Multidrug Resistance-associated Protein mRNA Levels Research Articles

Induction of multidrug resistance-associated protein 3 expression by diesel exhaust particle extract in human bronchial epithelial BEAS-2B cells

Diesel exhaust particles (DEPs) are common environmental air pollutants known to impair expression and activity of drug detoxifying proteins, including hepatic ATP-binding cassette (ABC) drug transporters. The present study was designed to determine whether organic DEP extract (DEPe) may also target ABC drug transporters in bronchial cells. DEPe (10 μg/mL) was demonstrated to induce mRNA and protein expression of the multidrug resistance-associated protein (MRP) 3 in cultured bronchial epithelial BEAS-2B cells, whereas mRNA levels of other MRPs, multidrug resistance gene 1 or breast cancer resistance protein were unchanged, reduced or not detected. DEPe also increased MRP3 mRNA expression in normal human bronchial epithelial cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway by AhR antagonist or AhR silencing, as well as the silencing of nuclear-factor-E2-related factor 2 (Nrf2) repressed DEPe-mediated MRP3 induction. This underlines the implication of the AhR and Nrf2 signaling cascades in DEPe-mediated MRP3 regulation. DEPe was additionally demonstrated to directly inhibit MRP activity in BEAS-2B cells, in a concentration-dependent manner. Taken together, these data indicate that DEPs may impair expression and activity of MRPs, notably MRP3, in human bronchial cells, which may have consequences in terms of lung barrier and toxicity for humans exposed to diesel pollution.

SKP2 siRNA inhibits the degradation of P27<sup>kip1</sup> and down-regulates the expression of MRP in HL-60/A cells

S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

Reversal of the multidrug resistance in human hepatocellular carcinoma cells by recombinant adenoviral delivery of the MRP antisense RNA in vitro

To explore the effect of multidrug resistance-associated protein (MRP) antisense RNA mediated by recombinant adenoviruses on reversal of the multidrug resistance in human drug-resistant hepatocellular carcinoma (HCC) cells. The HCC cells were first treated in vitro by the recombinant adenoviruses containing antisense MRP. Chemosensitivity of all of the cells to ADM and DNR was determined by MTT assay. The levels of the MRP mRNA and its product P190 were measured by RT-PCR and flow cytometry (FCM) respectively according to the time after transfection. The accumulation of the DNR in these cells was analyzed by FCM simultaneously. IC50 of the cells infected by Ad-Asmrp to ADM and DNR was 0.487 microg/ml and 0.328 microg/ml respectively, with the factors of resistance (RF) decreased by 36.8 and 35.4 respectively (P < 0.05). Continuous decrease in levels of MRP mRNA and P190 was observed 24 h and 48 h after infection respectively (P < 0.01). The intracellular DNR accumulation was increased simultaneously in the Ad-Asmrp transfectants (P < 0.001). MRP antisense RNA mediated by recombinant adenoviruses could increase chemosensitivity and partially reverse the MDR in HCC cells in vitro by inhibit expression of MRP gene. The study would provide experimental basis for the reversal of MDR in HCC by antisense RNA technology mediated by recombinant adenoviruses.

Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction

A resistant descendant of the human stomach carcinoma cell line EPG85-257 was selected in the presence of increasing concentrations of daunorubicin (DRC). To avoid the expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), cells were cultured in the presence of verapamil. The resulting cells were used to evaluate an induced carbonyl reduction as a new determinant in DRC resistance. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) toxicity assay was performed to estimate sensitivity to DRC in both cell lines. ic 50 values of DRC increased almost 8-fold in the resistant descendants compared to the parental cell line. P-gp transcripts were detectable in both cell lines at only very low levels, and no significant alterations between sensitive and resistant cells were observed. MRP mRNA expression was markedly higher compared to P-gp mRNA, but, as was the case with P-gp, MRP mRNA levels in sensitive and resistant cells showed no alteration. This was probably due to the effect of the presence of verapamil during cell selection. Another known drug resistance factor, the lung resistance-related protein (LRP), was not at all detectable. Interestingly, resistant cells possessed 6-fold higher levels of DRC carbonyl-reducing activity, leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL). The 6-fold higher DRCOL formation roughly parallels the 8-fold increase in DRC ic 50 values during cell selection, and therefore may account for DRC resistance in these cells. The determination of specific carbonyl reducing enzymes, known to be involved in DRC detoxification, revealed that mRNA expression of carbonyl reductase (EC 1.1.1.184), aldose reductase (EC 1.1.1.21), and dihydrodiol dehydrogenase 2 (EC 1.3.1.20) increased in the resistant descendant. In contrast, the phase II-conjugating enzyme activities of glutathione S-transferases were significantly lower in resistant than in sensitive cells, whereas those of glucuronosyl transferase were not detectable in either cell line. Apparently, conjugating enzymes are not involved in DRC resistance in human stomach carcinoma cells. These studies indicate that DRC resistance in human stomach carcinoma cells may appear as a result of an induction of metabolic DRC inactivation via carbonyl reduction to the less active 13-hydroxy metabolite DRCOL.

The multidrug resistance-associated protein (MRP) is over-expressed and functional in rat hepatoma cells.

Expression of multidrug-resistance-associated protein (MRP), a drug efflux pump transporting a wide range of xenobiotics, including anti-cancer drugs and chemical carcinogens, and present at low levels in normal hepatocytes, was investigated in rat hepatoma cells. Northern-blot analysis allowed detection of high levels of MRP mRNA in rat diethylnitrosamine-induced hepatocarcinomas when compared with normal liver. Similarly, elevated expression of MRP transcripts were evidenced in 6 rat hepatoma cell lines of different origins, especially in HTC cells, that, in contrast, failed to express mRNA of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump sharing numerous substrates with MRP. HTC cells were also found by Western blotting to display much higher amounts of MRP than those observed in normal hepatocytes. In contrast, the MRP gene copy number was similar both in hepatoma HTC cells and in hepatocytes, as assessed by Southern blotting. Analysis of MRP-related transport using 3 types of MRP substrates, namely, the fluorescent glutathione-bimane, the anionic dye calcein and the cationic anti-cancer drug vincristine, demonstrated that HTC cells displayed cellular efflux of these 3 compounds, an efflux strongly inhibited by MRP modulators such as indomethacin. These results indicate that MRP is over-expressed and functional in rat hepatoma cells and may therefore be included in the de-toxifying pathways that are altered during hepatocarcinogenesis and are thus thought to contribute to the known multidrug resistance of liver tumors.

Five putative drug resistance parameters (MDR1/P‐glycoprotein, MDR‐associated protein, glutathione‐S‐transferase, bcl‐2 and topoisomerase IIα) in 57 newly diagnosed acute myeloid leukaemias

Using a modified quantitative reverse transcriptase (RT) PCR assay in 57 patients with acute myeloid leukaemia (AML) from a Swiss Phase III multicentre study (SAKK 30/85), we measured the m-RNA expression of the genes from the multidrug resistance gene 1 (MDR1), the multidrug resistance associated protein (MRP), glutathione-S-transferase (GST) pi, bcl-2 and topoisomerase (topo) IIalpha. P-glycoprotein (p-gp) was measured by Western blot, and GST activity by functional assays. To analyse progression-free (PFS) and overall survival (OS), parameters were prospectively divided into "low" and "high" groups, according to their median values (exceptions: MDR1 and p-gp). Median follow-up was 60 months. MDR1- and MRP mRNA levels correlated with each other (r=0.54, Spearman), FABM4/M5 and extramedullary disease. "Low" bcl-2-mRNA predicted longer PFS: 22 months vs. 7 months (median,p=0.02, log rank), and longer OS: 64 months vs. 14 months (p=0.06). "Low" topo IIalpha predicted poorer outcome: median PFS 9 vs. 19 months (p=0.03); median survival 12 months vs. "not reached" (p=0.03). An improved outcome tendency, albeit nonsignificant, was seen in p-gp-negative patients. In a Cox model adjusted for age, performance status, presence of Auer rods, FAB type and clinical response, bcl-2 and topo IIalpha mRNA levels retained their predictive values.

Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines.

Multidrug resistance (MDR) in human cancer cells is multifactorial. Previously, we reported on the association between expression of P-glycoprotein (Pgp), the multidrug resistance-associated protein (MRP), and the lung resistance protein (LRP) with the MDR phenotype in the NCI panel of 60 human cancer cell lines used for in vitro anticancer drug screening. Eight cell lines from this panel, manifesting widely divergent levels of in vitro drug resistance were chosen to investigate the role of MRP and LRP expression at the molecular level. LRP mRNA levels, as determined by ribonuclease protection assay, varied significantly among the 8 cell lines, and correlated closely with in vitro drug resistance to both MDR and non-MDR related drugs. LRP mRNA expression was determined to be a stronger correlate of drug sensitivity than protein expression. In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity. The rates of newly transcribed LRP or MRP mRNA did not correlate with mRNA levels, indicating that mRNA stability or other features of processing may be important in regulation of LRP and MRP mRNA levels. Using Southern blot analysis, LRP gene amplification was shown not to be associated with LRP overexpression. These data suggest that LRP expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents.

Possible role of the multidrug resistance-associated protein (MRP) in chemoresistance of human melanoma cells.

Human malignant melanoma is characterised by unresponsiveness to conventional chemotherapy. Melanoma-derived cell lines are often markedly chemoresistant, suggesting that cellular mechanisms mediate the multidrug resistance (MDR) phenotype. The multidrug resistance-associated protein (MRP) is a drug transporter protein associated with resistance to a broad spectrum of lipophilic drugs. To investigate whether MRP is involved in intrinsic drug resistance of human melanoma, we analysed expression and functional activity of MRP as well as its impact on chemoresistance in 40 melanoma cell lines (35 established by us from primary and metastatic lesions and 5 obtained from international sources), as well as in one dysplastic naevus-derived cell line and in normal melanocytes. By reverse transcriptase-polymerase chain reaction various levels of MRP mRNA were detected in all melanoma cell lines, and by immunoblot the corresponding protein in a high percentage of them. Functional activity of MRP was assayed by analysing cellular accumulation of 3H-daunomycin (3H-DM) and calcein in response to MRP-modulators by beta-spectrometric and fluorescence-activated cell sorter analysis, respectively. Probenecid (PRO), N-ethylmaleimide (NEM) and benzbromarone (BB) moderately (< or = 1.43-fold) but significantly enhanced intracellular accumulation of MRP substrate probes corresponding to MRP expression. Moreover, the sensitivity of melanoma cell lines to daunomycin (DM) and doxorubicin (DOX), but not to vinblastine (VBL), etoposide (VP-16) and cisplatin (CDDP), analysed by an MTT-based survival assay, were inversely correlated with MRP-gene expression. Our results imply that MRP may be a component of the intrinsic chemoresistance phenotype characteristic of human malignant melanoma.

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.

Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line.

Prior studies have shown that, in some human tumour cells, increased expression of the multidrug resistance gene MDR1 can be induced in response to certain stress conditions such as a transient exposure to cytotoxic agents. Little is known about the possibility of increasing the expression of the recently cloned multidrug resistance-associated protein (MRP) in response to a transient exposure to cytotoxic drugs. In order to examine this possibility, we have used sensitive assays (RT-PCR, flow cytometry) and the sensitive large cell lung cancer cell line, COR-L23/P, and the revertant line (COR-L23/Rev), generated by growing the doxorubicin-selected, MRP-overexpressing resistant variant COR-L23/R without drug exposure for 24-28 weeks. COR-L23/Rev overexpresses MRP, but to a lesser extent than COR-L23/R. COR-L23/Rev rapidly recovered similar levels of MRP mRNA, protein expression, resistance and drug accumulation deficit as COR-L23/R after a 48-72 h exposure to cytotoxic concentrations of doxorubicin or vincristine but not cisplatin. The increase in MRP mRNA could only be detected 3 to 4 days after the transient exposure to drugs. However, when the parental line, COR-L23/P, was exposed to equitoxic doses of doxorubicin, vincristine or cisplatin, no increase in the levels of MRP mRNA could be observed at higher doses (5- to 10-fold the IC50) of doxorubicin or vincristine (but not of cisplatin), we detected a transient increase in the levels of MDR1 mRNA immediately after short-term exposure. In conclusion, we have shown that a human revertant lung cancer cell line (COR-L23/Rev) has the ability to recover quickly, similar levels of MRP expression and resistance as COR-L23/R after a transient exposure to the MDR-drugs doxorubicin and vincristine.

Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in normal peripheral blood and bone marrow haemopoietic cells.

We studied the expression of multidrug resistance-associated protein (MRP) in normal haemopoietic cells from peripheral blood and bone marrow. The MRP mRNA levels were estimated by RT/PCR and in situ hybridization (ISH) assay, and the protein levels by flow cytometry. 21 samples of peripheral blood and 21 samples of bone marrow (11 normal bone marrow donors, 10 patients in complete remission after chemotherapy for large cell lymphoma or acute myeloid leukaemia) were analysed. In peripheral blood the mean MRP mRNA level in CD3+ cells was statistically higher than in the other cells (3-fold by the methods used). The levels of MRP in CD3+ varied from one individual to another (4.5-34.8 units by RT/PCR and 5-23 grains/cell by ISH); however, this was proportional to the variation in all the cell lineages of same individual (r = 0.84). In bone marrow the mean MRP levels of the various cell lineages (including CD34+) were similar to the basal level in HL60 cells. Individual expression levels were again variable; however, there was no difference between untreated normal bone marrow and post chemotherapy normal bone marrow. MRP protein expression was determined by flow cytometry with the monoclonal antibody MRPm6. The CD4+ lymphocytes exhibited a higher MRP protein expression than the other cell lineages, including CD8+ cells. There was a good correlation between the three methods used (RT/PCR and ISH, P = 0.0001, r = 0.87; RT/PCR and flow cytometry, P = 0.0001, r = 0.85; ISH and flow cytometry, P = 0.002, r = 0.67).

Coordinated Induction of MRP/GS-X Pump and γ-Glutamylcysteine Synthetase by Heavy Metals in Human Leukemia Cells

We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.

Alterations in expression of the multidrug resistance-associated protein (MRP) gene in high-grade transitional cell carcinoma of the bladder

Expression of the MRP gene has been demonstrated in vitro to be a casual factor in non-P-glycoprotein-mediated multidrug resistance, and is implicated in resistance to a number of the chemotherapeutic agents currently used in the treatment of high-grade transitional cell carcinoma (TCC) of the bladder (doxorubicin, epirubicin and vinblastine). Using a sensitive RT-PCR-based technique, we have quantified MRP mRNA levels in a series of untreated TCC (n=24), normal bladder (n=5) and control tissue and cell line samples. MRP mRNA was widely expressed and detectable in all samples analysed, with considerable (up to 190-fold) variation observed between individual tumour samples. MRP mRNA levels found in TCC samples were lower than those determined for normal peripheral mononucleocyte (2.3-fold) and testis (4.1-fold) samples, previously reported to be high-expressing tissues, and varied over a similar range to that observed in normal bladder samples. Results indicate that MRP mRNA levels in a greater proportion of high-grade (G3) bladder tumours (55%, 6/11) are significantly reduced (P=0.018) compared with low- and moderate-grade (G1/2) bladder tumours (8%, 1/13), and suggest that MRP mRNA levels frequently become reduced as a consequence of tumour progression to advanced, poorly differentiated disease. No correlation was apparent between MRP and MDR1 mRNA levels, thus providing no evidence to suggest common regulation of the two genes. In a limited number of patients, no evidence was found to support a role for MRP mRNA levels as a determinant of response to chemotherapy in patients being uniformly treated with either cisplatin-methotrexate-vinblastine (n=6) or epirubicin-cisplatin-methotrexate (n=4) regimens. Similarly, no overall pattern of altered MRP mRNA expression was observed following chemotherapy in four patients from whom post chemotherapy biopsies were taken. This study provides a useful pilot investigation regarding the level, variation and pattern of MRP mRNA expression in TCC of the bladder, and suggests that further studies to establish the clinical significance of these variations are required.

Expression of the multidrug resistance-associated protein (MRP) gene in primary non-small-cell lung cancer

One of the major problems in the cure of advanced non-small-cell lung cancer (NSCLC) is its lack of response to cytotoxic drug treatment, and the mechanisms underlying this intrinsic drug resistance are unclear. We determined the expression of a newly recognised drug resistance gene, the Multidrug Resistance-associated Protein (MRP) gene, in normal lung tissue and in tumour biopsies from 35 surgically resected NSCLCs (11 adenocarcinomas, 24 squamous cell carcinomas). MRP mRNA levels were quantitated by RNase protection assay and expression of the MRP Mr 190,000 glycoprotein was estimated by immunohistochemistry. Using the MRP-specific monoclonal antibody MRPr1, MRP expression was detected by immunohistochemistry in epithelial cells lining the bronchi in normal lung. In NSCLC approximately 35% of the samples showed elevated MRP mRNA levels. Based on MRP-specific immunohistochemical staining the tumours were divided into 4 groups: 12% were scored as negative (-), 14% showed weak cytoplasmic staining of the tumour cells (+/-), 40% had a clear cytoplasmic staining (+), and in 34% a strong cytoplasmic as well as membranous staining was observed (++). MRP expression, as estimated by immunohistochemistry, correlated with the MRP mRNA levels quantitated by RNase protection assay (correlation coefficient = 0.745, p = 0.0009), with MRP mRNA levels (mean +/- SD) of 3.0 +/- 1.0 U, 3.5 +/- 0.7 U, 7.5 +/- 5.9 U, and 19.3 +/- 10.7 U, in the (-), (+/-), (+), and (++) immunohistochemistry expression groups, respectively. Among the squamous cell carcinomas a correlation was observed between MRP staining and tumour cell differentiation: the strongest MRP staining was predominantly found in the well differentiated tumours. Hyperexpression of MRP is frequently observed in primary NSCLC, especially in the well differentiated squamous cell carcinomas. Further studies are needed to assess the role of MRP in the mechanism of clinical drug resistance in NSCLC.

6 O - Intrinsic multidrug resistance-associated protein in human malignant melanoma cell lines

Disseminated malignant melanoma is characterized by unresponsiveness to chemotherapy without overexpressing corresponding P-glycoprotein. Another ABC-transporter protein functioning as transmembrane drug-efflux pump and involved in multidrug resistance (MDR), is the multidrug resistance-associated protein (MRP). We investigated wether MRP plays a role in malignant melanoma chemoresistance. Using the RT-PCR (two different oligonucleotid primer sets) we determined mRNA-expression in 33 cell lines established from primary tumors (n=11), metastatic lesions (n=17) and malignant effusions (n=5). Protein expression was determined by Western blot analysis (MRPm6, QCRL-1) and confirmed by immunocytochemistry. All cell lines scored positiv for MRP mRNA at different levels (5high, 13 intermediate, 15low). Levels of MRP mRNA were significantly higher in primary tumors as compared to metastases and malignant effusions. The respective protein was detectable in Western blot and in immunocytochemistry. Accumulation studies using ³H-daunomycin as MRP substrate and genistein as specific MRP modulator were perfornled and revealed - in contrast to the control cell models GLC4/ADR and HL601/AR - no drug efflux activily of MRP. In conclusion melanoma cell lines express MRP mRNA and protein at different levels. The biological role has to be investigated in further studies.

Expression of multidrug resistance-associated protein (MRP), MDR1 and DNA topoisomerase II in human multidrug-resistant bladder cancer cell lines

The acquisition of the multidrug resistance phenotype in human tumours is associated with an overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with a 190 kDa membrane ATP-binding protein encoded by a multidrug resistance-associated protein (MRP) gene. Human bladder cancer is a highly malignant neoplasm which is refractory to anti-cancer chemotherapy. In order to understand the mechanism underlying multidrug resistance in bladder cancer, we established three doxorubicin-resistant cell lines, T24/ADM-1, T24/ADM-2 and KK47/ADM, and one vincristine-resistant cell line, T24/VCR, from human bladder cancer T24 and KK47 cells respectively. Both T24/ADM-1 and T24/ADM-2 cells which had elevated MRP mRNA levels showed both a cross-resistance to etoposide and a decreased intracellular accumulation of etoposide. T24/VCR cells which had elevated levels of MDR1 mRNA and P-glycoprotein but not of MRP mRNA, showed cross-resistance to doxorubicin. On the other hand, KK47/ADM cells, which had elevated levels of both MRP and MDR1 mRNA and a decreased level of topoisomerase II mRNA, were found to be cross-resistant to etoposide, vincristine and a camptothecin derivative, CPT-11. Our present study demonstrates a concomitant induction of increased levels of MRP mRNA, decreased levels of topoisomerase II mRNA and decreased drug accumulation during development of multidrug resistance in human bladder cancer cells. The enhanced expression of the MRP gene is herein discussed in a possible correlation with the decreased expression of the topoisomerase II gene.

High expression of the multidrug resistance-associated protein (MRP) in chronic and prolymphocytic leukaemia.

The expression of the multidrug resistance-associated protein (MRP), a new glycoprotein involved in drug resistance, was investigated in tumour samples from 80 patients with chronic B-cell malignancies by a quantitative RNase protection assay. In B-cell chronic lymphocytic leukaemia (B-CLL) (n = 32), either treated (n = 18) or untreated (n = 14), a high percentage of patients (20/32: 63%) had relatively high expression levels of the MRP gene (25U or more). In addition, hyperexpression of the MRP gene was demonstrated in 4/10 (40%) untreated patients with B-cell prolymphocytic leukaemia (B-PLL). In contrast, low MRP mRNA expression levels were detected in hairy cell leukaemia (n = 7), non-Hodgkin's lymphoma (n = 13) and multiple myeloma (n = 18). Statistical analysis of MRP expression in untreated CLL (mean +/- SD 29.2 +/- 18.5 U) versus treated CLL (mean +/- SD 26.7 +/- 13.7 U) did not show significant differences in MRP expression between the two groups. Southern blot analysis did not reveal amplification of the MRP gene in the leukaemia samples with elevated MRP mRNA levels. We conclude that B-PLL and B-CLL frequently display high MRP expression and that this hyperexpression is probably due to transcriptional activation and/or increased mRNA stability.

Possible involvement of multidrug‐resistance‐associated protein (MRP) gene expression in spontaneous drug resistance to vincristine, etoposide and adriamycin in human glioma cells

The multidrug-resistance phenotype in human tumors is partly associated with over-expression of the 170 kDa-P-glycoprotein encoded by the multidrug-resistance-1 (MDR1) gene. Another related, but non-P-glycoprotein, multidrug-resistance-associated protein (MRP) gene encodes a 190 kDa-membrane ATP-binding protein. Glioblastoma multiforme is a highly malignant primary neoplasm of the central nervous system which is refractory to anti-cancer chemotherapy, but the mechanism underlying this drug resistance is unknown. Out of glioma cell lines, 2, namely IN500 and T98G, which had elevated MRP mRNA levels, showed the highest resistance to multiple anti-cancer agents such as etoposide, vincristine and adriamycin, and decreased intracellular accumulation of etoposide. In the remaining 5 cell lines, various degrees of sensitivity to adriamycin and etoposide appeared to correlate with their respective MRP mRNA levels. Our study proposes that MRP may be involved in spontaneous multidrug resistance in human gliomas.