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Anti-cancer-prostaglandin-induced cell-cycle arrest and its modulation by an inhibitor of the ATP-dependent glutathione S-conjugate export pump (GS-X pump).

ABSTRACT: The A and J series of prostaglandins (PGs) accumulate in the nuclei to suppress the proliferation of cancer cells. Here we report that Delta7-PGA1 methyl ester, a synthetic anti-cancer PG, increased the level of mRNA for the cyclin-dependent kinase inhibitor p21 in human leukaemia HL-60 cells. The induction of p21 was associated with the accumulation of hypophosphorylated retinoblastoma protein (pRB) and the suppression of c-myc gene expression. Since the p53 gene is deleted in HL-60 cells, the anti-cancer PG is suggested to inhibit cancer cell growth by inducing p21 via a p53-independent pathway. Unlike HL-60 cells, cisplatin-resistant HL-60/R-CP cells were insensitive to Delta7-PGA1 methyl ester. While c-myc expression was transiently suppressed, neither G1 arrest nor hypophosphorylation of pRB was observed with the anti-cancer PG. Plasma membrane vesicles from HL-60/R-CP cells showed an enhanced level of GS-X pump (ATP-dependent glutathione S-conjugate export pump) activity towards the glutathione S-conjugate of Delta7-PGA1 methyl ester (Km 110 nM). GIF-0019 ¿N-carbomethoxy-S-[5-(4-benzoylphenyl)pentyl]glutathione dimethyl ester¿, a specific inhibitor of the GS-X pump, dose-dependently enhanced the cellular sensitivity of HL-60/R-CP cells to Delta7-PGA1 methyl ester and induced G1 arrest. The GS-X pump is suggested to play a pivotal role in modulating the biological action of the anti-cancer PG.

SUBMITTER: Ishikawa T

PROVIDER: S-EPMC1219906 | biostudies-other | 1998 Dec

REPOSITORIES: biostudies-other

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ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump.

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| S-EPMC1217068 | biostudies-other

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Characterisation of Glutathione Export from Human Donor Lenses.

Project description:PurposeTo investigate whether human donor lenses are capable of exporting reduced glutathione.MethodsHuman lenses of varying ages were cultured in artificial aqueous humor for 1 hour under hypoxic conditions to mimic the physiologic environment and reduced glutathione (GSH) and oxidized glutathione (GSSG) levels measured in the media and in the lens.ResultsHuman donor lenses released both GSH and GSSG into the media. Donor lenses cultured in the presence of acivicin, a γ-glutamyltranspeptidase inhibitor, exhibited a significant increase in GSSG levels (P < 0.05), indicating that GSSG undergoes degradation into its constituent amino acids. Screening of GSH/GSSG efflux transporters revealed Mrp1, Mrp4, and Mrp5 to be present at the transcript level, but only Mrp5 was expressed at the protein level. Blocking Mrp5 function with the Mrp inhibitor MK571 led to a significant decrease in GSSG efflux (P < 0.05), indicating that Mrp5 is likely to be involved in mediating GSSG efflux. Measurements of efflux from the anterior and posterior surface of the lens revealed that GSH and GSSG efflux occurs at both surfaces but predominantly at the anterior surface.ConclusionsHuman lenses export GSH and GSSG into the surrounding ocular humors, which can be recycled by the lens to maintain intracellular GSH homeostasis or used by neighboring tissues to maintain GSH levels.Translational relevanceEarly removal of a clear lens, as occurs to treat myopia and presbyopia, would eliminate this GSH reservoir and reduce the supply of GSH to other tissues, which, over time, may have clinical implications for the progression of other ocular diseases associated with oxidative stress.

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Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

Project description:We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.

| S-EPMC2120605 | biostudies-other

Identification of mutation-prone points in bile salt export pump.

Project description:BACKGROUND: The bile salt export pump mediates uphill canalicular bile acid secretion. Inherited dysfunction of the bile salt export pump causes a progressive and a benign form of familial intrahepatic cholestasis. Mutations within the bile salt export pump gene are reported. Presently, prediction of protein nanostructure and function is a great challenge in the proteomics and structural genomics era. Identification of the point vulnerable to mutation is a new trend to expand knowledge on disorders at the genomic and proteomic level of diseases. MATERIALS AND METHODS: A bioinformatic analysis was performed to study the positions that determine peptide motifs in the amino acid sequence of the bile salt export pump. To identify the weak linkage in bile salt export pump, a new bioinformatic tool named GlobPlot was used. RESULTS: The positions 16-34, 119-127, 451-459, 550-561, 1084-1099, 1108-1118, 1135-1140, 1211-1217, and 1314-1321 were identified as the positions prone to mutation. CONCLUSION: Based on this study, the weak linkages in the bile salt export pump can be identified and can provide good information for expectation of possible new mutations that can lead to cross species jumping. In addition, the results from this study provide useful information for further research on the bile salt export pump.

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