Increased levels of alpha‐class and pi‐class glutathione S‐transferases in cell lines resistant to 1‐chloro‐2,4‐dinitrobenzene

Abstract

Glutathione S-transferase (GST) enzymes are often over-expressed in tumor cells made resistant to cytotoxic drugs but it is unclear whether GST over-expression is directly linked to the resistance mechanism. We have made a human lung tumor cell line resistant to 1 -chloro-2,4-dinitrobenzene (CDNB) in order to establish whether selection for resistance with a model GST substrate results in selection of a cell line with higher GST levels. The resistant line (CDNB'), although only twofold more resistant to this compound, exhibited a marked (15-fold) increase in GST activity compared to the wild-type cell line (28 -+ 10 versus 357 -+ 30 nmol CDNB conjugated . min-' . mg-' protein, respectively). Resistance to CDNB was associated with a marked increase in the level of both alphaclass and pi-class GST. Resolution of the GST by reverse-phase HPLC demonstrated that the increase in the expression of the alpha-class enzymes was due to elevated levels of both the B, and B, subunits. The increased levels of alpha-class and pi-class GST in the CDNB' cells was not due to either gene amplification or increased mRNA levels and appears to involve either altered mRNA utilization or protein stabilization. In addition to being resistant to CDNB, the CDNB' cell line also showed a 2.5-fold resistance to cumene hydroperoxide but was not cross-resistant to the anticancer drug chlorambucil. To demonstrate that the increased GST level was part of the resistance mechanism the alpha-class GST B, cDNA under control of the p-actin promoter was stably expressed in the breast tumor cell line MCF-7. The cell lines generated were twofold more resistant to CDNB relative to the parental line.Many cancer patients who initially respond to treatment with anti-tumour drugs subsequently become resistant to chemotherapy. To help increase the clinical effectiveness of chemotherapy considerable effort has been made to identify the biochemical and genetic changes responsible for the drugresistant phenotype. Such work has demonstrated the existence of numerous resistance mechanisms [l], including alterations in drug transport, decreased activation of pro-drugs, increased drug detoxification, increased drug sequestration and mutation in drug target site(s). Although tumor cells can develop tolerance to chemotherapeutic agents by diverse processes, most resistance mechanisms are drug specific. For example, increased levels of P-glycoprotein, a transmembrane efflux pump of broad specificity, can confer resistance against anthracyclines, vinca alkaloids and podophylotoxin derivatives but not alkylating agents nor cis-platinum [I].The glutathione S-transferases (GST), a supergene family of detoxication enzymes, have been shown to become increased in many models of drug resistance (reviewed in [2-41). The GST are represented by several gene families, DDI 9SYAbbreviations. CDNB, 1 -chloro-2,4-dinitrobenzene; Me,SO, dimethyl sulphoxide; GST, glutathione S-transferase; MTT, 3-(4,sdimethylthiazol-2-yl)-2,S-diphenyl tetrazolium bromide; SV40, simian sarcoma virus 40;...

Overexpression of GSTA2 protects against cell cycle arrest and apoptosis induced by the DNA inter‐strand crosslinking nitrogen mustard, mechlorethamine

“…The absence of resistance to chlorambucil was attributed to the possible need for multiple factors [Wareing et al, 1993]. The possibility that resistance to nitrogen mustard compounds requires multiple factors has also been proposed to explain the difficulty in using DNA transfection of single GST genes to demonstrate responsibility for resistance to nitrogen mustards [Tew, 1994].…”

“…Escalating doses of the model GST substrate, CDNB, were used to elicit resistance in a human lung cancer cell line [Wareing et al, 1993]. The resistant cells expressed 20-fold higher levels of GSTA1 and GSTA2.…”

In Vivo Regulation of Human Glutathione Transferase GSTP by Chemopreventive Agents

“…Glutathione S -transferase P (GSTP) has been reported to be significantly elevated in a range of animal and human tumour types, although the role of this enzyme in the tumorigenic process is still unclear; indeed, the endogenous role(s) of GSTP have yet to be fully elucidated (2, 3). Cell lines made resistant to toxic chemicals, including anti-cancer drugs, express high levels of GSTP (4); likewise, cell lines in which GSTP is over-expressed are found to be resistant to a range of drugs and chemicals (5), even though many of these compounds are not known to be substrates of the enzyme. It appears, therefore, that GSTP is likely to have actions which are not mediated by its primary catalytic function (6).…”

Increased Skin Papilloma Formation in Mice Lacking Glutathione Transferase GSTP

“…Glutathione S-transferases (GST) are a multi-gene family of dimeric enzymes playing an important role in chemical detoxification due to their capacity to catalyse the addition of reduced glutathione to reactive electrophiles [1]. GSTP has received particular attention because of its association with carcinogenesis, drug resistance and chemical toxicity [2-4]. In order to define the in vivo functions of this protein, we have generated Gstp -null mice; these mice develop normally, are fertile and show no obvious abnormalities [5].…”