Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn387, Gln398, Asn629, and Thr642 with Ala, Thr402 with Ala and Arg, and Tyr645 with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr402 within transmembrane 1 may be important for helical interactions, and Asn629 may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity

Glucuronidation as a mechanism of intrinsic drug resistance in colon cancer cells: contribution of drug transport proteins

We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-beta-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport protein

Glucuronidation as a Mechanism of Intrinsic Drug Resistance in Human Colon Cancer: Reversal of Resistance by Food Additives

Abstract We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-b-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport proteins.

Role of Basic Residues within or near the Predicted Transmembrane Helix 2 of the Human Breast Cancer Resistance Protein in Drug Transport

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys452, Lys453, His457, Arg465, and Lys473 were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in Vmax/Km for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His457 and Arg465 might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP

Between secularization and religious reorganization – the religiosity of Polish and Turkish new immigrants in Germany

Dieser Beitrag befasst sich mit der Bedeutung von Religion im Prozess der Integration von Einwanderern in Deutschland. Gestützt auf neueste Daten (SCIP 2010/11) einer Befragung unter polnischen und türkischen Neuzuwanderern wird untersucht, welchen Einfluss das Migrationsereignis als solches auf Religiosität ausübt und wie sich religiöse Partizipation und private religiöse Praxis in der Frühphase der Integration verändern. Die Untersuchung bestätigt, erstens, dass beide Gruppen einen markanten Rückgang religiöser Partizipation erfahren. Dass dieser Rückgang bei muslimischen Türken stärker ausfällt als bei katholischen Polen und sich insbesondere beim Kirchgang oder Moscheebesuch, weniger dagegen bei der privaten Gebetspraxis nachweisen lässt, deutet auf die Bedeutung religiöser Opportunitätsstrukturen hin. Zweitens zeigt sich, dass bei den polnischen Neuzuwanderern der Rückgang an Religiosität mit starken Bindungen in die säkulare deutsche Aufnahmegesellschaft verknüpft ist, während dies bei den türkischen Neuankömmlingen nicht der Fall ist. Nur für jene lässt sich, drittens, beobachten, dass dem anfänglichen Einbruch der Religiosität mit dem Aufenthalt in Deutschland ein allmählicher Wiederanstieg folgt. Die Befunde deuten darauf, dass assimilationstheoretische Argumente um Überlegungen zu Dynamiken symbolischer Grenzziehung zu erweitern sind, die gruppenspezifische Muster des Wandels von Religiosität zu erfassen gestatten. Aufgrund solcher komplexer Dynamiken religiösen Wandels im Migrationsgeschehen ist insgesamt damit zu rechnen, dass öffentlich sichtbare religiöse Diversität ein dauerhaftes Merkmal moderner Einwanderungsgesellschaften darstellen wird.publishe