We have shown previously that tumor cell resistance to cisplatin is associated with elevated intracellular levels of glutathione, which is accomplished at least in part by increased expression of the heavy subunit of γ-glutamylcysteine synthetase (γ-GCS). To investigate the mechanism by which γ-GCS expression is elevated, we have examined four related human ovarian cancer cell lines with increasing cisplatin resistance. Relative amounts of steady-state γ-GCS mRNA in CP70, C30, and C200 were 4.8, 6.0, and 10.6, respectively, compared to the parental A2780 cell line, and a proportional increase in the transcriptional rate but not RNA stability was demonstrated. In contrast, no increase in mRNA for the γ-GCS light subunit was found. To determine the mechanism of up-regulation of this mRNA, we have cloned the promoter of the gene that encodes the heavy subunit of γ-GCS. This region contains AP-1, NF-κB, XRE, AP-2, EpRE, CAAT, and TATA box elements upstream of the transcription initiation site and two MREs between this site and the start codon for the protein. Using gel mobility shift assays, we have found nuclear extract binding activity to the AP-1 response element to be closely associated with the level of γ-GCS gene expression. A supershift assay showed that the AP-1 DNA-binding complexes are predominantly formed by dimers of JUN family members. Consistent with this finding, the expression of c-JUN was found to be elevated in the resistant cells. In contrast to AP-1 binding, AP-2 and NF-κB binding were inversely related to resistance. Furthermore, we have examined a partial revertant of the C200-resistant cells, which shows lower glutathione levels, and found decreased γ-GCS expression associated with decreased AP-1 binding activity.
This work was supported in part by National Cancer Institute Grants CA51175 and CA51228.