MAB4147 Sigma-AldrichAnti-MRP1 Antibody, clone MRPm5

Cancer stem cells (CSCs) in gliomas are associated with resistance to radio- and chemotherapy, based on O(6)-methylguanine-DNA methyltransferase (MGMT) hypermethylation and the Multidrug resistance (MDR) system activation.

We studied the expression of O(6)-methylguanine-DNA methyltransferase (O(6)-MGMT), P-glycoprotein (Pgp), and multidrug resistance protein-1 (MRP-1) in 23 glioblastomas using RT-PCR, methylation-specific PCR, and immunohistochemistry, and analyzed their association with overall patient survival. Univariate analysis of collected data demonstrated that the expressions of O(6)-MGMT and MRP-1 detected by immunohistochemistry, in addition to the consistent factors, including preoperative Karnofsky performance scale (KPS), radical surgery, and tumor location and extension, were significant prognostic factors for the overall survival (OS) of patients with glioblastoma, who received nimustine (ACNU)-based chemotherapy in association with surgery and radiotherapy. Among them, following multivariate analysis, preoperative KPS, radical surgery, tumor location, and the expression of O(6)-MGMT remained as significant prognostic factors. These findings suggest that immunohistochemical analysis of O(6)-MGMT in patients with glioblastoma can be a useful method to predict the effects of chemotherapy and identify alternative chemotherapeutic regimens for O(6)-MGMT-positive patients.

Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1). MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established. The MRP1 protein belongs to a family of at least six members. Three of these, i.e., MRP1, MRP2, and MRP3, can transport MDR drugs and could be involved in MDR. The substrate specificity of the other family members remains to be defined. Specific monoclonal antibodies are required for wide-scale studies on the putative contribution of these closely related transporter proteins to MDR. In this report, we describe the extensive characterization of a panel of monoclonal antibodies (Mabs) detecting several MDR-related transporter proteins in both human and animal tissues. The panel consists of P3II-1 and P3II-26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M2I-4, M2II-12, M2III-5 and M2III-6 for MRP2; M3II-9 and M3II-21 for MRP3; and M5I-1 and M5II-54 for MRP5. All Mabs in the panel appeared to be fully specific for their cognate antigens, both in Western blots and cytospin preparations, as revealed by lack of cross-reactivity with any of the other family members. Indeed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunohistochemical analyses, distinct differences in reactivity and suitability were noted. These Mabs should become valuable tools in studying the physiological functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.

The human multidrug resistance protein (MRP) is a 190 kd membrane glycoprotein that can cause resistance of human tumor cells to various anticancer drugs, by extruding these drugs out of the cell. Three different monoclonal antibodies, directed against different domains of MRP, allowed us to determine the localization of MRP in a panel of normal human tissues and malignant tumors. Whereas in malignant tumors strong plasma membrane MRP staining was frequently observed, in normal human tissues MRP staining was predominantly cytoplasmatic. Here, MRP was detected in several types of epithelia, muscle cells, and macrophages. From the presence of MRP in many epithelia we infer that MRP, like MDR1 P-glycoprotein, may have an excretory function in protecting the organism against xenobiotics. Recent studies indicate a role for MRP as a carrier for transport of glutathione-conjugated endo- and xenobiotics. The presence of MRP in bronchiolar epithelium, heart muscle, and macrophages would agree with the glutathione S-conjugate carrier activity previously detected in these cells. Furthermore, in 46 of 119 untreated tumors from various histogenetic origins MRP staining was seen. In these tumors MRP may contribute to the intrinsic resistance against treatment with chemotherapeutic drugs.

BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.