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Michael M. Gottesman - One of the best experts on this subject based on the ideXlab platform.
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P-GlycoProtein is not Present in mitochondrial membranes
Experimental Cell Research, 2007Co-Authors: Jill K. Paterson, Michael M. GottesmanAbstract:Recent rePorts have indicated the Presence of P-glycoProtein in crude mitochondrial membrane fractions, leading to the assumPtion that P-glycoProtein is Present in mitochondrial membranes, and may be involved in transPort across these membranes. To determine the validity of this claim, two cell lines overexPressing endogenous P-glycoProtein were investigated. Using various centrifugation stePs, mitochondria were Purified from these cells and analyzed by Western blot reaction with the anti-P-glycoProtein antibody C219 and organelle-sPecific antibodies. While P-glycoProtein is Present in crude mitochondrial fractions, these fractions are contaminated with Plasma membranes. Further Purification of the mitochondria to remove Plasma membranes revealed that P-glycoProtein is not exPressed in mitochondria of the KB-V1 (vinblastine-resistant KB-3-1 cells) or MCF-7(ADR) (adriamycin-resistant MCF-7 cells) cell lines. To further substantiate these findings, we used confocal microscoPy and the anti-P-glycoProtein antibody 17F9. This demonstrated that in intact cells, P-glycoProtein is not Present in mitochondria and is Primarily localized to the Plasma membrane. These findings are consistent with the role of P-glycoProtein in conferring multidrug resistance by decreasing cellular drug accumulation. Therefore, contrary to Previous sPeculation, P-glycoProtein does not confer cellular Protection by residing in mitochondrial membranes.
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P glycoProtein and multidrug resistance
Current Opinion in Genetics & Development, 1996Co-Authors: Michael M. Gottesman, Ira Pastan, Suresh V. AmbudkarAbstract:Although the Phenomenon of simultaneous resistance to multiPle cytotoxic drugs (multidrug resistance) in cancer cells has been discussed for more than two decades, and the human and mouse genes encoding an energy-dePendent transPorter (the multidrug transPorter or P-glycoProtein) resPonsible for multidrug resistance were cloned 10 years ago, there is still considerable controversy about the mechanism of action of this efflux PumP and its true biological function. This review summarizes the current research on the mechanism of action of the multidrug transPorter, including the hydroPhobic cleaner and altered Partitioning models, the Possible function of P-glycoProtein as a chloride and/or ATP channel, the role of PhosPhorylation in its function and fact and sPeculation about its Physiological role.
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Effects of PhosPhorylation of P-glycoProtein on multidrug resistance
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Ursula A. Germann, Suresh V. Ambudkar, Timothy C. Chambers, Ira Pastan, Michael M. GottesmanAbstract:Cells exPressing elevated levels of the membrane PhosPhoProtein P-glycoProtein exhibit a multidrug resistance PhenotyPe. Studies involving Protein kinase activators and inhibitors have imPlied that covalent modification of P-glycoProtein by PhosPhorylation may modulate its biological activity as a multidrug transPorter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells indePendent of, or in addition to their effects on the state of PhosPhorylation of P-glycoProtein. The Protein kinase(s) resPonsible for P-glycoProtein PhosPhorylation has(ve) not been unambiguously identified, although several Possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of PhosPhorylation are clustered within the linker region that connects the two homologous halves of P-glycoProtein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from PhosPhorylation- and dePhosPhorylation-defective mutants suggest that PhosPhorylation of P-glycoProtein is not essential to confer multidrug resistance.
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Characterization of the azidoPine and vinblastine binding site of P-glycoProtein.
The Journal of biological chemistry, 1992Co-Authors: Edward P. Bruggemann, Michael M. Gottesman, S J Currier, I PastanAbstract:To determine the number of drug binding sites that exist on the multidrug transPorter, P-glycoProtein, we used azidoPine, a dihydroPyridine Photoaffinity comPound that reverses multidrug resistance and labels P-glycoProtein. AzidoPine labels P-glycoProtein in two distinct locations: one labeled site is within the amino half of P-glycoProtein between amino acid residues 198 and 440, and the other site is within the carboxy half of the Protein. Vinblastine is a cytotoxic drug that is used in cancer chemotheraPy and is a substrate for transPort by P-glycoProtein. We found that vinblastine inhibits azidoPine labeling to aPProximately the same extent at each labeled site on P-glycoProtein. Because several studies have shown that amino acid residue 185 of P-glycoProtein Plays a critical role in some asPects of drug binding and transPort, we also studied the effect that amino acid residue 185 has on azidoPine labeling. These studies show that azidoPine labels both sites equivalently in both wild-tyPe (G185) and mutant (V185) P-glycoProteins. We conclude from our results that the two halves of P-glycoProtein aPProach each other to form a single binding site for these drugs.
Suresh V. Ambudkar - One of the best experts on this subject based on the ideXlab platform.
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P glycoProtein and multidrug resistance
Current Opinion in Genetics & Development, 1996Co-Authors: Michael M. Gottesman, Ira Pastan, Suresh V. AmbudkarAbstract:Although the Phenomenon of simultaneous resistance to multiPle cytotoxic drugs (multidrug resistance) in cancer cells has been discussed for more than two decades, and the human and mouse genes encoding an energy-dePendent transPorter (the multidrug transPorter or P-glycoProtein) resPonsible for multidrug resistance were cloned 10 years ago, there is still considerable controversy about the mechanism of action of this efflux PumP and its true biological function. This review summarizes the current research on the mechanism of action of the multidrug transPorter, including the hydroPhobic cleaner and altered Partitioning models, the Possible function of P-glycoProtein as a chloride and/or ATP channel, the role of PhosPhorylation in its function and fact and sPeculation about its Physiological role.
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Purification and reconstitution of functional human P-glycoProtein.
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Suresh V. AmbudkarAbstract:The overexPression of the P-glycoProtein, theMDR1 gene Product, has been linked to the develoPment of resistance to multiPle cytotoxic natural Product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoProtein, a member of the ATP-binding cassette (ABC) suPerfamily of transPorters, is believed to function as an ATP-dePendent drug efflux PumP with broad sPecificity for chemically unrelated hydroPhobic comPounds. We review here recent studies on the Purification and reconstitution of P-glycoProtein to elucidate the mechanism of drug transPort. P-glycoProtein from the human carcinoma multidrug resistant cell line, KB-V1, was Purified by sequential chromatograPhy on anion exchange followed by a lectin (wheat germ agglutinin) column. ProteoliPosomes reconstituted with Pure Protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dePendent [3H]vinblastine accumulation. Both the ATPase and vinblastine transPort activities of the reconstituted P-glycoProtein were inhibited by vanadate. In addition, the vinblastine transPort was inhibited by veraPamil and daunorubicin. These studies Provide strong evidence that the human P-glycoProtein functions as an ATP-dePendent drug transPorter. The develoPment of the reconstitution system and the availability of recombinant Protein in large amounts due to recent advances in overexPression of P-glycoProtein in a heterologous exPression system should facilitate a better understanding of the function of this novel Protein.
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Effects of PhosPhorylation of P-glycoProtein on multidrug resistance
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Ursula A. Germann, Suresh V. Ambudkar, Timothy C. Chambers, Ira Pastan, Michael M. GottesmanAbstract:Cells exPressing elevated levels of the membrane PhosPhoProtein P-glycoProtein exhibit a multidrug resistance PhenotyPe. Studies involving Protein kinase activators and inhibitors have imPlied that covalent modification of P-glycoProtein by PhosPhorylation may modulate its biological activity as a multidrug transPorter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells indePendent of, or in addition to their effects on the state of PhosPhorylation of P-glycoProtein. The Protein kinase(s) resPonsible for P-glycoProtein PhosPhorylation has(ve) not been unambiguously identified, although several Possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of PhosPhorylation are clustered within the linker region that connects the two homologous halves of P-glycoProtein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from PhosPhorylation- and dePhosPhorylation-defective mutants suggest that PhosPhorylation of P-glycoProtein is not essential to confer multidrug resistance.
Victor Ling - One of the best experts on this subject based on the ideXlab platform.
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Reconstitution of Drug TransPort by Purified P-glycoProtein
Journal of Biological Chemistry, 1995Co-Authors: Adam B. Shapiro, Victor LingAbstract:Abstract P-glycoProtein confers multidrug resistance uPon cells in which it is highly exPressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotheraPy of cancer. Although P-glycoProtein is widely believed to function as an ATP-dePendent drug efflux PumP, the unusually broad substrate sPecificity of P-glycoProtein has engendered the ProPosal of other, less direct mechanisms. None of the hyPothetical mechanisms has been definitively tested, however, in a Purified system where other cellular comPonents and Processes are absent. We have used a fluorescent substrate of P-glycoProtein, Hoechst 33342, to measure transPort activity in real-time of highly Purified P-glycoProtein in a reconstituted liPosome system in which the P-glycoProtein has a uniformly inside-out orientation. Using this system, we demonstrated MgATPdePendent, chemosensitizer-inhibitable transPort of Hoechst 33342. TransPort was Prevented by omission of Mg, by substitution of nonhydrolyzable adenylyl-β,-imidodiPhosPhate for ATP, by inhibition of the ATPase activity of P-glycoProtein with vanadate and N-ethylmaleimide, and by the chemosensitizers veraPamil and amiodarone. Measurements of intraliPosomal PH during Hoechst 33342 transPort detected no large PH changes in P-glycoProtein-containing liPosomes. These results are inconsistent with a mechanism in which P-glycoProtein affects drug accumulation by directly altering intracellular PH. The Hoechst 33342 transPort assay results are consistent with mechanisms in which P-glycoProtein alone is sufficient to transPort drugs out of the membrane bilayer.
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Using Purified P-glycoProtein to understand multidrug resistance
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Adam B. Shapiro, Victor LingAbstract:Since P-glycoProtein was discovered almost 20 years ago, its causative role in multidrug resistance has been established, but central Problems of its biochemistry have not been definitively resolved. Recently, major advances have been made in P-glycoProtein biochemistry with the use of Purified and reconstituted P-glycoProtein, as well as membranes from nonmammalian cells containing heterologously exPressed P-glycoProtein. In this review we describe recent findings using these systems which are elucidating the molecular mechanism of P-glycoProtein-mediated drug transPort.
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Identification of distinct P-glycoProtein gene sequences in rat.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1992Co-Authors: Kathryn L. Deuchars, Monika Duthie, Victor LingAbstract:Abstract In higher vertebrates, P-glycoProtein is usually encoded by a small family of genes. We have determined that the rat contains three P-glycoProtein genes and have cloned distinct genomic fragments containing the Putative 3′ untranslated regions of these P-glycoProtein genes. Sequence analysis indicates that the rat P-glycoProtein genes belong to the three P-glycoProtein classes identified in mammals. These cloned sequences will be useful for delineating the exPression of P-glycoProtein genes in the rat. We have also isolated a fourth clone which contains only a short, but highly conserved P-glycoProtein domain. This clone aPPears not be a member of the P-glycoProtein gene family, and its relationshiP to P-glycoProtein is unknown.
Tohru Komano - One of the best experts on this subject based on the ideXlab platform.
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CePharanthin, a multidrug resistant modifier, is a substrate for P- glycoProtein
The Journal of pharmacology and experimental therapeutics, 1995Co-Authors: Maretoshi Hirai, Kazumitsu Ueda, Yusuke Tanigawara, Ryohei Hori, K. Tanaka, Takao Shimizu, Masato Yasuhara, Yoshiyuki Kakehi, Osamu Yoshida, Tohru KomanoAbstract:P-glycoProtein modulators are resPected to be multidrug resistance reversing agents in cancer chemotheraPy. Some calcium channel blockers, calmodulin inhibitors or immunosuPPressive agents have been used in clinical studies, although the dose of these drugs required to test in vitro exPerimental data might cause Potent Pharmacological effects which are not desirable in Patients. By using LLC-GA5-COL150 cells that exPress P-glycoProtein sPecifically on the aPical membranes, we examined the transPort of anticancer drugs mediated by P-glycoProtein. CePharanthin, a biscoclaurine alkaloid, Potently inhibits the transPort of vinblastine and daunorubicin, both commonly used anticancer agents. The 50% inhibitory concentration of cePharanthin on daunorubicin transPort was 2.06 microM. Combined inhibitory effects on daunorubicin transPort were observed when cePharanthin was used together with cyclosPorin A, a Potent immunosuPPressive agent and P-glycoProtein modulator. CePharanthin itself was transPorted by P-glycoProtein. Transcellular transPort of cePharanthin across LLC-GA5-COL150 cell monolayers was saturable when its concentration was under 5 microM, and the transPort was inhibited by P-glycoProtein modulators. These results indicate that cePharanthin can reverse multidrug resistance, and ProPer combination with other P-glycoProtein modulators could Potentiate its inhibitory effect on exPelling the anticancer drugs out of the cell via P-glycoProtein.
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P glycoProtein mediated transcellular transPort of mdr reversing agents
FEBS Letters, 1993Co-Authors: Tohru Saeki, Kazumitsu Ueda, Yusuke Tanigawara, Ryohei Hori, Tohru KomanoAbstract:Understanding of the interactions between P-glycoProtein and multidrug resistance (MDR) reversing agents is imPortant in designing more effective MDR modulators. We examined transcellular transPort of several MDR modulators by using a drug-sensitive ePithelial cell line, LLC-PK1 and its transformant cell line, LLC-GA5-COL300, which exPresses human P-glycoProtein on the aPical surface. Basal-to-aPical transPorts of azidoPine and diltiazem across the LLC-GA5-COL300 monolayer were increased and aPical-to-basal transPorts were decreased comPared to those across the LLC-PK1 monolayer, indicating that P-glycoProtein transPorts azidoPine and diltiazem. Movements of nitrendiPine and staurosPorine across the ePithelial monolayer were not affected by P-glycoProtein. These results suggests that some MDR modulators exert their inhibitory effect not only by blocking the initial binding of anticancer drugs but throughout the course of the transPort Process.
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Human P-glycoProtein transPorts cyclosPorin A and FK506.
The Journal of biological chemistry, 1993Co-Authors: Tohru Saeki, Kazumitsu Ueda, Yusuke Tanigawara, Ryohei Hori, Tohru KomanoAbstract:CyclosPorin A, a cyclic undecaPePtide, and FK506 are efficient immunosuPPressive agents. They also attract attention as effective P-glycoProtein modulators that inhibit P-glycoProtein from binding to anticancer drugs and overcome multidrug resistance. CyclosPorin A itself interacts with a common binding site of P-glycoProtein to which Vinca alkaloids and veraPamil bind. We were interested to determine whether cyclosPorin A and FK506 are substrates for P-glycoProtein to transPort, and we studied their transcellular transPort. In LLC-PK1 cells, derived from Porcine kidney Proximal tubule and forming a highly Polarized ePithelium, cyclosPorin A was transPorted in a saturable manner. LLC-GA5-COL300, a transformant cell line derived by transfecting LLC-PK1 with human MDR1 cDNA isolated from normal adrenal gland, exPresses P-glycoProtein sPecifically on the aPical surface and shows a tyPical multidrug-resistant PhenotyPe. LLC-GA5-COL300 cells showed increased transPort of cyclosPorin A from the basal to the aPical side. Kinetic analysis showed that this transPort was a tyPical saturable transPort with the calculated aPParent Michaelis constant (KaPPm) and the maximum flux (Vmax) as 8.4 microM and 2.4 nmol/mg Protein/h, resPectively. LLC-GA5-COL300 also showed increased transPort of FK506 from the basal to the aPical side. These results indicate that P-glycoProtein transPorts the immunosuPPressive agents cyclosPorin A and FK506.
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human P glycoProtein transPorts cortisol aldosterone and dexamethasone but not Progesterone
Journal of Biological Chemistry, 1992Co-Authors: Kazumitsu Ueda, Tohru Saeki, Tohru Komano, Yusuke Tanigawara, Maretoshi Hirai, N Okamura, Noriyuki Kioka, Ryohei HoriAbstract:Abstract We exPressed human MDR1 cDNA isolated from the human adrenal gland in Porcine LLC-PK1 cells. A highly Polarized ePithelium formed by LLC-GA5-COL300 cells that exPressed human P-glycoProtein sPecifically on the aPical surface showed a multidrug-resistant PhenotyPe and had 8.3-, 3.4-, and 6.5-fold higher net basal to aPical transPort of 3H-labeled cortisol, aldosterone, and dexamethasone, resPectively, comPared with host cells. But Progesterone was not transPorted, although it inhibited azidoPine Photoaffinity labeling of human P-glycoProtein and increased the sensitivity of multidrug-resistant cells to vinblastine. An excess of Progesterone inhibited the transePithelial transPort of cortisol by P-glycoProtein. These results suggest that cortisol and aldosterone are Physiological substrates for P-glycoProtein in the human adrenal cortex and that substances that efficiently bind to P-glycoProtein are not necessarily transPorted by P-glycoProtein.
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ExPression of human P-glycoProtein in yeast cells--effects of membrane comPonent sterols on the activity of P-glycoProtein.
Agricultural and Biological Chemistry, 1991Co-Authors: Tohru Saeki, Tohru Komano, Alfredo M. Shimabuku, Yoshinao Azuma, Yuji Shibano, Kazumitsu UedaAbstract:A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoProtein Produced was situated in its native orientation in the yeast Plasma membrane. Drug-binding activities of the recombinant P-glycoProteins were markedly decreased comPared to that of the authentic P-glycoProtein. To identify the bases of decreased binding we studied the effects of membrane comPonent sterols on the azidoPine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is Produced from ergosterol by UV irradiation, inhibited azidoPine binding. These sterols in yeast membrane Probably inhibit the function of human P-glycoProtein as a multidrug transPorter in yeast cells, because exPression of P-glycoProtein in yeast cells did not confer resistance to doxorubicin.
Christopher F. Higgins - One of the best experts on this subject based on the ideXlab platform.
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communication between multiPle drug binding sites on P glycoProtein
Molecular Pharmacology, 2000Co-Authors: Catherine Martin, Georgina Berridge, Prakash Mistry, Christopher F. Higgins, Peter Charlton, Richard CallaghanAbstract:P-glycoProtein, a member of the ATP-binding cassette transPorter family, is able to confer resistance on tumors against a large number of functionally and chemically distinct cytotoxic comPounds. Several recent investigations suggest that P-glycoProtein contains multiPle drug binding sites rather than a single site of broad substrate sPecificity. In the Present study, radioligand-binding techniques were used to directly characterize drug interaction sites on P-glycoProtein and how these multiPle sites interact. The drugs used were classified as either 1) substrates, which are known to be transPorted by P-glycoProtein (e.g., vinblastine) or 2) modulators, which alter P-glycoProtein function but are not themselves transPorted by the Protein (e.g., XR9576). Drug interactions with P-glycoProtein were either comPetitive, at a common site, or noncomPetitive, and therefore at distinct sites. Based on these data, we can assign a minimum of four drug binding sites on P-glycoProtein. These sites fall into two categories: transPort, at which translocation of drug across the membrane can occur, and regulatory sites, which modify P-glycoProtein function. Intriguingly, however, some modulators interact with P-glycoProtein at a transPort site rather than a regulatory site. The Pharmacological data also demonstrate that both transPort and regulatory sites are able to switch between high- and low-affinity conformations. The multiPle sites on P-glycoProtein disPlay comPlex allosteric interactions through which interaction of drug at one site switches other sites between high- or low-affinity conformations. The data are discussed in terms of a model for the mechanism of transPort by P-glycoProtein.
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The multidrug resistance P-glycoProtein
Current Opinion in Cell Biology, 1993Co-Authors: Christopher F. HigginsAbstract:Abstract P-glycoProtein Plays an imPortant role in the resistance of cancers to chemotheraPy. Thus, an understanding of the mechanism by which it functions, and its ‘normal’ Physiological role, is of clinical relevance as well as intrinsic interest. Considerable Progress towards this goal has been made in the last year or so. In addition, the finding that P-glycoProtein is associated with both a channel and a transPorter activity has, Potentially, far-reaching imPlications for an understanding of the nature of channels and transPortes.
