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Abstract: Accumulation of methotrexate (MTX) and its polyglutamates (PGs) has been recognized as an important factor in MTX efficacy. We have previously described a multidrug-resistant human breast cancer cell line, MCF7/MX, that exhibits reduced accumulation of total MTX as well as MTX-PGs, and that is resistant to continuous MTX exposure [Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, et al. Methotrexate cross-resistance in a mitoxantrone selected multidrug-resistant MCF7 breast cancer cell line is due to enhanced energy-dependent drug efflux. Cancer Res 2000;60:3514–21]. These cells express high levels of the breast cancer resistance protein (BCRP/ABCG2) that has been shown to actively transport MTX and short-chain MTX-PGs in vitro. However, the effect of BCRP on MTX-PG accumulation in intact cells was unclear. Here, we show that MTX transport by BCRP is required for the observed lower levels of MTX-PGs in the resistant cells. When BCRP was inhibited with fumitremorgin C, or in cells expressing a mutated form of BCRP that is unable to transport MTX, MTX-PG accumulation was similar or even higher than that in the parental cells that do not express BCRP. Concomitantly, there was increased inhibition of thymidylate synthase. It had previously been suggested that BCRP-mediated efflux of MTX-PGs contributed to the reduced MTX-PG accumulation. However, we found no evidence of BCRP-mediated efflux of MTX-PGs from intact cells, suggesting that direct efflux of MTX-PGs does not play a major role in MTX resistance. Together, these data show that BCRP overexpression can cause a reduction in total MTX accumulation as well as a reduction in the proportion of long-chain MTX-PGs. In contrast, BCRP overexpression did not affect natural folate accumulation or the relative distribution of folylpolyglutamates in the resistant, as compared to the parental, cells. Thus, it appears that BCRP overexpression affects the metabolism of the antifolate MTX, but not that of natural folates, although indirect effects cannot be excluded. [Copyright &y& Elsevier]

Background: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone.Methods: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively.Results: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells.Conclusions: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC) n , located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC) 2 alleles were observed; however, in western gorilla, (GGGGC) n alleles with n = 2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC) n was n = 4 > 5 ≫ 2, 6. When frequencies of the (GGGGC) n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC) 2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC) n short tandem repeats are inherited, and that the (GGGGC) 2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility. [ABSTRACT FROM AUTHOR]