Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.     

Expression of the multidrug resistance-associated protein (MRP) gene in human cancers

We determined the expression of a newly recognized drug resistance gene, the multidrug resistance-associated protein (MRP) gene, [Cole et al., Science (Washington DC), 258: 1650-1654, 1992], in normal human tissues and in >370 human tumor biopsies using a quantitative RNase protection assay and immunohistochemistry. MRP mRNA appeared to be ubiquitously expressed at low levels in all normal tissues, including peripheral blood, the endocrine glands (adrenal and thyroid), striated muscle, the lymphoreticular system (spleen and tonsil), the digestive tract (salivary gland, esophagus, liver, gall bladder, pancreas, and colon), the respiratory tract (lung), and the urogenital tract (kidney, bladder, testis, and ovary). The human cancers analyzed could be divided into three groups with regard to MRP expression. Group 1 consists of tumors that often exhibit high to very high MRP mRNA levels (e.g., chronic lymphocytic leukemia). Group 2 comprises the tumors that often exhibit low, but occasionally exhibit high MRP mRNA expression (e.g., esophagus squamous cell carcinoma, non-small cell lung cancer, and acute myelocytic leukemia). Group 3 comprises the tumors with predominantly low levels of MRP mRNA, comparable to the levels found in normal tissues (e.g., other hematological malignancies, soft tissue sarcomas, melanoma, and cancers of the prostate, breast, kidney, bladder, testis, ovary, and colon). Using the MRP-specific mAbs MRPr1 and MRPm6, we confirmed the elevated MRP mRNA levels in tumor tissues by immunohistochemistry. We conclude that hyperexpression of MRP is observed in several human cancers, and that additional studies are needed to assess the clinical relevance of MRP.     

Expression of the multidrug resistance-associated protein (MRP) gene in urothelial carcinomas

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.     

Expression of multidrug resistance-associated protein (MRP) in head and neck squamous cell carcinoma

Vascular parkinsonism is thought to be a distinct parkinsonian syndrome associated with small deep infarcts and white matter lesions (WMLs). We studied the prevalence of parkinsonian features (bradykinesia, rigidity, tremor, and gait disorder) in relation to small deep or territorial infarcts and WMLs on computed tomography (CT) in 62 lacunar and 41 territorial stroke patients, at 3.0 (median) years of follow up. One or more parkinsonian signs were found in 36% of these patients; 11% clinically had parkinsonism. Parkinsonian signs were found more frequently in lacunar than in territorial stroke patients: bradykinesia in 45% and 7%, rigidity in 13% and 7%, tremor in 6% and 7%, and gait disorder in 16% and 7%, respectively. Patients with WMLs at study entry (n = 16) were compared with those without WMLs (n = 87): 56% and 25% had bradykinesia, 25% and 8% rigidity, 25% and 3% tremor, and 38% and 8% gait disorder, respectively. Regression analysis with adjusted odds ratios ([a]OR) showed that WMLs at study entry were associated with bradykinesia ([a]OR 8.0, 95% confidence interval [CI] 1.6-41.6), gait disorder ([a]OR 7.1, 95% CI 1.5-33.7), and tremor ([a]OR 7.0, 95% CI 1.2-40.3). Bradykinesia was associated with lacunar stroke at study entry ([a]OR 11.5, 95% CI 2.4-54.9). Thus, one third of our stroke patients had one or more parkinsonian signs, and 10% clinically had a parkinsonian syndrome that differed from Lewy body parkinsonism: infrequent resting tremor, but frequent gait disorder. Parkinsonian signs were associated with WMLs and lacunar stroke. Therefore, this study favors a distinct vascular parkinsonian syndrome.     

Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents

Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents. The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP). As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells. We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells. Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells. The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide. Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function. These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.     

Difloxacin reverses multidrug resistance in HL-60/AR cells that overexpress the multidrug resistance-related protein (MRP) gene

In this study, we have examined the in vitro chemosensitizing activity of difloxacin, a quinolone antimicrobial agent, in the multidrug-resistant human myeloid leukemia HL-60/AR cell line. HL-60/AR cells were found to overexpress multidrug resistance-associated protein (MRP) mRNA as compared to HL-60 cells. Difloxacin, in a concentration-dependent manner, increased the sensitivity of HL-60/AR cells to daunorubicin, adriamycin, and vincristine, and partially corrected the altered drug transport. In addition, difloxacin corrected subcellular distribution of adriamycin by inducing redistribution of the drug from the perinuclear region to the nucleus in HL-60/AR cells. The chemosensitizing effect of difloxacin was observed at clinically achievable concentrations. We conclude that difloxacin is an effective chemosensitizer of MRP-associated multidrug-resistant tumor cells and is a potential candidate for clinical use to reverse multidrug resistance.     

Expression of human MRP6, a homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells

Arachidonic acid and eicosapentaenoic acid are important precursors for the production of prostaglandins and other hormone-like eicosanoid molecules. These fatty acids are synthesized by animals by elongating and desaturating precursor fatty acids such as linoleic acid (18:2Delta9,12) and alpha-linolenic acid (18:3Delta9, 12,15). We have identified a Delta5 fatty acid desaturase gene (fat-4) from the nematode Caenorhabditis elegans. We have expressed this gene product in Saccharomyces cerevisiae and demonstrate that it readily converts di-homo-gamma-linolenic acid (20:3Delta8,11,14) to arachidonic acid (20:4Delta5,8,11,14). The FAT-4 Delta5-desaturase also acts on a number of other substrates, including fatty acids that do not contain a double bond at the Delta8 position.     

Changes in multidrug transporter protein expression in endothelial cells cultured from isolated human brain microvessels

Retinol and proguanil are metabolised by the same family of hepatic cytochrome P450, i.e. CYP2C. We used proguanil as a probe to study CYP2C activity, and by implication retinol metabolism, in psoriasis. In vitro studies showed that retinol competitively inhibited the hepatic metabolism of proguanil to cycloguanil. Proguanil metabolism was assessed in 82 patients with chronic plaque psoriasis. Following proguanil orally (200 mg), urine was analysed for proguanil and cycloguanil. A proguanil to cycloguanil ratio < 1 signified extensive metabolism and a ratio > 10 poor metabolism. A wider range of ratios was observed in psoriasis than previously reported for normal subjects. The proguanil to cycloguanil ratio was assessed in 10 cases each of know severe and mild psoriasis. Low CYP2C activity was associated with severe psoriasis, poor metaboliser status occurring in 50% of the severe group, but in none of the mild cases, p < 0.01. These findings may indicate differences in retinoid metabolism in psoriasis.     

Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP)

HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains. Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents. The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16). Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants. Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants. The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance. The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.     

Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide

Immunohistochemical assessment was made of nm23 protein expression in pulmonary adenocarcinoma. Of the 147 adenocarcinomas 67% (99/147) were weakly and 33% (48/147) strongly positive for nm23 protein. nm23 protein expression in primary tumors was shown to correlate inversely with advancing pathologic stage and the degree of metastasis in regional lymph nodes (P < 0.05). The staining of tumors without nodal metastasis was more intense than with nodal metastasis (P < 0.02). Nodal metastasis was seen in 37% (55/147) cases examined. The immunoreactivity to nm23 protein in tumor cells of nodal metastasis was essentially the same as in those of primary tumors (P < 0.01). Significant correlation between patient prognosis and immunoreactivity for nm23 in primary tumors (P < 0.05) was demonstrated. But none could be found between immunoreactivity and other parameters such as histologic grading, distant metastasis, tumor size or disease-free survival. Neither was there any significant correlation between pathologic parameters examined and the expression of nm23 in any histologic subtype. Multivariate analysis using Cox's proportional hazards regression model with five variables indicated nm23 and lymph node metastasis to contribute to overall patient survival. Based on risk ratio disadvantageous state/advantageous states, the gravity of prognostic factors was assessed for lymph node metastasis as 9.25, nm23 expression as 2.06, distant metastasis as 1.23, pathologic stage as 0.78 and tumor size as 0.77. The results suggested that in pulmonary adenocarcinoma a reduced expression of nm23 protein was associated with lymph node metastasis and poor patient survival.     

Membrane topology and glycosylation of the human multidrug resistance-associated protein

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.     

Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells

The multidrug resistance-associated protein Mrp2 is expressed in liver, kidney, and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells. We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat Mrp2. To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules. Both 17beta-estradiol-17-beta-D-glucuronide ([3H]E217betaG, 50 nM) and [3H]leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion. Uptake of [3H]E217betaG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 microM). Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of [3H]E217betaG transport. The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide did not inhibit transport. In conclusion, we show direct ATP-dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity.     

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.     

Hypoxia reoxygenation-induced injury of cultured pulmonary microvessel endothelial cells

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.     

Carbamoylation of glutathione reductase by N,N-bis(2-chloroethyl)-N- nitrosourea associated with inhibition of multidrug resistance protein (MRP) function

The physiopathology of radiation-induced bone damage is no completely elucidated. Ionizing radiation may induce an inhibition or an impairment of growing bone. This fact is of particular importance in children, and represents one of the most important dose-limiting factors in the radiotherapeutic management of children with malignant diseases. Scoliosis, epiphyseal slippage, avascular necrosis, abnormalities of craniofacial growth may be observed after radiation. Child's age at the time of treatment, location of irradiated bone and irradiation characteristics may influence the radiation-related observed effects. In adults, pathological analysis of mature bone after ionizing radiation exposure are rare, suggesting that it is difficult to draw a clear feature of the action of radiation on the bone. Osteoporosis, medullary fibrosis and cytotoxicity on bone cells lead to fracture or necrosis. Various factors can influence bone tolerance to radiation such as bone involvement by tumor cells or infection, which is frequent is mandibulary osteoradionecrosis. Technical improvements in radiation techniques have also decreased radio-induced bone complications: the volume, fractionation and total dose are essential to consider. The absence of a consistent radiation-induced late effects evaluation scale has hampered efforts to analyze the influence of various therapeutic maneuvers and the comparison of results from different reported series. The currently proposed evaluation scale may help harmonizing the classification of radiation-induced bone late effects.     

Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells

To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred approximately 4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.     

A possible role for multidrug resistance-associated protein in the secretion of basic fibroblast growth factor by osteogenic sarcoma cell line (MG-63)

Basic fibroblast growth factor (bFGF) lacks signal sequence and therefore the mechanism of its secretion is unknown. Multidrug resistance-associated protein (MRP) has been shown to transport a variety of molecules. Therefore, in this study we examined the role of MRP in the secretion of bFGF by osteogenic sarcoma MG-63 cells which spontaneously secrete bFGF. We show that MG-63 cells express MRP both at the protein and at the mRNA level. Furthermore, probenecid (a putative inhibitor of transport activity of MRP), in a concentration-dependent manner, inhibited secretion of bFGF from MG-63 cells with concomitant increase in intracellular contents of bFGF. These results suggest that MRP may have a possible role in the secretion of bFGF.