Time course of glutathione S-transferase elevation in Walker mammary carcinoma cells following chlorambucil exposure.

Resistance of Walker 256 rat mammary carcinoma cells to chlorambucil has been shown to be accompanied by a specific increase in the A2-2 subunit of glutathione S-transferase (GST) (Buller et al., Mol Pharmacol 31: 575-578, 1987). Analysis of the time course of GST activity following chlorambucil exposure revealed a 7.5- and 3-fold elevation on day 7 post-treatment in Walker-sensitive (WS) and Walker-resistant (WR) cells, respectively. Flow activated cell sorting (FACS) analyses using antibodies specific for rat liver cytosolic GST supported these results and demonstrated the heterogeneous response of WS cells to chlorambucil exposure. The range of GST levels in drug-treated cells was very broad as compared to that of untreated cells. Transcripts for each class of GST (alpha, mu and pi) were quantified for days 1-9 post-treatment from densitometric scans of RNA slot blots. Elevations in GST alpha RNA preceded increases in GST activity (day 7) in both WS and WR cells. Because fluctuations in GSTA1-1 transcripts were not observed, it was concluded that the increased expression of the alpha class must be attributed to increases in GSTA2-2 transcripts. Amplification of the GST genes in drug-treated cells was not present. These results support the role of GSTA2-2 in the detoxification of chlorambucil. The time course of the cellular response to chlorambucil suggests that the elevation of GSTA2-2 transcripts following alkylating agent exposure may represent only one component of a series of events which collectively confer protection and lead to the establishment of drug resistance.