ABSTRACT:The aim of the present study was to assess the contribution of polymorphisms in the breast cancer resistance protein/ATP-binding cassette transporter G2 (BCRP/ABCG2) gene to the placental expression from a new perspective, allelic imbalance. Polymorphisms were screened by polymerase chain reaction (PCR)-singlestrand conformation polymorphism analysis followed by sequencing with DNA extracted from 100 placentas. To evaluate whether the C421A polymorphism acts as a cis-element in BCRP transcription, allelic imbalance was determined using informative lymphoblasts and 56 samples of placental cDNA. In most of the placental samples we tested, the difference in expression levels between the two alleles was small, and only two samples indicated a monoallelic expression (i.e., preferential expression of one allele). These results suggest that 1) the predominant allelic expression pattern of BCRP in placental samples is biallelic, and 2) the mutation C421A is not a genetic variant acting in cis, but is considered to influence the translation efficiency.Breast cancer resistance protein (BCRP), also called mitoxantroneresistant protein, is the second member of the G family of ATPbinding cassette transporters (ABCG2) (Allikmets et al., 1998;Doyle et al., 1998;Miyake et al., 1999;Doyle and Ross, 2003). The BCRP gene is located at 4q22 and encodes a 72-kDa membrane protein composed of 655 amino acids (Allikmets et al., 1998;Doyle et al., 1998;Allen et al., 1999;Bailey-Dell et al., 2001). In contrast to many other ABC transporters, BCRP has only one ATP-binding region and one transmembrane domain. Therefore, BCRP is referred to as a half-transporter, and its homodimerization may be necessary to transport substrates .In normal human tissues, BCRP is highly expressed in the placenta, colon, small intestine, and liver (Maliepaard et al., 2001). On the basis of its tissue distribution and findings in knockout mice, BCRP is speculated to have a major influence on the pharmacokinetic and pharmacodynamic profiles of certain xenobiotics and endogenous substrates. For example, inhibition of mouse Bcrp 1 by GF120918, a dual inhibitor for BCRP and P-glycoprotein, has been demonstrated to increase the bioavailability of topotecan when GF120918 was administered orally to mdr1a/1b(Ϫ/Ϫ) mice (Jonker et al., 2000). In a clinical study, coadministration of GF120918 was also associated with a marked increase in the bioavailability of and systemic exposure to topotecan (Kruijtzer et al., 2002).Recent clinical studies indicate that the large interindividual variability in drug response occurs as a result of molecular alterations to various proteins such as drug-metabolizing enzymes, drug targets and receptors, and drug transporters. Most studies on molecular alterations have focused on the impact of single-nucleotide polymorphisms (SNPs) on the expression and function of these proteins (Evans and Relling, 1999;Evans and Johnson, 2001). Several groups have reported naturally occurring SNPs in the BCRP gene. G34A and C421A occur at relative...

These results suggest that Q141K SNPs may associate with a lower expression level, and S441N SNPs may affect both the expression level and cellular localization. It is possible that subjects with these polymorphisms may have lower expression level of BCRP protein and, consequently, a reduced ability to export these substrates.

We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 g/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 g/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 M. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r ‫؍‬ ؊0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.Candidiasis is a life-threatening disease in patients with immune suppression. Azoles are used widely for the treatment of candidiasis, but some clinical isolates show resistance (11,28). Further investigation of the mechanisms underlying the antifungal effects of azoles may aid in the development of new treatment strategies.Azoles exert a cytostatic or cytotoxic effect via inhibition of synthesis reactions in the metabolic pathways of essential fungal cell membrane components including ergosterol (29,30). Their primary target is the cytochrome P450-catalyzed 14␣-demethylation of ergosterol precursors. Mutations or overexpression of 14␣-demethylase, encoded by the ERG11 gene, as well as changes in the ergosterol synthesis pathway reportedly participates in the induction of resistance (14,19,20,25).Since it was found that miconazole, ketoconazole, and deacetylated ketoconazole were inserted in a lipid layer (6) and miconazole induced the release of K ϩ and intracellular ATP from Candida species (3, 4, 7), these agents may cause direct membrane damage.Recent studies clarified that a decrease in drug concentration brought about by energy-dependent efflux pumps is usually involved in multidrug resistance including azole resistance (1,2,16,26). This pumping system closely resembles a system in cancer cells based on the P-glycoprotein encoded by the MDR-1 gene. Thus, common biologic mechanisms shared with other eukaryotic cells are involved in the antifungal effects of azoles.In eukaryotic cells, mitochondria are common organelles that represent an important source of reactive oxygen species (ROS). Many cellular stresses such as irradiation and cytotoxic drugs cause growth inhibition and the death of mammalian cells via endogenous ROS production (5, 15, 27). ROS produced by granulocytes or monocytes are known to exert activity against fungi (9, 31). Furthermore, recent studies demonstrated that Candida albicans possesses an ROS scavenger, superoxide dismutase; this suggests that fungi may require a cytoprotective mechanism against not only exogenous ROS but also endogenous, fungus-derived ROS (1...

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We examined whether survivin acts as a constitutive and inducible radioresistance factor in pancreatic cancer cells. Using a quantitative TaqMan reverse transcription-polymerase chain reaction for survivin mRNA in five pancreatic cancer cell lines, we found an inverse relationship between survivin mRNA expression and radiosensitivity. PANC-1 cells, which had the highest survivin mRNA levels, were most resistant to X-irradiation; MIAPaCa-2 cells, which showed the least survivin mRNA expression, were the most sensitive to X-irradiation. Our results suggested that survivin could act as a constitutive radioresistance factor in pancreatic cancer cells. To determine whether radioresistance is enhanced by induction of survivin expression by irradiation, PANC-1 and MIAPaCa-2 cells were subjected to sublethal doses of X-irradiation followed by a lethal dose. Survivin mRNA expression was increased significantly in both PANC-1 and MIAPaCa-2 cell lines by pretreatment with a sublethal dose of X-irradiation, as was cell survival after exposure to the lethal dose. In this system, enzymatic caspase-3 activity was significantly suppressed in cells with acquired resistance. These results suggest that survivin also acts as an inducible radioresistance factor in pancreatic cancer cells. Survivin, then, appears to enhance radioresistance in pancreatic cancer cells; inhibition of survivin mRNA expression may improve the effectiveness of radiotherapy.