The human multidrug resistance gene 1 (MDR1) encodes the plasma membrane P-glycoprotein (P-gp/ABCB1) that functions as an efflux pump for various anticancer agents. We recently reported that estrogens down-regulate the expression of breast cancer resistance protein (BCRP/ABCG2). In our present study we demonstrate that estrogens also down-regulate P-gp expression in the MDR1-transduced, estrogen receptor α α α α (ER-α α α α)-positive human breast cancer cells, MCF-7/MDR and T-47D/MDR. The P-gp expression levels in MCF-7/MDR cells treated with 100 pM estradiol were found to be 10-20-fold lower than the levels in these same cells that were cultured without estradiol. In contrast, estradiol did not affect the P-gp expression levels in the ER-α α α α-negative cancer cells, MDA-MB-231/MDR and NCI/ ADR-RES. Estrone and diethylstilbestrol were also found to downregulate P-gp in MCF-7/MDR cells, but progesterone treatment did not produce this effect. Tamoxifen reversed the estradiol-mediated down-regulation of P-gp in MCF-7/MDR cells, suggesting that ER-α α α α activity is necessary for the effects of estradiol upon P-gp. However, estradiol was found not to alter the MDR1 transcript levels in either MCF-7/MDR and T-47D/MDR cells, suggesting that posttranscriptional mechanisms underlie its effects upon P-gp downregulation. MCF-7/MDR cells also showed eight-fold higher sensitivity to vincristine when treated with 100 pM estradiol, than when treated with 1 pM estradiol. These results may serve to provide a better understanding of the expression control of ABC transporters, and possibly allow for the establishment of new cancer chemotherapy strategies that would control P-gp expression in breast cancer cells and thereby increase their sensitivity to MDR1-related anticancer agents. (Cancer Sci 2006; 97: 1198-1204)

The human multidrug resistance gene 1 (MDR1) encodes a plasma membrane P-glycoprotein (P-gp) that functions as an efflux pump for various structurally unrelated anticancer agents. We have identified two nonsynonymous germ-line mutations of the MDR1 gene, C3583T MDR1 and T3587G MDR1, in peripheral blood cell samples from Japanese cancer patients. Two patients carried the C3583T MDR1 allele that encodes H1195Y P-gp, whereas a further two carried T3587G MDR1 that encodes I1196S P-gp. Murine NIH3T3 cells were transfected with pCAL-MDR-IRES-ZEO constructs carrying either wild-type (WT), C3583T, or T3587G MDR1 cDNA and selected with zeocin. The resulting zeocin-resistant mixed populations of transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. The cell surface expression of I1196S P-gp in 3T3/I1196S cells could not be detected by fluorescence-activated cell sorting, although low expression of I1196S P-gp was found by Western blotting. H1195Y P-gp expression levels in 3T3/H1195Y cells were slightly lower than the corresponding WT P-gp levels in 3T3/WT cells. By immunoblotting analysis, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was visualized as a 140-kDa protein. 3T3/I1196S cells did not show any drug resistance unlike 3T3/H1195Y cells. Moreover, a vanadate-trap assay showed that the I1196S P-gp species lacks ATP-binding activity. Taken together, we conclude from these data that T3587G MDR1 expresses a nonfunctional P-gp and this is therefore the first description of such a germ-line mutation. We contend that the T3587G MDR1 mutation may affect the pharmacokinetics of MDR1-related anticancer agents in patients carrying this allele. [Mol Cancer Ther 2006; 5(4):877 -84]