Influence of chemosensitizers on resistance mechanisms in daunorubicin-resistant Ehrlich ascites tumour cells.

Aim: To determine whether treatment with chemosensitizers influences the development of the drug-resistant phenotype.

Methods: Three sublines were developed from the sensitive Ehrlich ascites tumour cell line (EHR2) and six sublines from the EHR2/DNR cell line positive for P-glycoprotein (PGP) by treatment with daunorubicin (DNR), a combination of DNR and verapamil (VER), or a combination of DNR and cyclosporin A (CsA). A clonogenic assay was used to determine resistance, the expression of PGP, the multidrug resistance associated protein ( Mrp1) and topoisomerase IIalpha and beta were measured by Western blotting, and reverse transcriptase-polymerase chain reaction was used for determination of mdr1a and b, and Mrp1 mRNA.

Results: Compared with the EHR2 cell line, the amounts of mdr1a mRNA increased significantly in all sublines except EHR2/DNR, whereas mdr1b mRNA levels were unchanged. Compared with the EHR2 subline selected in DNR alone, the levels of mdr1a mRNA and PGP were significantly lower in the EHR2 sublines selected in the presence of chemosensitizer. Furthermore, mdr1a mRNA and PGP were unchanged in all cotreated sublines selected from the PGP-positive EHR2/DNR cell line. The mRNA and protein levels of Mrp1 did not change significantly in any of the cell lines. Only one DNR plus VER-selected subline showed a decrease in topoisomerase IIalpha (one-third as compared with EHR2). All DNR plus CsA-selected sublines showed significantly less resistance than the corresponding DNR- and DNR plus VER-selected sublines. The effect of VER and CsA on cytotoxicity was retained in all cell lines treated with chemosensitizer.

Conclusions: Selection in chemosensitizer resulted in a decrease in the expression of mdr1a and PGP. These chemosensitizers do not seem to influence Mrp1 expression or topoisomerase II. Selection in CsA may retard the development of resistance.