EXPRESSION AND CLINICAL SIGNIFICANCE OF MULTIDRUG RESISTANCE GENE AND MULTIDRUG RESISTANCE-ASSOCIATEDPROTEIN GENE IN ACUTE LEUKEMIA

Objective: To evaluate the expression and clinical significance of multidrug resistance gene (mdr1) and multidrug resistance-associated protein (MRP) gene in acute leukemia. Methods: The expression of mdr1 and MRP assay in 55 patients with acute leukemia (AL) by reverse transcription polymerase chain reaction (RT-PCR). Results: The mdr1 and MRP gene expression levels in the relapsed AL and the blastic plastic phases of CML were significantly higher than those in the newly diagnostic AL and controls. The mdr1 and MRP gene expression levels in the clinical drug-resistant group were significantly higher than those in the non-drug-resistant group. The complete remission (CR) rate in patients with high mdr1 expression (14.3%) was significantly lower than that with low mdr1 expression (57.5%); similarly the CR rate in patients with high MRP level was also lower than that with low MRP level. Using both high expression of mdr1 and MRP gene as the indicator for evaluating multidrug resistance (MDR), the positive predictive value and accuracy increased in comparison with single gene high expression. Conclusion: Elevated level of mdr1 or MRP gene expression might be unfavorable prognostic factors for AL patient and may be used as an important index for predicting drug-resistance and relapse in AL patient. Measuring both mdr1 and MRP gene expression would increase accuracy and sensibility of evaluating MDR in acute leukemia.

EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) AND ITS RELATIONSHIP WITH CLINICOPATHOLOGICAL FACTORS IN NON-SMALL CELL LUNG CANCER

Objective: To investigate the relationship between the expression of multidrug resistance-associated protein (MRP) and clinicopathological factors and prognosis. Methods: The expression of MRP in 62 cases with non-small cell lung cancer (NSCLC) was detected using immunohistochemistry method. The expression of MRP in 30 cases of NSCLC and corresponding normal lung tissues were detected using immunohistochemistry and Western Blot. Results: this study of tumor tissues confirmed the plasma membrane and/or cytoplasm locations of MRP. There was apparent difference between normal lung tissues and NSCLC in MRP. The survival analysis of 62 NSCLC showed that the mean survival time of the patients with negative MRP expression was 69.8117.41 months and that of patients with positive MRP expression, 25.384.46 months. Log-rank test suggested that the difference between them was significant (P=0.0156). It was also found that in squamous cell lung cancer the statistically significant difference between the mean survival time of patients with positive MRP expression and those with negative MRP expression (P=0.0153). Multivariate Cox model analysis suggested that the survival time was significantly related to expression of MRP (P=0.035) and lymphatic metastasis (P=0.038). Conclusion: MRP expression in NSCLC is significantly higher compared with normal lung tissues. The mean survival time of patients with negative MRP was relative longer and expression of MRP was an independent factor for prognosis.

Expression and significance of multi-drug resistance-associated protein 3 in different tumor cell lines

Objective To investigate the expression and meaning of MRP3 in different tumor cells. MethodsThe monoclonal antibody against MRP3 was used to identify the expression of MRP3 by flow cytometer in seven tumor cells and human embryo kidney cell lines 293T.And RT-PCR was used to detect the mRNA of MRP3 in eight cell lines. ResultsThe mRNA of MRP3 was expressed in three pancreatic carcinoma cell lines.MRP3 protein was observed in BxPC-3 and AsPC-1 cells. ConclusionMRP3 may express in different tumor in tissue-specific manner.BxPC-3 and AsPC-1 may serve as cellular models for in vitro studies on multidrug resistance of pancreatic carcinoma.

JNK1,JNK2,and JNK3 are involved in P-glycoprotein-mediated multidrug resistance of hepatocellular carcinoma cells

BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,

Effects of Hypoxia on Expression of P-gp and Mutltidrug Resistance Protein in Human Lung Adenocarcinoma A549 Cell Line

To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein was immunohistochemically detected by culturing human lung adenocarcinoma A549 cell under hypoxia (2 % O_2) for 24 h. After interaction with adriamycin or cisplatin under hypoxia (2 % O_2) for 24 h, the cell survival rate was detected by MTT. Our results showed that the expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein under hypoxia were higher than the expression under normoxia, and correlations between the expression of HIF-1α and P-gp or multidrug resistance-associated protein was observed (P<0.05). The resistance of adriamycin of A549 cell was enhanced under hypoxia. It is concluded that the resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1α is obviously correlated with the expression of P-gp and mutltidrug resistance protein.

In silico pharmacophore models to predict endogenous substrates for human multidrug resistance-associated proteins

Multidrug resistance-associated proteins （MRPs） can effiux structurally diverse drugs, drug conjugates, drug metabolites, as well as other small molecules out of the cells, and this is the main cause of producing multidrug resistance （MDR） of some anticaneer drugs. Therefore, it is crucial to uncover the molecular features of MRPs substrates in developing anti-MDR cancer therapy. In the present study, common feature pharmacophore models were developed by employing CATALYST Pharmacophore Modeling and Analysis tools using substrates of MRPs, including MRP1, -2, -3, -4, -5, -6, -8 and MRPs family, respectively. The models were validated using independent decoy sets generated in DUD-E, and the ones with best A UC （area under the curve） scores were chosen to predict endogenous substrates by screening the Human Metabolome Database （HMDB）. A number of molecules obtained by pharmacophore screening have been validated in the literatures. By comparing physical properties （ALOGP, Molecular_PolarSurfaceArea, Molecular_Volume, Molecular_Weight, Num H Acceptors, Num H Donors） and scaffold features of the screened candidates with the known substrates, we found that： 1） The two sets have consistent ALOGP, Molecule_Volume and Molecule_Weight distribution trend; 2） Substrates of MRP1 have a better lipophilicity than the other subtypes, which is consistent with the two hydrophobic centers on the MRP1 pharmacophore; 3） In the aspect of the scaffold structures, they have the identical or similar backbone fragments.

Effect of Histone Deacetylase Inhibition on the Expression of Multidrug Resistance-associated Protein 2 in a Human Placental Trophoblast Cell Line

Background： Placental multidrug resistance-associated protein 2 （MRP2）, encoded by ABCC2 gene in human, plays a significant role in regulating drugs＇ transplacental transfer rates. Studies o11 placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases （HDACs） inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC 1/2/3 are preliminarily involved in this process. Methods： The human choriocarcinoma-derived trophoblast cell line （Bewo cells） was treated with the HDAC inhibitors-trichostatin A （TSA） at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA （siRNA） specific for HDACI/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC 1/2/3/ABCC2 messenger RNA （mRNA） and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively. Results： TSA could inhibit total HDAC activity and HDAC 1/2/3 expression in company with increase ofM RP2 expression in Bewo cells. Reduction of HDAC 1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L （vs. vehicle group, all P 〈 0.001 ）, accompanied with dose-dependent induction of MRP2 expression （P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P 〈 0.001 for 5.0 μmol/L）, whereas no significant diferences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells weir week, and no significant differences were noticed among these three groups （all P 〉 0.05）. However, MRP2 expression was remarkably elevated in H DAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells （P 〈 0.001 ）. Conclusions： HDACs inhibition could up-regulate placental MRP2 expression in ritzy, and HDAC 1 was probably to be involved in this process.

应用蛋白质芯片对汉防己甲素单用及与屈洛昔芬伍用逆转白血病细胞耐药机理的研究

本研究的目的是应用蛋白质芯片检测汉防己甲素(Tet)单独及与屈洛昔芬(Drol)伍用对作用的白血病细胞表面的耐药蛋白,包括P糖蛋白(Pgp)、多药耐药相关蛋白(MRP1)、乳腺癌耐药蛋白(BCRP)表达的作用,为逆转剂的临床应用提供理论依据。选择位于膜表面Pgp、MRP1、BCRP耐药蛋白及其相应的抗体为研究体系,制备蛋白芯片,直接对逆转剂作用12、24和48小时的K562/A02细胞进行检测。结果表明:Tet和Drol联合作用24小时时检测到Pgp表达下调(Tet+Drol组:85.27±3.095,对照组:93.67±2.748,P<0.05)。经逆转剂单独及联合作用K562/A02细胞48小时时均检测到Pgp表达下调,且联合应用两药对Pgp表达下调作用明显(Tet+Drol:82.62±3.227,Tet:86.44±2.906,Drol:87.23±2.049,对照组:93.67±2.748,P<0.05)。检测结果与流式细胞仪检测结果一致。结论:逆转剂Tet和Drol对K562/A02细胞的Pgp下调呈时间依赖性。联合作用24小时时出现下调Pgp表达,单独作用48小时均下调Pgp表达,联合用药时下调作用明显。不同检测时间均未见下调MRP1和BCRP的表达。

非小细胞肺癌COX-2的表达与多药耐药性关系的研究

目的:探讨环氧合酶-2(COX-2)在非小细胞肺癌(NSCLC)组织中的表达,并探讨COX-2对多药耐药相关蛋白(MRP)表达的影响。方法:应用免疫组化技术检测62例非小细胞肺癌及10例正常肺组织中COX-2及MRP的表达。结果:正常肺组织未见COX-2及MRP的表达。NSCLC组织中COX-2的阳性表达率为61.3%(37/62),其阳性表达与NSCLC的组织学类型、肺癌组织不同临床分期、有无淋巴结转移、组织分化程度有关,与肺癌患者的性别无关。COX-2阳性组织中MRP的表达阳性率为89.2%(33/37),而COX-2阴性组织中MRP同时阴性率为68.0%(17/25),相关关系分析显示肺癌COX-2表达与多药耐药相关蛋白MRP表达呈显著正相关。结论:COX-2的表达参与NSCLC的发生及恶化进展,并可能调控MRP的表达。

非小细胞肺癌组织中MRP1和LRPmRNA的表达及其临床意义

目的探讨多药耐药相关蛋白1(MRP1)和肺耐药蛋白(LRP)基因在非小细胞肺癌(NSCLC)中的表达及其临床意义。方法应用RT-PCR半定量法检测60例NSCLC患者癌组织及癌旁组织中MRP1和LRPmRNA的表达。结果NSCLC患者的癌组织中MRP1、LRPmRNA表达量显著高于癌旁组织(P≤0.02),且两者在癌组织中的表达呈正相关;MRP1mRNA在肿瘤直径>3cm组表达量明显高于肿瘤直径≤3cm组(P=0.013);LRPmRNA表达量NSCLC腺癌组高于鳞癌组(P=0.02),中高分化组高于低分化组(P=0.033)。结论MRP1、LRP在NSCLC的多药耐药(MDR)中可能起重要作用,且在形成MDR的过程有某种程度的关联,联合检测MRP1、LRPmRNA对于预测NSCLC患者化疗效果、协助临床选择化疗方案可能具有一定的意义。

氧化砷对MR_2细胞耐药蛋白的调节作用

目的 :研究氧化砷 (三氧化二砷 ,As2 O3 )对肿瘤耐药蛋白的作用。方法 :以耐维A酸 (ATRA)早幼粒白血病细胞株MR2 为研究对象 ,以非耐药早幼粒白血病细胞株NB4为对照组 ,用免疫组化法观察P 糖蛋白 (Pgp)、多药耐药相关蛋白 (MRP)的表达。结果 :MR2 细胞Pgp表达 (30 %～ 4 0 % )较NB4细胞 (10 %～ 2 0 % )明显增强 (P <0 .0 0 1) ,MR2 细胞MRP表达 (6 0 .4± 4 .0～ 6 6 .5± 4 .4 )较NB4细胞 (2 8.3± 5 .6～ 31.2± 5 .1)明显增强 (P <0 .0 0 1)。 0 .5～ 2 .0μmol/L氧化砷能显著降低MR2 细胞Pgp、MRP表达。MR2 细胞Pgp、MRP表达的降低与氧化砷作用浓度和作用时间成负相关。结论 :MR2 细胞耐药蛋白Pgp、MRP表达的下调 ,可能是氧化砷克服耐药的敏感性指标。维A酸 (ATRA)可能是Pgp、MRP的转运底物。

人胎盘ATP结合盒转运蛋白对格列本脲胎盘转运的影响

目的研究成熟胎盘ATP结合盒转运蛋白对格列本脲胎盘透过率的影响。方法建立人胎盘体外循环灌注模型40例,分为4组(n=10),分别用格列本脲、格列本脲和维拉帕米(PGP抑制剂组)、格列本脲和尼卡地平(BCRP抑制剂组)、格列本脲和吲哚美辛(MRPs抑制剂组)循环灌注3 h,计算各组格列本脲的胎盘透过率和清除指数。结果3 h循环结束后,4组格列本脲的平均胎盘透过率分别为(0.78±0.66)%、(2.17±0.90)%、(1.45±0.70)%、(3.65±2.10)%,与格列本脲组相比,加入抑制剂后各组格列本脲胎盘透过率均有增加(P<0.05),MRPs抑制剂组透过率高于PGP抑制剂组和BCRP抑制剂组(P<0.05);4组格列本脲相对透过率分别为(0.03±0.02)、(0.08±0.04)、(0.05±0.02)、(0.13±0.05),PGP抑制剂组和MRPs抑制剂组的清除指数均高于格列本脲组(P<0.05),且MRPs抑制剂组高于PGP抑制剂组(P<0.05)。结论PGP、BCRP、MRPs抑制剂均不同程度地参与了格列本脲的胎盘转运,其中,MRPs抑制剂的影响可能更为显著。

突变型p53蛋白与P-糖蛋白在涎腺粘液表皮样癌中的表达及意义

目的:探讨突变型p53蛋白与多药耐药蛋白P-糖蛋白在涎腺粘液表皮样癌(MEC)的表达及意义。方法:应用免疫组织化学技术(SP法)检测突变型p53蛋白和P-糖蛋白在37例涎腺MEC及12例正常涎腺组织中的表达,分析二者表达与涎腺MEC临床病理特点的联系及二者表达之间的相关性。结果:37例涎腺MEC中突变型p53蛋白阳性表达率为54.1%(20/37),正常涎腺组织无表达,差异有统计学意义(P<0.001);涎腺MEC中P-糖蛋白阳性表达率为91.9%(34/37),正常涎腺组织阳性表达率为58.3%(7/12),差异有统计学意义(P<0.05)。37例涎腺MEC中突变型p53蛋白的阳性表达与临床分期、分化程度、有无淋巴结转移有关,差异有统计学意义(P<0.05)。37例涎腺MEC中突变型p53蛋白的阳性表达与P-糖蛋白的阳性表达有明显相关性(P<0.05)。结论:突变型p53蛋白和P-糖蛋白在涎腺MEC中均有较高水平表达,突变型p53蛋白过表达与涎腺MEC的生长、分化及淋巴结转移的生物学行为关系密切。检测涎腺MEC中突变型p53蛋白和P-糖蛋白的表达可为临床拟定化疗方案提供依据,也可为判断涎腺MEC的预后提供参考。

降低细胞膜角蛋白8和乳腺癌耐药蛋白的表达逆转耐药性

目的研究细胞膜角蛋白8(CK8)和乳腺癌耐药蛋白(breast cancer resistant protein,BCRP)作为多靶点治疗逆转耐药的可行性。方法共转染特异的CK8-siRNAs和BCRP-siRNAs至人乳腺癌多药耐药细胞MCF-7/MX,用Western blot方法检测siRNAs对CK8和BCRP蛋白表达的抑制,荧光染色用激光共聚焦显微镜观察细胞膜表面CK8表达量的变化,并用Sulforhodamine B的方法检测转染前后细胞对多种化疗药敏感性的变化。结果MCF-7/MX细胞导入CK8-siRNAs和BCRP-siRNAs后,CK8和BCRP的表达水平均明显降低,且细胞表面CK8染色也明显降低,同时对米托蒽醌等化疗药的敏感性明显提高,耐药表型明显逆转。结论CK8和BCRP在MCF-7/MX耐药细胞中共同高表达且在乳腺癌多药耐药表型的形成中起重要作用,共同抑制CK8和BCRP的表达可以有效逆转乳腺癌的多药耐药,为治疗肿瘤多药耐药提供了一条新的思路。

Evaluation of the Mrp2-mediated flavonoid-drug interaction potential of quercetin in rats and in vitro models

Quercetin is a biologically active flavonoid that has been used as a popular health supplement.It is reported that quercetin may cause flavonoid-drug interaction mediated by P-glycoprotein,the most predominant efflux transporter.In this study,we comprehensively evaluated the potential of the pharmacokinetic interaction of quercetin mediated by multidrug resistance-associated protein 2(MRP2),another major efflux transporter.MRP2-transfected MDCKII cells and LS174T cells were used to evaluate the potential inhibition and induction of MRP2 by quercetin in vitro.To evaluate the induction effect of quercetin on Mrp2 in vivo,Mrp2 mRNA expression in rat liver,kidney,and small intestinal tissues was determined after the oral administration of quercetin(50,100,or 250 mg/kg)for seven days.Mrp2-mediated interaction potential was also evaluated by the pharmacokinetic study of phenolsulfonphthalein in rats after single or multiple doses of quercetin.Additionally,the effect of quercetin on absorption of docetaxel,a P-glycoprotein and CYP3A4 substrate,was also evaluated.Quercetin inhibited the function of MRP2 at 10μM and induced the mRNA expression of MRP2 at 50μM in vitro.Additionally,at 100 mg/kg,quercetin markedly increased Mrp2 expression in the small intestine of rats.However,there was no significant change in phenolsulfonphthalein pharmacokinetics due to single-(50,100,or 250 mg/kg)or multiple-dose(50,100,or 250 mg/kg for seven days)quercetin co-administration.By contrast,a significant interaction caused by quercetin(100 mg/kg)was observed in the absorption of docetaxel.The results suggested that although quercetin modulates the function and expression of MRP2 in vitro,it may have a low potential of Mrp2-mediated interaction and present negligible safety concerns related to the interaction.

EXPRESSION OF MDRII MRP AND LRP GENES IN GASTRIC CARCINOMA AND THEIR CLINICAL SIGNIFICANCE

Objective: To explore the expression of mdrl,multidrug resistance-associated protein (MRP) and lungresistance protein (LRP) genes in human gastric cancerand their clinical significance. Methods: The mdrlmRNA was assayed by RT-PCR, the MRP and LRPwere detected by flow cytometry. Results: The positiverate of mdrl mRNA was 44.4% (12/27), and the meanMRP and LRP expression were independent uponpatient histologic type, nodal involvement, and TNMstage. The mdrl mRNA expression in patients withserosa invasion was 30.0% (6120), much lower than thatwithout serosa invasion (85.7%). Conclusion: Themultidrug resistance cells are present in primary gastriccarcinomas prior to chemotherapy, and analysis of mdrlgene, MRP, LRP may have guiding significance in thetreatment of gastric carcinoma.

Different gap junction-propagated effects on cisplatin transfer result in opposite responses to cisplatinin normal cells versus tumor cells

OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.

Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRPl（196/210） substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the [Coat＇A196Rz/Coat＇B210Rz]5 is more significantly superior to that of the [Coat＇A 196Rz/Coat＇B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat＇A 196Rz/Coat＇B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg^2＋ on cleavage efficiency indicate that cleavage reaction is dependent on Mg^2＋ ions concentration. The plot of lg（kobs） vs. lgc（Mg^2＋） displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg^2＋. It suggests that Mg^2＋ ions play a crucial role in multi-ribozyme cleavage on the substrate.

EFFECTS OF NEOADJUVANT CHEMOTHERAPY ON MDR1 AND MRP GENE EXPRESSION IN PRIMARY BREAST CANCER

Objective: To investigate the effects of neoadjuvant chemotherapy on the expression of drug resistance genes, multidrug resistance-1 (MDR1) and multidrug resistance-associated protein (MRP), in patients with primary breast cancer. Methods: MDR1 and MRP expression were detected by semi-quantitative RT-PCR in 20 patients with primary breast cancer, before and after chemotherapy. Results: Before chemotherapy, MDR1 and MRP expression can be detected in 15 cases (75%) and 18 cases (90%) respectively. After chemotherapy, expression of MDR1 is not significantly different from that before chemotherapy, but expression of MRP is significantly different from that before chemotherapy. Conclusion: Expression of drug resistance gene MRP, but not MDR1, is enhanced in patients with primary breast cancer submitted to neoadjuvant chemotherapy.