Phosphorylation of the Multidrug Resistance Encoded Protein P190

Ma, Liandong; Nandigama, Krishnamachary; Center, Melvin S.

doi:10.1021/bi00010a024

Cited by 53 publications

(23 citation statements)

References 8 publications

(8 reference statements)

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“…Several studies report an increase in PKC activity in cells with an overexpression of P-gP or MRP (Beck et al, 1998;Ratnasinghe et al, 1998). Pglycoprotein and MRP are phosphorylated (Ma et al, 1995;Clavy et al, 1997); however, it is a matter of debate whether phosphorylation modulates the pump function (Ma et al, 1995;Smith and Zilfou, 1995;Clavy et al, 1997;Ratnasinghe et al, 1998). Since dCK may be phosphorylated by PKC, and exhibits a higher activity in the phosphorylated state (Wang and Kucera, 1994), PKC might play a role in the collateral sensitivity to gemcitabine of MDR cells.…”

Section: Discussionmentioning

confidence: 99%

Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines

et al. 2003

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Gemcitabine (2 0 ,2 0 -difluorodeoxycytidine) is a deoxycytidine analogue that is activated by deoxycytidine kinase (dCK) to its monophosphate and subsequently to its triphosphate dFdCTP, which is incorporated into both RNA and DNA, leading to DNA damage. Multidrug resistance (MDR) is characterised by an overexpression of the membrane efflux pumps P-glycoprotein (P-gP) or multidrug resistance-associated protein (MRP). Gemcitabine was tested against human melanoma, non-small-cell lung cancer, smallcell lung cancer, epidermoid carcinoma and ovarian cancer cells with an MDR phenotype as a result of selection by drug exposure or by transfection with the mdr1 gene. These cell lines were nine-to 72-fold more sensitive to gemcitabine than their parental cell lines. The doxorubicin-resistant cells 2R120 (MRP1) and 2R160 (P-gP) were nine-and 28-fold more sensitive to gemcitabine than their parental SW1573 cells, respectively (Po0.01), which was completely reverted by 25 mM verapamil. In 2R120 and 2R160 cells, dCK activities were seven-and four-fold higher than in SW1573, respectively, which was associated with an increased dCK mRNA and dCK protein. Inactivation by deoxycytidine deaminase was 2.9-and 2.2-fold decreased in 2R120 and 2R160, respectively. dFdCTP accumulation was similar in SW1573 and its MDR variants after 24 h exposure to 0.1 mM gemcitabine, but dFdCTP was retained longer in 2R120 (Po0.001) and 2R160 (Po0.003) cells. 2R120 and 2R160 cells also incorporated four-and six-fold more [ 3 H]gemcitabine into DNA (Po0.05), respectively. P-glycoprotein and MRP1 overexpression possibly caused a cellular stress resulting in increased gemcitabine metabolism and sensitivity, while reversal of collateral gemcitabine sensitivity by verapamil also suggests a direct relation between the presence of membrane efflux pumps and gemcitabine sensitivity.

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Section: Discussionmentioning

confidence: 99%

Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines

et al. 2003

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“…(It is extremely unlikely to play a major role in intrinsic resistance of LoVo/C7 cells anyway, as P-gp expression is undetectable in these cells.) On the other hand, recent reports indicate that MRP is also a substrate for PKC (Gekeler et al, 1995;Ma et al, 1995), and the possibility that phosphorylation might positively modulate MRP activity could be more relevant to the resistant phenotype of LoVo/C7 cells. However, this hypothesis awaits further experimental support.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a clonal human colon adenocarcinoma line intrinsically resistant to doxorubicin

Dolfini

Dasdia²,

Arancia³

et al. 1997

Br J Cancer

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Summary Intrinsic low-level resistance to anti-cancer drugs is a major problem in the treatment of gastrointestinal malignancies. To address the problem presented by intrinsically resistant tumours, we have isolated two monoclonal lines from LoVo human colon adenocarcinoma cells: LoVo/C7, which is intrinsically resistant to doxorubicin (DOX); and LoVo/C5, which shows the same resistance index for DOX as the mixed parental cell population. For comparison, we have included in the study a LoVo-resistant line selected by continuous exposure to DOX and expressing a typical multidrug resistant (MDR) phenotype. In these cell lines we have studied the expression and/or activity of a number of proteins, including P-glycoprotein 170 (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione (GSH)-dependent enzymes and protein kinase C (PKC) isoforms, which have been implicated in anti-cancer drug resistance. Intracellular DOX distribution has been assessed by confocal microscopy. The results of the present study indicate that resistance in LoVo/C7 cells cannot be attributed to alterations in P-gp, LRP or GSH/GSH-dependent enzyme levels. Increased expression of MRP, accompanied by alterations in the subcellular distribution of DOX, has been observed in LoVo/C7 cells; changes in PKC isoform pattern have been detected in both intrinsically and pharmacologically resistant cells.

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“…This is in contrast to the molecular weights observed for the C2 fragments generated from KB/ MRP, Sf/MRP, and for the MRP1-(1264 -1531) fragment when analyzed by SDS-PAGE in which they migrate as a 35-, 36-, and 36-kDa band, respectively. The slower migration of these peptides on SDS-PAGE compared with the predicted molecular weight may perhaps be attributed to post-translational modifications (25).…”

Section: Gsh Inhibits the Generation Of A C-terminal Tryptic Fragmentmentioning

confidence: 96%

GSH Inhibits Trypsinization of the C-terminal Half of Human MRP1

Ren

Furukawa

Nakajima

et al. 2005

Journal of Biological Chemistry

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MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an ϳ35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and coexpressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L 0 region of MRP1. Thus the L 0 region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates. MDR1 is the major obstacle to successful cancer chemotherapy and is mediated by membrane proteins whose mechanism of action is not yet completely understood (1). MRP1 (multidrug resistance protein 1) is a 190-kDa glycoprotein that belongs to a family of membrane proteins referred to as ATP-binding cassette (ABC) transporters that typically contain two transmembrane domains (TMD) and two nucleotide binding domains (NBD) (4). In most ABC transporters, hydrolysis of ATP by the NBDs is believed to provide energy for substrate transport (4). MRP1 is distinguished from the other ABC transporter, P-glycoprotein (P-gp), by an extra N-terminal TMD that is connected to the core region (⌬MRP) by a cytoplasmic linker region (L 0 ) (5). The human MRP1 is frequently overexpressed in cells whose MDR is not mediated by P-gp (2, 3). As an organic anion transporter, MRP1 actively transports a wide variety of diverse anionic compounds (6 -8). It was also found that although GSH is a poor substrate for MRP1, it can stimulate the ATP-dependent transport of certain non-anionic organic drugs such as vincristine (VCR) (9), adriamycin (ADM) (10, 11), and aflatoxin B 1 (12) as well as certain endogenous hydrophilic anionic conjugates such as estrone 3-sulfate (13) and a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (14). Loe et al. (9) have shown that GSH is required for vesicular transport of VCR by MRP1 and for inhibition by VCR of the transport of organic anions by MRP1. ATP-dependent uptake of [ 3 H]GSH into membrane vesicles from MRP1-transfected cells was stimulated by VCR in a dose-dependent manner (9). This experiment, however, did not...

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Phosphorylation of the Multidrug Resistance Encoded Protein P190

Cited by 53 publications

References 8 publications

Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines

Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines

Characterization of a clonal human colon adenocarcinoma line intrinsically resistant to doxorubicin

GSH Inhibits Trypsinization of the C-terminal Half of Human MRP1

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