Human ATP-binding cassette G2 (ABCG2, also known as mitoxantrone resistance protein, breast cancer-resistance protein, ABC placenta) is a member of the superfamily of ATP-binding cassette (ABC) transporters that have a wide variety of substrates. Overexpression of human ABCG2 in model cancer cell lines causes multidrug resistance by actively effluxing anticancer drugs. Unlike most of the other ABC transporters which usually have two nucleotide-binding domains and two transmembrane domains, ABCG2 consists of only one nucleotide-binding domain followed by one transmembrane domain. Thus, ABCG2 has been thought to be a half-transporter that may function as a homodimer. In this study, we characterized the oligomeric feature of human ABCG2 using non-denaturing detergent perfluoro-octanoic acid and Triton X-100 in combination with gel filtration, sucrose density gradient sedimentation, and gel electrophoresis. Unexpectedly, we found that human ABCG2 exists mainly as a tetramer, with a possibility of a higher form of oligomerization. Monomeric and dimeric ABCG2 did not appear to be the major form of the protein. Further immunoprecipitation analysis showed that the oligomeric ABCG2 did not contain any other proteins. Taken together, we conclude that human ABCG2 likely exists and functions as a homotetramer.
Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.
Overexpression of human ATP-binding cassette transporter ABCG2 in cancer cells causes multidrug resistance by effluxing anticancer drugs. ABCG2 is considered as a half transporter and is thought to function as a homodimer. However, recent evidence suggests that it may exist as a higher form of oligomer consisting of 12 subunits. In this study, we mapped the oligomerization domain of human ABCG2 to its transmembrane domain consisting of TM5-loop-TM6. This oligomerization domain, when expressed alone in HEK293 cells, also forms a homododecamer. Furthermore, this domain has activity that inhibits drug efflux and resistance function of the full-length ABCG2 likely by disrupting the formation of the homo-oligomeric full-length ABCG2. These findings suggest that human ABCG2 may exist and work as a homo-oligomer by interactions located in TM5-loop-TM6, and that ABCG2 oligomerization may be used as a target for therapeutic development to circumvent ABCG2-mediated drug resistance in cancer treatment. [Cancer Res 2007;67(9):4373-81]
BackgroundMultidrug resistance (MDR) is a major problem in successful treatment of cancers. Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in MDR and an important role in protecting cancer stem cells. Knockout of ABCG2 had no apparent adverse effect on the mice. Thus, ABCG2 is an ideal target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and helping eradicate cancer stem cells.Methods/Preliminary FindingsUsing rational screening of representatives from a chemical compound library, we found a novel inhibitor of ABCG2, PZ-39 (N-(4-chlorophenyl)-2-[(6-{[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]amino}-1,3-benzothiazol-2-yl)sulfanyl]acetamide), that has two modes of actions by inhibiting ABCG2 activity and by accelerating its lysosome-dependent degradation. PZ-39 has no effect on ABCB1 and ABCC1-mediated drug efflux, resistance, and their expression, indicating that it may be specific to ABCG2. Analyses of its analogue compounds showed that the pharmacophore of PZ-39 is benzothiazole linked to a triazine ring backbone.Conclusion/SignificanceUnlike any previously known ABCG2 transporter inhibitors, PZ-39 has a novel two-mode action by inhibiting ABCG2 activity, an acute effect, and by accelerating lysosome-dependent degradation, a chronic effect. PZ-39 is potentially a valuable probe for structure-function studies of ABCG2 and a lead compound for developing therapeutics targeting ABCG2-mediated MDR in combinational cancer chemotherapy.
Human ABCG2, a member of the ATP-binding cassette transporter superfamily which transports a wide variety of substrates, is highly expressed in placental syncytiotrophoblasts, in the canalicular membranes of liver, in the apical membrane of the small intestine epithelium, and at the luminal surface of the endothelial cells of human brain micro vessels. This strategic tissue localization indicates that ABCG2 plays an important role in absorption, distribution, and elimination of xenobiotics and drugs. High ABCG2 expression has also been detected in many hematological malignancies and solid tumors, indicating that ABCG2 is likely responsible also for the multidrug resistance in cancer chemotherapy. Indeed, ABCG2 can actively transport structurally diverse conjugated- or unconjugated-organic molecules and various anticancer drugs. Many chemo-sensitizing agents have been discovered, which can be developed for increasing drug adsorption and reversing drug resistance in cancer chemotherapy by inhibiting ABCG2 function or expression. This review summarizes current knowledge on ABCG2, its relevance to multidrug resistance and drug disposition, and its ever-growing numbers of substrates and inhibitors.
Background: NQO2 protects against ␥ radiation-induced myeloproliferative disease, but the mechanism remains unknown. Results: Radiation-induced NQO2, independent of NQO1, competes with the 20 S proteasome for interaction with C/EBP␣ region Ser-268 to Val-279 to stabilize C/EBP␣, leading to protection against myeloproliferative disease. Conclusion: NQO2 stabilizes C/EBP␣ against 20 S degradation to protect against myeloproliferative disease. Significance: Stress-responsive NQO2 functions as an endogenous factor against myeloproliferative diseases.
Background: NQO1 protects against myeloproliferative diseases. Results: Radiation-induced NQO1 competes with 20S proteasome for binding with myeloid differentiation factor C/EBP␣ leading to stabilization of C/EBP␣ and protection against myeloproliferative diseases. Conclusion: NQO1 is an endogenous factor in stabilization of C/EBP␣ and protection against myeloproliferative diseases during radiation and chemical stress. Significance: NQO1 might serve as therapeutic target for prevention of hematological diseases.
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that protects cells against radiation and chemical-inducedoxidative stress. Disruption of NQO1 gene in mice leads to increased susceptibility to myeloproliferative disease. In this report, we demonstrate that NQO1 controls the stability of myeloid differentiation factor C/EBP␣ against 20S proteasomal degradation during radiation exposure stress. Co-immunoprecipitation studies showed that NQO1, C/EBP␣, and 20S all interacted with each other. C/EBP␣ interaction with 20S led to the degradation of C/EBP␣. NQO1 in presence of its cofactor NADH protected C/EBP␣ against 20S degradation. Deletion and site-directed mutagenesis demonstrated that NQO1 and 20S competed for the same binding region 268 SGAGAG-KAKKSV 279 in C/EBP␣. Mutagenesis studies also revealed that NQO1Y127/Y129 required for NADH binding is essential for NQO1 stabilization of C/EBP␣. Exposure of mice and HL-60 cells to 3 Grays of ␥-radiation led to increased NQO1 that stabilized C/EBP␣ against 20S proteasomal degradation. This mechanism of NQO1 regulation of C/EBP␣ may provide protection to bone marrow against adverse effects of radiation exposure. The studies have significance for human individuals carrying hetero-or homozygous NQO1P187S mutation and are deficient or lack NQO1 protein.
NAD(P)H:quinone oxidoreductase 1 (NQO1)2 is a stress-inducible flavoprotein involved in cellular defense against the electrophilic and oxidizing metabolites (1-2). Induction or depletion of NQO1 levels are associated with decreased and increased susceptibilities to oxidative stress, respectively (3-4). NQO1 is known to bind to and stabilize tumor suppressor p53, p73␣, and p33 against 20S proteasomal degradation (5-6). These findings suggest that NQO1 exercises a selective "gatekeeping" role in regulating the proteasomal degradation of certain proteins.Hematopoiesis is a highly orchestrated multi-step process that turns the hematopoietic stem cells into various specialized and distinct blood cell types (7). This process is regulated by transcription factors including C/EBP␣ and PU.1 (7). C/EBP␣ is found predominantly in immature myeloid cells, whereas both lymphoid and myeloid cells express PU.1 (8 -9). C/EBP␣ is a leucine zipper factor (10 -11). Deregulation of C/EBP␣ has been found to be associated with myeloid transformation (12-13). The PU.1 gene contains two enhancer elements located 14 kilobases upstream of its promoter that bind C/EBP␣ and regulate expression of PU.1 (14).A cytosine to thymidine (C3 T)...
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