Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans.

Acquired resistance of mammalian cells to multiple chemotherapeutic drugs can result from enhanced expression of the multidrug resistance-associated protein (MRP), which belongs to the ABC transporter superfamily. ABC transporters play a role in the protection of organisms against exogenous toxins by cellular detoxification processes. We have identified four MRP homologues in the soil nematode Caenorhabditis elegans, and we have studied one member, mrp-1, in detail. Using an mrp::lacZ gene fusion, mrp-l expression was found in cells of the pharynx, the pharynx-intestinal valve and the anterior intestinal cells, the rectum-intestinal valve and the epithelial cells of the vulva. Targeted inactivation of mrp-l resulted in increased sensitivity to the heavy metal ions cadmium and arsenite, to which wild-type worms are highly tolerant. The most pronounced effect of the mrp-1 mutation is on the ability of animals to recover from temporary exposure to high concentrations of heavy metals. Nematodes were found to be hypersensitive to heavy metals when both the MRP homologue, mrp-1, and a member of the P-glycoprotein (Pgp) gene family, pgp-1, were deleted. We conclude that nematodes have multiple proteins, homologues of mammalian proteins involved in the cellular resistance to chemotherapeutic drugs, that protect them against heavy metals.     

斑马鱼Abcc2介导微囊藻毒素解毒功能探究

研究克隆了斑马鱼（Danio rerio）abcc2基因序列,探讨了其在微囊藻毒素（MC-LR）解毒中的潜在功能。结果表明:斑马鱼abcc2具有同哺乳动物ABCC2相似的介导荧光底物MCB外排的转运活性,MC-LR处理可显著诱导其在斑马鱼幼体中的转录表达;过表达Abcc2蛋白能显著增强ZF4细胞和斑马鱼胚胎对MC-LR的耐受性;Abcc2作为MC-LR的主要耐受因子,在组织防御和有毒物质的排泄中起重要作用,但其解毒功能还不清楚。研究结果为进一步揭示鱼类抗MC-LR积累的分子机理及培育低MC-LR残留的养殖新品种提供理论基础。     

An Active Efflux System for Heavy Metals in Cisplatin-Resistant Human KB Carcinoma Cells

The mechanism for cisplatin resistance in cisplatin-resistant KCP-4 cells was studied. Although multidrug resistance-associated protein (MRP) was not detected in KCP-4 cells, the cells were more resistant to heavy metals than multidrug-resistant C-A120 cells that overexpressed MRP. KCP-4 cells expressed metallothionein, but it was scarcely involved in cisplatin resistance in these cells. KCP-4 cells did not express canalicular multispecific organic anion transporter (cMOAT). The glutathione(GSH) level was 4.7-fold higher in KCP-4 cells than in KB-3-1 cells. When the GSH level in KCP-4 cells was decreased by treating the cells with buthionine sulfoximine and nitrofurantoin, the accumulation of and sensitivity to cispaltin in the cells were increased. C-A120 cells were only 3.0-fold more resistant to cisplatin than KB-3-1 cells and this resistance was not affected by the increased glutathione level. The accumulation of platinum in C-A120 and KCP-4 cells was 68.5 and 20.4% of that in KB-3-1 cells, respectively, while the intracellular levels of antimony potassium tartrate in C-A120 and KCP-4 cells were 13.2 and 9.9% of that in KB-3-1 cells, respectively. The ATP-dependent efflux of antimony was enhanced in both C-A120 and KCP-4 cells. These results, taken together, suggest an efflux pump for heavy metals different from MRP and cMOAT is involved in cisplatin resistance in KCP-4 cells.     

Structure-activity studies of verapamil analogs that modulate transport of leukotriene C(4) and reduced glutathione by multidrug resistance protein MRP1

The 190-kDa multidrug resistance protein MRP1 is an ATP-binding cassette protein that confers resistance to multiple antineoplastic agents and actively transports conjugated organic anions. We have previously shown that MRP1-mediated GSH transport is stimulated by verapamil but transport of verapamil in the presence or absence of GSH is not observed. We have now examined 20 sulfur-containing verapamil analogs for their ability to inhibit MRP1-mediated leukotriene C(4) (LTC(4)) transport and stimulate GSH uptake into inside-out membrane vesicles. All of the derivatives were poor inhibitors of LTC(4) uptake. However, the inhibitory potency of the more lipophilic dithiane compounds could be enhanced by coincubation with GSH whereas this was not the case for the more hydrophilic dithiane tetraoxides. The dithiane derivatives stimulated GSH transport whereas, with one exception, the dithiane tetraoxides did not. One pair of dithiane stereoisomers differed significantly in their ability to stimulate GSH transport although their ability to inhibit LTC(4) uptake in the presence of GSH was comparable. Our findings indicate that the GSH transport activity of MRP1 can be dissociated from its conjugated organic anion transport activity.     

Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1

Glutathione (GSH) depletion is an important hallmark of apoptosis. We previously demonstrated that GSH depletion, by its efflux, regulates apoptosis by modulation of executioner caspase activity. However, both the molecular identity of the GSH transporter(s) involved and the signaling cascades regulating GSH loss remain obscure. We sought to determine the role of multidrug resistance protein 1 (MRP1) in GSH depletion and its regulatory role on extrinsic and intrinsic pathways of apoptosis. In human lymphoma cells, GSH depletion was stimulated rather than inhibited by pharmacological blockage of MRP1 with MK571. GSH loss was dependent on initiator caspases 8 and 9 activity. Genetic knock-down (>60 %) of MRP1 by stable transfection with short hairpin small interfering RNA significantly reduced MRP1 protein levels, which correlated directly with the loss of MRP1-mediated anion transport. However, GSH depletion and apoptosis induced by both extrinsic and intrinsic pathways were not affected by MRP1 knock-down. Interestingly, stimulation of GSH loss by MK571 also enhanced the initiator phase of apoptosis by stimulating initiator caspase 8 and 9 activity and pro-apoptotic BCL-2 interacting domain cleavage. Our results clearly show that caspase-dependent GSH loss and apoptosis are not mediated by MRP1 proteins and that GSH depletion stimulates the initiation phase of apoptosis in lymphoid cells.     

The role of a novel copper complex in overcoming doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

One of the important pathways of resistance to anthracyclines is governed by elevated levels of glutathione (GSH) in cancer cells. Resistant cells having elevated levels of GSH show higher expression of multidrug-resistant protein (MRP); the activity of glutathione S-transferases (GSTs) group of enzymes have also been found to be higher in some drug-resistant cells. The general mechanism in this type of resistance seems to be the formation of conjugates enzymatically by GSTs, and subsequent efflux by active transport through MRP (MRP1-MRP9). MRPs act as drug efflux pump and can also co-transport drugs like doxorubicin (Dox) with GSH. Depletion of GSH in resistant neoplastic cells may possibly sensitize such cells, and thus overcome multidrug resistance (MDR). A number of resistance modifying agents (RMA) like DL-buthionine (S, R) sulfoxamine (BSO) and ethacrynic acid (EA) moderately modulate resistance by acting as a GSH-depleting agent. As most of the GSH-depleting agents have dose-related toxicity, development of non-toxic GSH-depleting agent has immense importance in overcoming MDR. The present study describes the resistance reversal potentiality of novel copper complex, viz., copper N-(2-hydroxy acetophenone) glycinate (CuNG) developed by us in Dox-resistant Ehrlich ascites carcinoma (EAC/Dox) cells. CuNG depletes GSH in resistant (EAC/Dox) cells possibly by forming conjugate with it. Depletion of GSH results in higher Dox accumulation that may lead to enhanced rate of apoptosis in EAC/Dox cells. In vivo studies with male Swiss albino mice bearing ascitic growth of EAC/Dox showed tremendous increase in life span (treated/control, T/C = 453%) for the treated group with apparent regression of tumor. Resistance to Dox in EAC/Dox cells is associated with over expression of GST-P1, GST-M1 (enzymes involved in phase II detoxification) and MRP1 (a transmembrane ATPase efflux pump for monoglutathionyl conjugates of xenobiotics). CuNG causes down regulation of all these three proteins in EAC/Dox cells. The effect of CuNG as RMA is better than BSO in many aspects.     

An ABC‐transporter of Arabidopsis thaliana has both glutathione‐conjugate and chlorophyll catabolite transport activity

An ABC-transporter of Arabidopsis thaliana exhibiting high sequence similarity to the human (MRP) and yeast (YCF) glutathione-conjugate transporters has been analysed and used to complement a cadmium-sensitive yeast mutant (DTY68) that also lacks glutathione-conjugate transport activity. Comparison of the hydrophobicity plots of this A. thaliana MRP-like protein with MRP and YCF demonstrates that the transmembrane domains are conserved, even at the N-terminus where sequence identity is low. Cadmium resistance is partially restored in the complemented ycf mutant, and glutathione-conjugate transport activity can be observed as well. The kinetic properties of the A. thaliana MRP-like protein (AtMRP3) are very similar to those previously described for the vacuolar glutathione-conjugate transporter of barley and mung bean. Furthermore, a hitherto undescribed ATP-dependent transport activity could be correlated with the gene product, i.e. vesicles isolated from the complemented yeast, but not from DTY68 or the wild type, take up the chlorophyll catabolite Bn-NCC-. The results indicate that the product of the MRP-like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.     

Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1)

The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.     

Comparative genotoxicity of aluminium and cadmium in embryonic zebrafish cells

Aluminium is a toxic metal whose genotoxicity has been scarcely studied in aquatic species and more generally in mammals. Recently, human and ecological disaster caused by the discharge of red mud in Hungary has revived questions about the toxicity of this metal particularly for the environment. On the contrary, cadmium is a highly toxic metal whose genotoxicity has been well characterized in various mammalian cells. However on non-human cells, little is known about its impact on DNA damage and repair.In this study, the genotoxic potential of both metals on embryonic zebrafish cells ZF4 was analyzed and particularly the impairment of the major DNA double strand breaks (DSB)-repair pathway, i.e. non-homologous end-joining (NHEJ).To this aim, DNA single strand breaks (SSB) and DSB were evaluated using the comet assay and the immunodetection of γ-H2AX proteins, respectively, in AlCl₃ or CdCl₂ exposed ZF4 cells. These exposures result in the production of DSBs a few hours after incubation. The DNA-PK kinase activity, essential for NHEJ, is more affected by the presence of aluminium than cadmium. Altogether our data provide evidence of the high toxicity induced by aluminium in zebrafish and indicates the pertinence of genotoxicity evaluation in organisms living in contaminated water.     

Reversal of MRP-mediated multidrug resistance in human lung cancer cells by the antiprogestatin drug RU486.

Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRP-overexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dose-dependent manner, through inhibition of cellular export of the dye; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 microM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function.     

Glutathione export during apoptosis requires functional multidrug resistance-associated proteins

GSH is released in cells undergoing apoptosis, and the present study indicates that the multidrug resistance-associated proteins (MRPs/ABCC) are responsible for this GSH release. Jurkat cells released approximately 75-80% of their total intracellular GSH during both Fas antibody- and staurosporine-induced apoptosis. In contrast, Raji cells, a lymphocyte cell line that is deficient in phosphatidylserine externalization, did not release GSH during apoptosis, and other apoptotic features appeared more slowly in these cells. Jurkat and Raji cell lines expressed comparable MRP and OATP/SLCO (organic anion-transporting polypeptide) mRNA levels, and MRP1 protein levels; however, differences existed in MRP1 localization and function. In Jurkat cells, MRP1 was largely localized to the plasma membrane, and these cells exported the MRP substrate calcein. Calcein release was enhanced during apoptosis. In contrast, Raji cells had little MRP1 at the plasma membrane and did not export calcein under basal or apoptotic conditions, indicating that these cells lack functional MRPs at the plasma membrane. GSH release in Jurkat cells undergoing apoptosis was inhibited by the organic anion transport inhibitors MK571, sulfinpyrazone, and probenecid, supporting a role for the MRP transporters in this process. Furthermore, when MRP1 expression was decreased with RNA interference, GSH release was lower under both basal and apoptotic conditions, providing direct evidence that MRP1 is involved in GSH export.