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 (CDNBr), 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-1 x mg-1 protein, respectively). Resistance to CDNB was associated with a marked increase in the level of both alpha-class 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 B1 and B2 subunits. The increased levels of alpha-class and pi-class GST in the CDNBr 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 CDNBr 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 B1 cDNA under control of the beta-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.