Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.
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PMID:A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). 1140 94

ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer. Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated membrane preparations. ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein. ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N-ethylmaleimide and cyclosporin A. Verapamil had no effect, while Fumitremorgin C, reversing ABCG2-dependent cancer drug resistance, strongly inhibited this ATPase activity. The functional expression of ABCG2 in this heterologous system indicates that no additional partner protein is required for the activity of this multidrug transporter, probably working as a homodimer. We suggest that the Sf9 cell membrane ATPase system is an efficient tool for examining the interactions of ABCG2 with pharmacological agents.
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PMID:Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. 1143 80

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.
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PMID:From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. 1149 41

This is the first report to show that a copper-transporting P-type adenosine triphosphatase, ATP7B, is expressed in certain breast carcinomas, and a priori knowledge of its expression is important for the choice of therapy. We investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain breast carcinomas. To test this hypothesis, ATP7B expression and protein level were examined in 41 breast carcinomas using RT-PCR and immunohistochemistry. ATP7B gene / protein could be detected in 22.0% (9 / 41) of breast carcinomas and ATP7B gene expression was correlated well with the protein expression. In nine ATP7B-positive tumors, adjacent normal breast tissue was similarly analyzed, revealing that ATP7B is upregulated in breast carcinoma. ATP7B gene expression in poorly differentiated carcinoma was significantly higher than that in well- / moderately differentiated carcinoma (P = 0.012). Furthermore, we found no association between the ATP7B gene / protein expression and that of MDR1, MRP1, LRP and BCRP. These findings suggested that ATP7B gene expression might be a chemoresistance marker for cisplatin in patients with poorly differentiated breast carcinoma.
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PMID:Copper-transporting P-type adenosine triphosphatase (ATP7B) is expressed in human breast carcinoma. 1180 10

Intrinsic or acquired resistance to chemotherapy is the major obstacle to overcome in the treatment of patients with solid carcinoma. Cisplatin is one of the most effective chemotherapeutic agents for treating ovarian carcinoma. Recently, copper-transporting P-type adenosine triphosphatase (ATP7B) has been demonstrated as one of the genes responsible for cisplatin resistance in vitro. We hypothesized that the expression of ATP7B gene increases resistance to cisplatin in ovarian carcinoma and a priori knowledge of its expression is important for the choice of therapy. The aim of our study was to assess the role of ATP7B gene in ovarian carcinoma and compare its expression with those of multidrug resistance-related transporters such as MDR1, MRP1, MRP2, LRP and BCRP genes. The transporters' gene expression profiles from 82 patients treated with cisplatin-based chemotherapy after surgery were assessed by RT-PCR. We did not observe any significant correlation between ATP7B gene expression and those of MDR1, MRP1, MRP2, LRP or BCRP. The expression level of ATP7B gene was significantly increased (p < 0.05) in patients with moderately-/poorly-differentiated ovarian carcinomas treated with cisplatin-based chemotherapy, thus ATP7B may serve as an independent prognostic factor in these patients. In contrast, the expression level of MDR1, MRP1, MRP2, LRP and BCRP genes were not prognostic indicators of disease. These findings suggest that ATP7B gene may be considered as a novel chemoresistance marker and that inhibitor(s) of ATP7B might be useful, in patients with ovarian carcinoma treated with cisplatin-based chemotherapy.
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PMID:Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cisplatin based chemoresistance marker in ovarian carcinoma: comparative analysis with expression of MDR1, MRP1, MRP2, LRP and BCRP. 1221 79

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the anti-estrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [3H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.
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PMID:Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis. 1266 85

ABCG2/MXR/ABCP1/BCRP is a member of the ATP-binding cassette membrane transporter, which consists of six transmembrane regions and one ATP-binding cassette. The transporter is known to be involved in the efflux of various anticancer compounds such as mitoxantrone, doxorubicin and topoisomerase I inhibitor. In this study, we analyzed the effects of polymorphisms in ABCG2, V12M and Q141K on transporter function. When polarized LLC-PK1 cells were transfected with variant ABCG2, drug-resistance to topoisomerase I inhibitor of cells expressing V12M or Q141K was less than 1/10 that of wild-type ABCG2 transfected cells, and was accompanied by increased drug accumulation and decreased drug efflux in the variant ABCG2-expressing cells. We further elucidated the molecular mechanisms of the transport dysfunction by investigating membrane localization and ATPase activity. Confocal microscopic analysis revealed that apical plasma membrane localization of V12M was disturbed, while the localization of wild-type transporters occurred specifically in the apical plasma membrane of polarized LLC-PK1 cells. Also, ATPase activities measured in the membrane of SF9 cells infected with variant ABCG2 showed that Q141K decreased activity by 1.3 below that of wild-type ABCG2. In addition, kinetic analysis of ATPase activity showed that the K(m) value in Q141K was 1.4-fold higher than that of wild-type ABCG2. These results indicated that naturally occurring SNPs alter transport functions of ABCG2 transporter and analysis of SNPs in ABCG2 may hold great importance in understanding the response/metabolism of chemotherapy compounds that act as substrates for ABCG2.
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PMID:Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2. 1475 Jan 75

Multidrug transporters influence drug distribution in vivo and are often associated with tumour drug resistance. Here we show that plant-derived polyphenols that interact with P-glycoprotein can also modulate the activity of the recently discovered ABC transporter, breast cancer resistance protein (BCRP/ABCG2). In two separate BCRP-overexpressing cell lines, accumulation of the established BCRP substrates mitoxantrone and bodipy-FL-prazosin was significantly increased by the flavonoids silymarin, hesperetin, quercetin, and daidzein, and the stilbene resveratrol (each at 30 microM) as measured by flow cytometry, though there was no corresponding increase in the respective wild-type cell lines. These compounds also stimulated the vanadate-inhibitable ATPase activity in membranes prepared from bacteria (Lactococcus lactis) expressing BCRP. Given the high dietary intake of polyphenols, such interactions with BCRP, particularly in the intestines, may have important consequences in vivo for the distribution of these compounds as well as other BCRP substrates.
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PMID:Interaction of the breast cancer resistance protein with plant polyphenols. 1504 79

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.
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PMID:Functional expression of the human breast cancer resistance protein in Pichia pastoris. 1524 Jan 9

ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.
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PMID:Mutational analysis of ABCG2: role of the GXXXG motif. 1526 Apr 87


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