumor necrosis factor-alpha, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.

Gamma glutamyltransferase (GGT) is a plasma membrane bound enzyme that initiates the degradation of glutathione. The presence of several promoters in the rat GGT gene indicates strict control and regulation of its expression. The aim of this study was to investigate whether the GGT gene was regulated differently after butyrate-induced differentiation and oxidative stress exposure of rat colon carcinoma cells and whether the regulation was related to the glutathione level. The activity of GGT was upregulated in a time-and-dose dependent manner after both butyrate and menadione incubations. The presence of antioxidants blocked the menadione but not the butyrate mediated induction of the enzyme. The level of intracellular glutathione was reduced after menadione, but not after butyrate incubations. Depletion of glutathione alone did not alter GGT activity. Reactive oxygen species (ROS) were not produced after incubations with butyrate, while menadione incubations produced ROS. The multiple GGT mRNA transcripts (mRNA I-V) that originate from the five distinct promoters were all present in the cell line. Incubations with butyrate enhanced mRNA II and IV transcripts whereas a reduction in mRNA IV-1 was noted during menadione incubations. The level of total GGT mRNA (I-V) was not altered when related to the amount of total beta-actin mRNA. We conclude that GGT activity can be upregulated by at least two distinct mechanisms during differentiation and oxidative stress. Apparently, the regulation of the enzyme is not directly linked to the intracellular level of glutathione.