This laboratory has been investigating the genetic and biochemical changes associated with drug resistance in human breast cancer. We have identified a number of changes associated with the development of multi-drug resistance in human breast cancer cells including over-expression of the mdr 1/P-glycoprotein drug efflux, as well as increased expression of the pi class isozyme of glutathione transferase in the selenium dependent glutathione peroxidase gene. In order to study the role of these genes in development of resistance, we have transfected expression vectors for the human mdr1 gene and for the alpha, mu, and pi class GST genes into drug sensitive breast cancer cells. We have also examined the possible interaction of GST and protein kinase C with P-glycoprotein through cotransfection experiments. In order to study the regulation of the genes overexpressed in drug resistant cells we have cloned the genomic promotor and flanking sequences for the human mdr1 gene, the glutathione transferase II gene, the selenium dependent glutathione peroxidase gene. The functional activity of the promoters were analyzed by fusing these elements to the CAT gene. Other studies from our lab have identified changes in drug accumulation in association with mitoxantrone resistance. Resistance in the latter appears to be due to markedly diminished methotrexate transport. In contrast resistance to mitoxantrone is associated with enhanced drug efflux in the absence of P-glycoprotein expression with enhanced drug efflux that is not mediated by mdr1/P-glycoprotein expression. However, membrane protein has been identified in the resistant cells which cross reacts with antibodies directed against the human P-glycoprotein. Another cell line selected for methotrexate resistance has developed a marked decrease in methotrexate accumulation. This defect is apparently due to a marked decrease in the reduced folate transport system in the resistant cells. Both of these cell lines provide useful models to study the molecular biology changes associated with the development of drug resistance in human breast cancer cells.
