Differential sensitivities of the MRP gene family and γ-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status

Abstract

1 Recent molecular cloning studies have identified six members in the multidrug-resistance protein ( MRP) gene family. However, the regulation of expression of these genes is largely unknown. We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and γ-GCSh, which encodes the heavy subunit of γ-glutamylcysteine synthetase (γ-GCS), could be up-regulated by prooxidants [Yamane et al., J Biol Chem 1998;273:31075–85]. In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction. A panel of colorectal cancer cell lines with different p53 status, i.e. HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC). MRP1 and γ-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes. We report here that induction of MRP1 and γ-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction. These results suggest that the effects of p53 on the induced expression of MRP1 and γ-GCSh depend on the environment of the cell and/or nature of p53 mutations. In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not. MRP6 mRNA was not detectable. Induction of MRP2 expression by prooxidants seemed to be independent of p53 status. Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.