INCREASED LEVELS OF ALPHA-CLASS AND PI-CLASS GLUTATHIONE S-TRANSFERASES IN CELL-LINES RESISTANT TO 1-CHLORO-2,4-DINITROBENZENE

Glutathione S-transferase (GST) enzymes are often over-expressed in tu mor 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-dini trobenzene (CDNB) in order to establish whether selection for resistan ce with a model GST substrate results in selection of a cell line with higher GST levels. The resistant line (CDNB(r)), although only two-fo ld more resistant to this compound, exhibited a marked (15-fold) incre ase in GST activity compared to the wild-type cell line (28 +/- 10 ver sus 357 +/- 30 nmol CDNB conjugated . min-1 . mg-1 protein, respective ly). Resistance to CDNB was associated with a marked increase in the l evel of both alpha-class and pi-class GST. Resolution of the GST by re verse-phase HPLC demonstrated that the increase in the expression of t he alpha-class enzymes was due to elevated levels of both the B1 and B 2 subunits. The increased levels of alpha-class and pi-class GST in th e CDNB(r) 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 CDN B(r) cell line also showed a 2.5-fold resistance to cumene hydroperoxi de but was not cross-resistant to the anticancer drug chlorambucil. To demonstrate that the increased GST level was part of the resistance m echanism the alpha-class GST B1 cDNA under control of the beta-actin p romoter was stably expressed in the breast tumor cell line MCF-7. The cell lines generated were twofold more resistant to CDNB relative to t he parental line.