Expression of the multidrug resistance gene product (P-glycoprotein) in myelodysplasia is associated with a stem cell phenotype

Previous studies have indicated relative resistance to chemotherapy in the myelodysplastic syndromes (MDS) and associated acute leukaemia. To determine if multidrug resistance may contribute to chemoresistance in these disorders, we studied bone marrow specimens for P-glycoprotein expression (P-GP) by immunocytochemical staining with monoclonal antibodies reactive with cytoplasmic (C219) or surface epitopes (MRK16) of P-GP. Forty-five case specimens from 43 patients were studied, including 32 cases of primary MDS, seven cases of acute myeloid leukaemia (AML) following MDS, and six therapy-related haematological disorders. Cytogenetic analysis was available on 36 specimens. Two staining patterns were detected: (1) cytoplasm and plasma membrane, and (2) staining restricted primarily to the nuclear-cytoplasmic junction. P-GP was detected in seven (22%) cases of primary MDS, four (57%) cases of AML evolving from MDS, and five (83%) cases of therapy-related haematological disorders. Expression of P-GP was restricted to blasts and leukaemic monocytes, and was otherwise absent from terminally differentiated blood cells. Analysis of the relation between P-GP expression and reactivity with the human progenitor cell antigen CD34, revealed a highly significant association (P = 0.001). P-GP reactivity was distributed equally among normal and abnormal karyotypes and did not correlate with specific cytogenetic abnormalities. These findings indicate that multidrug resistance in MDS and karyotypically-related haematological disorders is closely linked to a stem cell phenotype and may contribute to chemoresistance in these disorders.     

Modulation of BCRP mediated atypical multidrug resistance phenotype by RNA interference

Multidrug resistance (MDR) in human cancers is one of the major causes of failure of chemotherapy. The emergence of breast cancer resistance protein (BCRP), a member of the ABC transporter family, has necessitated the development of antagonists. To overcome the BCRP-mediated atypical multidrug drug resistance, two small interfering RNA constructs (RNAi) targeting two different regions of BCRP mRNA were designed to inhibit the atypical MDR expression by transfecting them into MCF-7/MX100 cell lines. The multidrug resistance index to mitoxantrone and the intensity of mitoxantrone fluorescence of MCF-7/MX100 decreased after transfected by pSUPER-BCRP-A and pSUPER-BCRP-B respectively; the BCRP mRNA level and the BCRP protein level of MCF-7/MX100 decreased after treated with pSUPER-BCRPs. The two constructed RNAi plasmids could reverse the atypical mutidrug resistance mediated by BCRP, but neither can reversed it completely, this may be due to low transfection efficiency and transient transfection.     

急性白血病多药耐药基因及多药耐药相关蛋白基因的表达及临床研究

目的探讨急性白血病（ＡＬ）多药耐药基因（ｍｄｒ１）及多药耐药相关蛋白基因（ＭＲＰ）的表达与预后的关系。方法采用逆转录聚合酶链反应（ＲＴＰＣＲ）的方法检测５５例ＡＬ患者的ｍｄｒ１及ＭＲＰ的表达。结果发现复发急变组的ｍｄｒ１及ＭＲＰ基因表达水平（０．７３５±０．２４９,１．１５７±０．４４７）较初治组（０．４０８±０．１８６,０．４６５±０．２５３）明显升高（Ｐ＜０．０１）。而且ｍｄｒ１及ＭＲＰ同时高表达者的完全缓解（ＣＲ）率明显低于ｍｄｒ１及ＭＲＰ同时低表达者（Ｐ＜０．０１）。结论ｍｄｒ１和ＭＲＰ同时高表达是判断ＡＬ患者耐药复发及不良预后的重要因素。     

Expression of multidrug resistance protein in metastatic colorectal carcinomas

To clarify the relationship between multidrug resistance protein (MRP) and clinicopathologic features, the influence of adjuvant chemotherapy, and prognosis of patients who underwent resection of metastatic liver carcinomas originating from colorectal carcinomas, we examined the expression of MRP in tumor tissues by immunostaining. Specimens of 38 primary colorectal tumors and 44 metastatic liver tumors of colorectal origin were examined (metastatic group). We also examined 28 nonmetastatic colorectal carcinomas. The percentages of nonmetastatic tumors and of primary and metastatic tumors of the metastasis group that expressed MRP were similar. MRP expression in primary and metastatic tumors did not correlate with any clinicopathologic features. The use of adjuvant chemotherapy after operation for primary colorectal carcinomas was associated with increased MRP expression among metastatic liver tumors. Expression of MRP in the tumor did not influence the prognosis or survival rate after resection of primary or metastatic tumors. Our data suggest that MRP expression in metachronous liver metastases from colorectal carcinomas may be induced by administration of anticancer drugs but is not associated with clinicopathologic features of the tumor, liver metastasis, or prognosis. Received: November 19, 1998 / Accepted: May 28, 1999     

人肝癌细胞株线粒体DNA缺失对多药耐药表型的影响

目的:探讨人肝癌细胞株mtDNA缺失对肿瘤MDR表型的影响.方法:采用MTT方法检测细胞药物敏感性,免疫组化方法检测P-gp,MRP表达.对比分析SK-Hep1细胞和rhooSK-Hep1细胞药物敏感性和P-gp,MRP表达的差异.结果:阿霉素(ADR)、顺铂CDDP对SK-Hep1细胞的12,24,36,48h抑制率分别为56%,61%,72%,75%和54%,60%,77%,81%,而对rhooSK-Hep1细胞的抑制率分别为10%,18%,20%,22%和19%,20.4%,21.3%,22.5%.SK-Hep1与rhooSK-Hep1细胞比较,药物敏感性显著降低(P<0.01).较之于SK-Hep1细胞,rhooSK-Hep1细胞P-gp,MRP表达明显增加(37%vs20%,P<0.01;33%vs18%,P<0.01).结论:mtDNA缺失的肿瘤细胞对化疗药物有显著抗性,P-gp,MRP表达增加可能是其MDR表型产生的重要原因.     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

Small molecules that dramatically alter multidrug resistance phenotype by modulating the substrate specificity of P-glycoprotein

By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug. Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters. Stimulation of drug efflux was detectable in the cells expressing P-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors. A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp. Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates. The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp. These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules.     

Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype.

The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P-glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P-glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.     

Expression of multidrug resistance gene mdr1 mRNA in a subset of normal bone marrow cells

The multidrug resistance gene mdr1, encoding P-glycoprotein (P-gp), can be expressed at high levels in tumour cells derived from normal tissues with constitutive high expression of this gene. In myelogenous leukaemia, the incidence of increased expression of mdr1 gene contrasts with the low expression of this gene in normal bone marrow (b.m.). To detect cells expressing mdr1 gene in normal and post-chemotherapy b.m., we used in situ RNA hybridization and RNA phenotyping by the polymerase chain reaction for mdr1 mRNA detection. The presence of P-gp was evaluated by immunocytochemistry with MRK16. Fifteen b.m. (eight normal and seven post chemotherapy) were tested by in situ hybridization and either PCR (three b.m.) or immunocytochemistry (11 b.m.) or both (one b.m.). With in situ mRNA hybridization, a subset (7.7% +/- 3.1%) of b.m. cells expressed mdr1 mRNA in all cases tested, with no significant differences between normal b.m. and post chemotherapy b.m. 18% of myeloid recognizable cells and 7% of the cells with lymphoid morphology expressed mdr1 mRNA. By RNA phenotyping, the four samples tested for in situ hybridization and two additional post chemotherapy b.m. expressed mdr1. MRK16 was unable to detect a significant number of cells expressing P-gp either by immunocytochemistry in the 12 b.m. tested for in situ hybridization (0% in nine cases; 0.4%, 1% and 3% of positive cells in three cases), or by flow cytometry in six additional normal b.m. (0-1.4% positive cells).     

Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia.

The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P =.01) and Pgp function (P =.03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P <.05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r =.90, P =.0001) and Pgp-positive blasts (r =.77, P =.0002). This increase in Pgp expression and function was inhibited by the addition of 1 micromol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.     

The HIV type 1 protease inhibitor saquinavir can select for multiple mutations that confer increasing resistance

Previous use of the HIV-1 protease inhibitor saquinavir resulted in the infrequent appearance of mutations in the HIV-1 protease gene associated with resistance. We have examined the ability of saquinavir to select for resistance mutations. In multiple selections of HIV-1 in cell culture with saquinavir, similar patterns of mutations were reproducibly observed and the number of mutations increased with increasing selective pressure. In a small number of subjects who showed an antiviral response when saquinavir was added to their therapeutic regimen, similar mutations were detected in viral genomic RNA in vivo after 30 to 40 weeks of therapy. These results indicate that saquinavir can select for resistance mutations and suggest that the infrequent appearance of these mutations in vivo is the result of low drug exposure. These results also predict that the use of higher levels of saquinavir will lead to an even greater frequency of resistance mutations in patients who fail therapy.     

Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias

The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.     

食管癌活检标本多药耐药基因表达及意义

目的 研究食管癌活检标本中多药耐药基因（multidrug resistance gene １,ＭＤＲ１基因）和多药耐药相关蛋白（multidrug resistance associatid pritein,ＭＲＰ）表达水平,探讨与食管癌临床特征及化疗的相关性。方法 应用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）技术定量分析标本中ＭＤＲ１基因和ＭＲＰ的表达水平。结果 ５８例标本中,ＭＤＲ１基因阳性表达率为８２．８％（４８/５８）,ＭＲＰ表达率为２９．３％（１７/５８）。