ABCMdb

PMID: 20159988

Pollex EK, Anger G, Hutson J, Koren G, Piquette-Miller M
Breast cancer resistance protein (BCRP)-mediated glyburide transport: effect of the C421A/Q141K BCRP single-nucleotide polymorphism.
Drug Metab Dispos. 2010 May;38(5):740-4. Epub 2010 Feb 16., [PubMed]

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0 ABCG2 p.Gln141Lys Breast Cancer Resistance Protein (BCRP)-Mediated Glyburide Transport: Effect of the C421A/Q141K BCRP Single-Nucleotide Polymorphism Erika K. Pollex, Gregory Anger, Janine Hutson, Gideon Koren, and Micheline Piquette-Miller Division of Clinical Pharmacology and Toxicology (E.K.P., J.H., G.K.) and Motherisk Program (E.K.P., J.H., G.K.), Hospital for Sick Children, Toronto, Ontario, Canada; and Department of Pharmaceutical Sciences (E.K.P., G.A., G.K., M.P.-M.) and Institute of Medical Science (J.H., G.K.), University of Toronto, Toronto, Ontario, Canada Received October 14, 2009; accepted February 16, 2010 ABSTRACT: The antidiabetic agent glyburide (glibenclamide) is frequently used for the treatment of type II diabetes and is increasingly being used for the treatment of gestational diabetes. Login to comment 3 ABCG2 p.Gln141Lys Associations between the Q141K (C421A) SNP and ABCG2 protein expression, membrane surface translocation, efflux activity, or ATPase activity have been shown. Login to comment 5 ABCG2 p.Gln141Lys The purpose of this study is to investigate whether the Q141K SNP causes alterations in ABCG2-mediated glyburide transport. Login to comment 6 ABCG2 p.Gln141Lys Glyburide accumulation assays were carried out with stably transfected human embryonic kidney (HEK)-293 cells expressing wild-type ABCG2 (Arg482) and polymorphic ABCG2 (Q141K). Login to comment 8 ABCG2 p.Gln141Lys The apparent Kt and Vmax values for the transfected HEK-293 cells expressing the polymorphic variant (Q141K) of ABCG2 were significantly higher than those values determined for the wild-type ABCG2-expressing cells (p < 0.05). Login to comment 9 ABCG2 p.Gln141Lys Our results indicate that the Q141K variant of ABCG2 may have the potential to alter the placental pharmacokinetics of glyburide used in pregnancy. Login to comment 28 ABCG2 p.Gln141Lys The results of these studies vary, showing inconsistencies in the effect of the C421A (Q141K) polymorphism on ABCG2 protein activity. Login to comment 29 ABCG2 p.Gln141Lys It has been suggested that the Q141K SNP may affect ABCG2 protein expression, membrane surface translocation, efflux activity, or ATPase activity. Login to comment 30 ABCG2 p.Gln141Lys Of particular concern, in vivo studies have shown patients expressing the Q141K variant who were exposed to the chemotherapeutic agents topotecan and diflomotecan achieved higher plasma drug levels, suggesting a significant reduction in ABCG2-mediated excretion (Sparreboom et al., 2004). Login to comment 34 ABCG2 p.Gln141Lys Because BCRP/ABCG2 is believed to protect the fetus against the accumulation of its substrates, it is important to determine the potential effect of the Q141K SNP on the placental transfer of drugs used in pregnancy. Login to comment 37 ABCG2 p.Gln141Lys Therefore, the purpose of this study is to investigate whether the Q141K SNP causes alterations in ABCG2-mediated glyburide transport. Login to comment 38 ABCG2 p.Gln141Lys Materials and Methods Stably transfected human embryonic kidney (HEK)-293 cells containing full-length ABCG2 encoding wild-type (Arg482) or the SNP variant (Q141K) of ABCG2, or an empty vector to serve as a control were obtained (Dr. Robert W. Robey, National Cancer Institute, Bethesda, MD), and expression was enforced by selection in G418 (Invitrogen, Carlsbad, CA). Login to comment 41 ABCG2 p.Gln141Lys Flow cytometry assays were performed to confirm surface expression of ABCG2 in the wild-type (Arg482) and polymorphic (Q141K) clones. Login to comment 51 ABCG2 p.Gln141Lys Transport activity was examined by measuring glyburide accumulation in stably transfected HEK-293 cells expressing wild-type ABCG2 (Arg482), polymorphic ABCG2 (Q141K), and an empty vector. Login to comment 72 ABCG2 p.Gln141Lys Cells were incubated for 30 min with either phycoerythrin-labeled negative control antibody or the phycoerythrin-labeled anti-ABCG2 antibody 5D3 (red, negative control; green, Arg482; blue, Q141K). Login to comment 77 ABCG2 p.Gln141Lys Data from the HEK-293 empty vector-transfected cell line and each of the ABCG2-transfected HEK-293 cell lines (Arg482, Q141K) were used simultaneously to fit the model parameters because the assumption was made that the permeability of the empty vector-transfected cell line and each of the ABCG2-transfected HEK-293 cell lines was the same. Login to comment 84 ABCG2 p.Gln141Lys The surface expression of ABCG2 in HEK-293 cells transfected with empty vector, wild-type (Arg482), or SNP variant (Q141K) of FIG. 2. Login to comment 85 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys A, glyburide accumulation in the presence and absence of FTC (10 ␮M) in HEK293 cells transfected with wild-type ABCG2 (Arg482), ABCG2 polymorphic variant (Q141K), and control (empty vector) after 60 min of exposure to 20 ␮M glyburide. Login to comment 87 ABCG2 p.Gln141Lys B, accumulation of glyburide in HEK-293 cells transfected with wild-type ABCG2 (Arg482), ABCG2 polymorphic variant (Q141K), and empty vector control after 20 min of exposure to various concentrations of glyburide in the presence and absence of FTC (10 ␮M). Login to comment 90 ABCG2 p.Gln141Lys As shown in Fig. 1, surface expression of ABCG2 was detected at similar levels in the ABCG2-transfected HEK-293 cell lines expressing wild-type (Arg482) and the polymorphic variant (Q141K) of ABCG2 (Fig. 1). Login to comment 94 ABCG2 p.Gln141Lys Inhibition of ABCG2/BCRP by fumitremorgin C (FTC) (10 ␮M) resulted in marked increases in intracellular accumulation of [3 H]glyburide in HEK-293 cells expressing the wild-type (Arg482) ABCG2 or SNP variant (Q141K) of ABCG2 after 60 min of exposure to 20 ␮M glyburide (Fig. 2A). Login to comment 96 ABCG2 p.Gln141Lys [3 H]Glyburide accumulation experiments carried out for 20 min at various concentrations of glyburide (0.2-100 ␮M) show inhibitable transport in both Arg482- and Q141K-transfected HEK-293 cells exposed to up to 20 ␮M glyburide (Fig. 2B). Login to comment 98 ABCG2 p.Gln141Lys Data obtained suggest that full FTC-mediated inhibition of BCRP/ ABCG2 is seen in the wild-type transfected cells at higher drug concentrations than in the SNP-transfected cells (Q141K) (Fig. 2B). Login to comment 101 ABCG2 p.Gln141Lys In the transfected HEK-293 cells expressing the polymorphic variant (Q141K) of ABCG2, the apparent Kt and Vmax values were 80.37 Ϯ 22.96 ␮M and 384.79 Ϯ 69.71 pmol/min ⅐ mg protein, respectively. Login to comment 102 ABCG2 p.Gln141Lys Both the apparent Kt and Vmax values for the transfected HEK-293 cells expressing the polymorphic variant (Q141K) of ABCG2 were found to be significantly higher than those values determined for the wild-type ABCG2-expressing cells (p Ͻ 0.05). Login to comment 106 ABCG2 p.Gln141Lys The recent identification of the nonsynonymous SNP, Q141K (Honjo et al., 2002; Imai et al., 2002), in the coding region of ABCG2 and the potential functional consequences on drug disposition has led to an increasingly important area of research. Login to comment 107 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys To elucidate the potential effects of the Q141K SNP on BCRP-mediated glyburide transport, we examined and compared the accumulation of glyburide in HEK293 cells stably transfected with full-length ABCG2 encoding wild-type or the SNP variant (Q141K) of ABCG2. Login to comment 110 ABCG2 p.Gln141Lys Regarding BCRP transport kinetics, the values obtained for apparent affinity (Kt) of glyburide for BCRP suggest that glyburide has greater affinity for the wild-type ABCG2 protein compared with the affinity determined for the Q141K ABCG2 variant. Login to comment 114 ABCG2 p.Gln141Lys Rate of glyburide transport in wild-type (Arg482), SNP (Q141K), and empty vector-transfected HEK-293 cells after exposure to various concentrations of glyburide (0.2-100 ␮M) for 20 min. Login to comment 119 ABCG2 p.Gln141Lys Metabolism of glyburide in this system is also thought to be minimal and similar in both wild-type and Q141K ABCG2-transfected cells; however, intracellular binding and glyburide metabolism could also confound the estimation of Kt values. Login to comment 120 ABCG2 p.Gln141Lys It has been previously reported that the Q141K variant is associated with increased sensitivity to chemotherapeutic agents as a result of a decrease in protein expression in transfected cells (Imai et al., 2002). Login to comment 121 ABCG2 p.Gln141Lys Data obtained in our study did not show reduced surface expression of Q141K ABCG2. Login to comment 122 ABCG2 p.Gln141Lys Thus, the impaired ability of the ABCG2 protein to efflux glyburide seen in cells transfected with Q141K ABCG2 was likely the result of SNP-induced alterations in the function of the protein itself. Login to comment 123 ABCG2 p.Gln141Lys Similar to our findings, Morisaki et al. (2005) and Zamber et al. (2003) did not observe a correlation between decreased expression of ABCG2 and the Q141K SNP. Login to comment 124 ABCG2 p.Gln141Lys However, Morisaki et al. (2005) noted that a larger proportion of the surface-localized Q141K ABCG2 protein is intracellular, implying less efficient post-translational processing in the SNP variant and thus providing a potential mechanism for reduced efficiency. Login to comment 125 ABCG2 p.Gln141Lys In summary, data from our accumulation studies indicate that the HEK-293 cell line expressing the Q141K polymorphic variant of the ABCG2/BCRP gene exhibits a reduced affinity for BCRP-mediated glyburide efflux. Login to comment 126 ABCG2 p.Gln141Lys Therefore, the Q141K variant of ABCG2 may have the potential to alter the placental pharmacokinetics of glyburide and other clinically used BCRP substrate drugs in pregnancy. Login to comment 128 ABCG2 p.Gln141Lys However, the results of this study provide sufficient reason to further investigate the impact of SNPs on ABCG2-mediated glyburide transport because the potential exists for increased fetal exposure to glyburide in pregnancy among patients carrying the Q141K polymorphic variant allele of ABCG2. Login to comment