Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are closely related mammalian ATP-binding cassette transporters that export organic ...anions from cells. Transfection studies have established that MRP confers resistance to natural product cytotoxic agents, and recent evidence suggests the possibility that cMOAT may contribute to cytotoxic drug resistance as well. Based upon the potential importance of these transporters in clinical drug resistance and their important physiological roles in the export of the amphiphilic products of phase I and phase II metabolism, we sought to identify other MRP-related transporters. Using a degenerate PCR approach, we isolated a cDNA that encodes a novel ATP-binding cassette transporter, which we designated MOAT-B. The MOAT-B gene was mapped using fluorescence in situ hybridization to chromosome band 13q32. Comparison of the MOAT-B predicted protein with other transporters revealed that it is most closely related to MRP, cMOAT, and the yeast organic anion transporter YCF1. Although MOAT-B is closely related to these transporters, it is distinguished by the absence of a approximately 200 amino acid NH2-terminal hydrophobic extension that is present in MRP and cMOAT and which is predicted to encode several transmembrane spanning segments. In addition, the MOAT-B tissue distribution is distinct from MRP and cMOAT. In contrast to MRP, which is widely expressed in tissues, including liver, and cMOAT, the expression of which is largely restricted to liver, the MOAT-B transcript is widely expressed, with particularly high levels in prostate, but is barely detectable in liver. These data indicate that MOAT-B is a ubiquitously expressed transporter that is closely related to MRP and cMOAT and raise the possibility that it may be an organic anion pump relevant to cellular detoxification.
NAD(P)H:Quinone Oxidoreductase1 (NQO1) also known as DT-diaphorase is a flavoprotein that catalyzes the two-electron reduction of quinones, quinone imines and azo-dyes and thereby protects cells ...against mutagenicity and carcinogenicity resulting from free radicals and toxic oxygen metabolites generated by the one-electron reductions catalyzed by cytochromes P450 and other enzymes. High levels of NQO1 gene expression have been observed in liver, lung, colon and breast tumors as compared to normal tissues of the same origin. The transcription of the NQO1 gene is activated in response to exposure to bifunctional (e.g. beta-naphthoflavone (beta-NF), 2, 3, 7, 8 tetrachorodibenzo-p-dioxin (TCDD)) and monofunctional (phenolic antioxidants/chemoprotectors e.g. 2(3)-tert-butyl-4-hydroxy-anisole (BHA)) inducers. The high level of expression of the NQO1 gene and its induction by beta-NF and BHA require the presence of an AP1 binding site contained within the human Antioxidant Response Element (hARE) and are mediated by products of proto-oncogenes, Jun and Fos. Induction of NQO1 gene expression involves transfer of a redox signal from xenobiotics to unknown 'redox protein(s)' which in turn, modify the Jun and Fos proteins for greater affinity towards the AP1 site of the NQO1 gene and activates transcription. The expression and regulation of the NQO1 gene is complex as many additional cis-elements have been identified in the promoter region and is a subject of great future interest. In addition to established tumors, NQO1 gene expression is also increased in developing tumors, indicating a role in cellular defense during tumorigenesis. It has been proposed that low molecular weight substance(s) can diffuse from tumor cells into surrounding normal cells and activate the expression of the NQO1 gene. Purification and characterization of such substance(s) may provide important information in regard to the mechanism of activation of NQO1 gene expression and the role of increased NQO1 expression in tumor development. In view of the general consensus that NQO1 is over-expressed in tumor cells and the realization that NQO1 may either activate or detoxify xenobiotics, it is important to establish the role of NQO1 in the activation, and the detoxification of xenobiotics and drugs and in the intrinsic sensitivity of tumors to bioreductive alkylating aziridinyl benzoquinones such as diaziquone (AZQ), mitomycin C (MMC), and indoloquinone EO9, as well as to the dinitrophenyl aziridine, CB1954, and the benzotriazine-di-N-oxide, SR 4233.
The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export ...into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostα-Ostβ exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostα-Ostβ have not been investigated. To determine the role of Ostalpha-Ostbeta in intestinal bile acid absorption, the Ostalpha gene was disrupted by homologous recombination in mice. Ostα... mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostα... vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostα..Mrp3... mice. The bile acid pool size was significantly reduced (>65%) in Ostα... mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostα... mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostα-Ostν is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis. (ProQuest: ... denotes formulae/symbols omitted.)
Antioxidant response elements (AREs) containing 12-O-tetradecanoylphorbol-13-acetate response element (TRE) (perfect AP1) and TRE-like (imperfect AP1) elements mediate high basal transcription of the ...NAD(P)H:quinone oxidoreductase1 (NQO1) and glutathione S-transferase Ya genes in tumor cells and its induction in response to xenobiotics and antioxidants. Mutations in the human NQO1 gene ARE (hARE) revealed the requirement for two TRE or TRE-like elements arranged in inverse orientation at the interval of three base pairs and a GC box for optimal expression and beta-naphthoflavone induction of the NQO1 gene. A single TRE element from the human collagenase gene failed to respond to beta-naphthoflavone. These results demonstrate that ARE (2 x TRE or TRE-like elements)-containing detoxifying enzyme genes and not genes that contain 1 x TRE are responsive to xenobiotics and antioxidants. Bandshift assays showed shifting of a complex of more or less similar mobility with hARE and TRE that could be competed by each other. Mutations in the 3'-TRE of the NQO1 gene hARE eliminated binding of nuclear proteins to the hARE and resulted in the loss of basal and induced expression, indicating that 3'-TRE is the most important element within the hARE. 5'-TRE-like element within the NQO1 gene hARE is required for xenobiotic response but may not bind to the nuclear proteins by itself. The GC box located immediately following the 3'-TRE is required for optimal expression and induction of the NQO1 gene. The comparison of AREs from several different genes indicated the requirement for specific arrangement and spacing of two TRE and TRE-like elements within the AREs.
Antioxidant response elements (AREs) containing 12- O -tetradecanoylphorbol-13-acetate response element (TRE) (perfect AP1) and TRE-like (imperfect AP1) elements mediate high basal
transcription of ...the NAD(P)H:quinone oxidoreductase 1 (NQO 1 ) and glutathione S -transferase Ya genes in tumor cells and its induction in response to xenobiotics and antioxidants. Mutations in the human
NQO 1 gene ARE (hARE) revealed the requirement for two TRE or TRE-like elements arranged in inverse orientation at the interval
of three base pairs and a GC box for optimal expression and β-naphthoflavone induction of the NQO 1 gene. A single TRE element from the human collagenase gene failed to respond to β-naphthoflavone. These results demonstrate
that ARE (2 Ã TRE or TRE-like elements)-containing detoxifying enzyme genes and not genes that contain 1 Ã TRE are responsive
to xenobiotics and antioxidants. Bandshift assays showed shifting of a complex of more or less similar mobility with hARE
and TRE that could be competed by each other. Mutations in the 3â²-TRE of the NQO 1 gene hARE eliminated binding of nuclear proteins to the hARE and resulted in the loss of basal and induced expression, indicating
that 3â²-TRE is the most important element within the hARE. 5â²-TRE-like element within the NQO 1 gene hARE is required for xenobiotic response but may not bind to the nuclear proteins by itself. The GC box located immmediately
following the 3â²-TRE is required for optimal expression and induction of the NQO 1 gene. The comparison of AREs from several different genes indicated the requirement for specific arrangement and spacing
of two TRE and TRE-like elements within the AREs.
Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues, with considerable intra- and interfamiliar phenotypic variability. PXE is caused ...by mutations in the ABCC6 gene, which encodes a transporter protein, MRP6, and targeted ablation of Abcc6 in mice recapitulates the manifestations of PXE. In this study, we examined the hypothesis that the expression of other members of the Abcc family may be altered in Abcc6 null mice, possibly explaining the phenotypic variability because of the functional overlap of these transporters. Analysis of the transcript levels of Abcc1-10 and 12 in the liver of Abcc6 super(---) mice by quantitative RT-PCR indicated that the levels of other C family mRNAs were not significantly different from wild-type mice. Next, we developed Abcc6-1 super(---) and Abcc6-3 super(---) double null mice and examined them for tissue mineralization. Histopathologic examination, coupled with computerized morphometric analysis, and chemical assay of calcium x phosphate product in the muzzle skin of Abcc1 super(---) and Abcc3 super(---) mice did not reveal evidence of mineralization. Abcc6-1 super(---) and Abcc6-3 super(---) double knock-out mice exhibited connective tissue mineralization similar to that in Abcc6 super(---) mice. These results emphasize the importance of the Abcc6 gene in the ectopic mineralization process and further suggest that other members of the Abcc family, particularly Abcc1 and Abcc3, do not modulate the effects of Abcc6 in this mouse model.
Overexpression of multidrug resistance (MDR1) P-glycoprotein (Pgp) remains an important barrier to successful chemotherapy in cancer patients and impacts the pharmacokinetics of many important drugs, ...thus evoking a need to noninvasively interrogate Pgp transport activity in vivo.
Cell tracer transport experiments as well as mouse biodistribution and microPET imaging studies were performed to characterize a nonmetabolized gallium(III) complex, gallium(III)-(bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-amino-propyl)ethylenediamine) (Ga-3-ethoxy-ENBDMPI)(+), as a candidate SPECT ((67)Ga) and generator-produced PET ((68)Ga) radiopharmaceutical recognized by MDR1 Pgp.
The (67)Ga-complex showed high membrane potential-dependent accumulation in drug-sensitive KB3-1 cells and modulator-reversible low accumulation in MDR KB8-5 cells. In KB8-5 cells, the median effective concentrations (EC(50)) of MDR modulators LY335979, PSC 833, and cyclosporin A were 69 nmol/L, 1 micromol/L, and 3 micromol/L, respectively. Using a variety of cells stably expressing MDR1 Pgp, multidrug resistance-associated proteins (MRP1-MRP6), or the breast cancer resistance protein (BCRP/MXR), the (67)Ga-complex was shown to be readily transported by MDR1 Pgp and, to a much lesser extent, by MRP1, but not MRP2-MRP6 or BCRP/MXR. In a nude mouse xenograft tumor model, the (67)Ga-complex produced a readily detected 3-fold difference between Pgp-expressing tumors and drug-sensitive tumors in the opposite flank. In mdr1a/1b(-/-) gene-deleted mice, the (67)Ga-complex showed 17-fold greater brain uptake and retention compared with wild-type mice with no net difference in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. This could be readily observed with microPET using the (68)Ga-complex. Incidentally, wild-type mice showed heart-to-blood ratios of >100 by 1 h after injection and heart-to-liver ratios of 2.2 by 120 min.
Molecular imaging of the functional transport activity of MDR1 Pgp with ((67/68)Ga-3-ethoxy-ENBDMPI)(+) may enable noninvasive SPECT/PET monitoring of the blood-brain barrier, chemotherapeutic regimens, and MDR1 gene therapy protocols in vivo. These Pgp-directed properties of the radiopharmaceutical may also translate favorably to myocardial perfusion imaging.
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