白血病多药耐药性及其逆转的研究
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摘要
研究目的
1．了解P糖蛋白(Pgp)、多药耐药基因(mdr1)、多药耐药相关蛋白基因(MRP)和DNA拓扑异构酶Ⅱ(TopoⅡ)是否为急性白血病临床耐药的预后因素。
2．确定Pgp、mdr1、MRP和TopoⅡ诊断急性白血病临床耐药的准确性。
3．研究发夹状小分子干扰RNA(hairpin siRNA)对白血病多药耐药细胞株K562／A02 mdr1和GSTπ基因的表达和功能的影响。
研究方法
1．45例急性白血病患者用单抗UIC2标记的流式细胞仪法测定Pgp；用RT-PCR方法检测其mdr1、MRP和TopoⅡ的表达，用单因素和多因素Logstic回归分析它们与白血病预后的关系。
2．以临床耐药作为金标准，用受试者工作特征(ROC)曲线下面积评价上述参数的准确性；在ROC曲线上确定各种参数的最佳临界点；为了提高这些参数的诊断价值，采用平行试验和系列试验计算其诊断临床耐药的灵敏度(Se)和特异度(Sp)。
3．根据mdr1 mRNA第79-99和GSTπmRNA第308-327核苷酸为作用靶点，合成针对靶区域序列的发夹状siRNA，克隆入pSilencer2．1-U6 neo，克隆产物为pSilence-mdr1和pSilence-GSTπ，转染K562／A02细胞株。用实时荧光定量PCR检测K562／A02 mdr1和GSTπmRNA的表达；用Westernblot和荧光免疫组化观察Pgp和GSTπ蛋白的表达；MTT法检测阿霉素对K562／A02细胞半数抑制浓度(IC_(50))。
研究结果：
1．耐药组Pgp、mdr1、MRP阳性率均高于敏感组(P＜0．01)，而TopoⅡ耐药组较低(P=0．049)。经单因素Logistic回归分析，Pgp、mdr1、MRP的过度表达，TopoⅡ的表达降低和55岁以上年龄组与临床耐药性显著相关；性别、发病时WBC、FAB亚型和骨髓原+(早)幼细胞比例与耐药性无关。多因素Logistic回归分析经以上变量校正后显示Pgp、mdr1、MRP、TopoⅡ和年龄仍与耐药性显著相关：Pgp(RR=14．87，P=0．003)；mdr1(RR=19．98，P=0．003)；MRP(RR=16．53，P=0．006)；TopoⅡ(RR=0．23，P=0．046)；年龄(RR=10．87，P=0．013)。将36例初发急性白血病患者单独分析，结果发现Pgp、mdr1和MRP亦与完全缓解密切相关。Pgp(RR=9．8，P=0．005)；mdr1(RR=12．1，P=0．005)；MRP(RR=33，P=0．002)。直线相关分析发现在所有病例组和临床耐药组中Pgp和mdr1；Pgp和MRP：mdr1和MRP具有相关性(P＜0．001)。
2．ROC曲线下面积(AUC~(ROC))：TopoⅡ明显低于Pgp、mdr1和MRP(P＜0．05)，而Pgp、mdr1和MRP的ROC曲线下面积差别无显著意义(P＞0．05)；Pgp、mdr1、MRP和TopoⅡ的最佳临界点分别为10％、0．8、1．0和0．9，在对应的灵敏度中，Pgp最高为74％，特异度中mdr1最高为86％；Pgp、mdr1和MRP的平行试验和系列试验计算，灵敏度和特异度均可提高至91％。
3．经pSilence-mdr1转染后的K562／A02细胞株mdr1 mRNA表达量下降了71．5％，从(2．8±1．65)×10~8拷贝／μgRNA下降至(3．9±2．37)×10~7拷贝／μgRNA，(P＜0．01)；同时pSilence-GSTπ作用后，K562／A02 GSTπmRNA表达量较mock下降了39．8％，从(2．3±1．14)×10~5拷贝／μgRNA下降至(5．4±2．45)×10~4拷贝／μgRNA(P＜0．01)。
4．Western blot结果显示pSilence-mdr1和pSilence-GSTπ分别转染K562／A02细胞株后与mock相比，Pgp和GSTπ的表达明显下降；而pSilence-mdr1和pSilence-GSTπ对α-tubulin的表达没有影响；K562／A02细胞株转染pSilence-laminA／C后与mock相比，laminA／C的表达明显下降，但对Pgp和GSTπ的表达没有作用；灰度扫描显示与内对照B-actin的比值，Pgp的表达量从K562／A02(mock)的0．75±0．019下降到K562／A02(pSilence-mdr1)的0．48±0．049(P＜0．001)；GSTπ的表达量从K562／A02(mock)的0．54±0．025下降到K562／A02(pSilence-GSTπ)的0．39±0．022(P＜0．01)。荧光免疫组化结果显示荧光显微镜下可见Pgp在K562／A02组有大点状的绿色荧光，阳性细胞比例从转染前的71．25±9．65％到pSilence-mdr1转染后的35．25±5．97％(P＜0．001)；GSTπ在K562／A02组有多量点状的红色荧光，阳性细胞从转染前的81．25±6．49％下降到pSilence-GSTπ转染后的41．25±4．43％(P＜0．001)。
5．MTT结果示对阿霉素的耐药指数从空载体转染后的23，到pSilence-mdr1转染后的8和pSilence-GSTπ转染后的10，差别有显著性。
研究结论：
1．Pgp、mdr1、MRP和TopoⅡ是急性白血病临床耐药的独立预后因素。mdr1和MRP具有相关性。
2．Pgp、mdr1和MRP诊断临床耐药价值较高；其联合试验可以提高灵敏度和特异度。
3．siRNA可有效、特异地逆转K562／A02 mdr1和GSTπ的多药耐药性。
A study of multidrug resistance andit's modulation in leukemia
Objectives:
1.To investigate whether Pgp,mdrl,MRP and TopoⅡare theprognostic factors of clinical resistance in acute leukemiapatients.
2.To determine the diagnostic value of Pgp,mdrl,MRP and TopoⅡof clinical resistance in acute leukemia patients.
3.To investigate the effectiveness and the specificity of hairpinsmall interference RNA(siRNA) on mdrl and GSTπexpression inmultidrug resistance human leukemia cell line K562/A02.
Methods:
1.45 cases were sequentially recruited.Flow cytometry usingmonoclonal antibody UIC2 was used to detect the expression ofPgp.RT-PCT was used to detect the expression of mdrl,MRP andTopoⅡ.Univariate and multivariate logistic regression wereused to analyse the relationship between the four factors andthe prognosis of acute leukemia.
2.According to the golden standard of clinical resistance,AreaUnder Receiver-Operating Characteristic Curve (AUC~(ROC)) was used to assess the accuracy of the four indicators and to determinethe cut-off point.
3.The Hairpin siRNA were synthesized according to the sequencetargeting mdrl and GSTπcorresponded to the coding region 79-99and 308-327,and cloned to pSilencer2.1-U6 neo,the clonedproducts were pSilence-mdrl and pSilence-GSTπ,transfected intoK562/A02.Expression of mdrl and GSTπmRNA were assayed by SYBRGreen I real-time PCR.Western blot analysis andimmunofluorescence test were used to detect the effectivenessand the specificity of the gene silence.50% inhibitionconcentration (IC_(50)) of doxorubicin(ADM) on K562/A02 wasdetermined by MTT method.
Results:
1.The expressions of Pgp,mdrl and MRP were significantly higherin resistant group than that in sensitive group (P＜0.01).Theexpression of TopoⅡwas lower in resistant group,but thedifference was not statistically significant.Logisticregression of univariate analysis showed that theover-expression of Pgp,mdrl and MRP,the lower expression ofTopoⅡand age over 55 were the prognostic factors of clinicalresistance in acute leukemia.Sex,initial white blood cellcount (WBC),bone marrow (BM) blast percentage and FAB subtypeswere not significant for the clinical resistance.Multivariateanalysis adjusted by above factors showed that theover-expression of Pgp,mdrl and MRP,the low expression of TopoⅡand age over 55 were still highly significant for clinicalresistance.Pgp:RR=14.87,P=0.003;mdrl:RR=19.98,P=0.003; MRP:RR=16.53,P=0.006;TopoⅡ:RR=0.23,P=0.046;Age:RR=10.87,P=0.013.
2.AUC~(ROC):Pgp 0.842±0.06,mdrl 0.729±0.079,MRP 0.739±0.077,TopoⅡ0.52±0.087.The cut-off point of Pgp,mdrl,MRP and TopoⅡwere 10%,0.8,1.0 and 0.9,respectively.The sensitivity andspecificity were Pgp 74% and 82%,mdrl 65% and 86%,MRP 61%and 86%,TopoⅡ65% and 41%.The parallel and serial testsof Pgp and mdrl;Pgp and MRP;Pgp,mdrl and MRP could increasethe sensitivity and the specificity up to 91%.
3.After transfected with pSilence-mdrl,the expression of mdrlmRNA in K562/A02 was reduced 71.5% compared to the mocktransfection,from (2.8±1.65)×10~8copy/μg RNAto (3.9±2.37)×10~7copy/μg RNA (P＜0.01);While the expression of GSTπmRNA inK562/A02 transfected with pSilence GSTπwas reduced 39.8%compared to the mock transfection,from (2.3±1.14)×10~5 copy/μgRNA to (5.4±2.45)×10~4 copy/μg RNA (P＜0.01).
4.Western blot results showed that mdrl and GSTπtargeted siRNAinhibited the expression of Pgp and GSTπprotein,with no effectonα-tubulin expression in comparison with mock treatment;lamin A/C siRNA decreased lamin A/C protein expression but hadno effect on the expression of Pgp and GSTπprotein.Immunofluorescence test also showed siRNA significantlyinhibited the expression of Pgp and GSTπprotein compared to themock treatment.
5.The resistance index after transfection was decreased to 8 and10 from 23 compared to the mock transfection,P＜0.01.
Conclusions:
1.The over-expression of Pgp,mdrl and MRP,and the lowerexpression of TopoⅡwere the unfavorable prognostic factorsof clinical resistance in acute leukemia patients.
2.The accuracy of Pgp,mdrl and MRP for diagnosing the clinicalresistance were high.The multiple tests could increase thesensitivity and the specificity.
3.The hairpin siRNA could effectively and specifically modulate themultidrug resistance in K562/A02 cell line.
1．了解P糖蛋白(Pgp)、多药耐药基因(mdr1)、多药耐药相关蛋白基因(MRP)和DNA拓扑异构酶Ⅱ(TopoⅡ)是否为急性白血病临床耐药的预后因素。
2．确定Pgp、mdr1、MRP和TopoⅡ诊断急性白血病临床耐药的准确性。
3．研究发夹状小分子干扰RNA(hairpin siRNA)对白血病多药耐药细胞株K562／A02 mdr1和GSTπ基因的表达和功能的影响。
研究方法
1．45例急性白血病患者用单抗UIC2标记的流式细胞仪法测定Pgp；用RT-PCR方法检测其mdr1、MRP和TopoⅡ的表达，用单因素和多因素Logstic回归分析它们与白血病预后的关系。
2．以临床耐药作为金标准，用受试者工作特征(ROC)曲线下面积评价上述参数的准确性；在ROC曲线上确定各种参数的最佳临界点；为了提高这些参数的诊断价值，采用平行试验和系列试验计算其诊断临床耐药的灵敏度(Se)和特异度(Sp)。
3．根据mdr1 mRNA第79-99和GSTπmRNA第308-327核苷酸为作用靶点，合成针对靶区域序列的发夹状siRNA，克隆入pSilencer2．1-U6 neo，克隆产物为pSilence-mdr1和pSilence-GSTπ，转染K562／A02细胞株。用实时荧光定量PCR检测K562／A02 mdr1和GSTπmRNA的表达；用Westernblot和荧光免疫组化观察Pgp和GSTπ蛋白的表达；MTT法检测阿霉素对K562／A02细胞半数抑制浓度(IC_(50))。
研究结果：
1．耐药组Pgp、mdr1、MRP阳性率均高于敏感组(P＜0．01)，而TopoⅡ耐药组较低(P=0．049)。经单因素Logistic回归分析，Pgp、mdr1、MRP的过度表达，TopoⅡ的表达降低和55岁以上年龄组与临床耐药性显著相关；性别、发病时WBC、FAB亚型和骨髓原+(早)幼细胞比例与耐药性无关。多因素Logistic回归分析经以上变量校正后显示Pgp、mdr1、MRP、TopoⅡ和年龄仍与耐药性显著相关：Pgp(RR=14．87，P=0．003)；mdr1(RR=19．98，P=0．003)；MRP(RR=16．53，P=0．006)；TopoⅡ(RR=0．23，P=0．046)；年龄(RR=10．87，P=0．013)。将36例初发急性白血病患者单独分析，结果发现Pgp、mdr1和MRP亦与完全缓解密切相关。Pgp(RR=9．8，P=0．005)；mdr1(RR=12．1，P=0．005)；MRP(RR=33，P=0．002)。直线相关分析发现在所有病例组和临床耐药组中Pgp和mdr1；Pgp和MRP：mdr1和MRP具有相关性(P＜0．001)。
2．ROC曲线下面积(AUC~(ROC))：TopoⅡ明显低于Pgp、mdr1和MRP(P＜0．05)，而Pgp、mdr1和MRP的ROC曲线下面积差别无显著意义(P＞0．05)；Pgp、mdr1、MRP和TopoⅡ的最佳临界点分别为10％、0．8、1．0和0．9，在对应的灵敏度中，Pgp最高为74％，特异度中mdr1最高为86％；Pgp、mdr1和MRP的平行试验和系列试验计算，灵敏度和特异度均可提高至91％。
3．经pSilence-mdr1转染后的K562／A02细胞株mdr1 mRNA表达量下降了71．5％，从(2．8±1．65)×10~8拷贝／μgRNA下降至(3．9±2．37)×10~7拷贝／μgRNA，(P＜0．01)；同时pSilence-GSTπ作用后，K562／A02 GSTπmRNA表达量较mock下降了39．8％，从(2．3±1．14)×10~5拷贝／μgRNA下降至(5．4±2．45)×10~4拷贝／μgRNA(P＜0．01)。
4．Western blot结果显示pSilence-mdr1和pSilence-GSTπ分别转染K562／A02细胞株后与mock相比，Pgp和GSTπ的表达明显下降；而pSilence-mdr1和pSilence-GSTπ对α-tubulin的表达没有影响；K562／A02细胞株转染pSilence-laminA／C后与mock相比，laminA／C的表达明显下降，但对Pgp和GSTπ的表达没有作用；灰度扫描显示与内对照B-actin的比值，Pgp的表达量从K562／A02(mock)的0．75±0．019下降到K562／A02(pSilence-mdr1)的0．48±0．049(P＜0．001)；GSTπ的表达量从K562／A02(mock)的0．54±0．025下降到K562／A02(pSilence-GSTπ)的0．39±0．022(P＜0．01)。荧光免疫组化结果显示荧光显微镜下可见Pgp在K562／A02组有大点状的绿色荧光，阳性细胞比例从转染前的71．25±9．65％到pSilence-mdr1转染后的35．25±5．97％(P＜0．001)；GSTπ在K562／A02组有多量点状的红色荧光，阳性细胞从转染前的81．25±6．49％下降到pSilence-GSTπ转染后的41．25±4．43％(P＜0．001)。
5．MTT结果示对阿霉素的耐药指数从空载体转染后的23，到pSilence-mdr1转染后的8和pSilence-GSTπ转染后的10，差别有显著性。
研究结论：
1．Pgp、mdr1、MRP和TopoⅡ是急性白血病临床耐药的独立预后因素。mdr1和MRP具有相关性。
2．Pgp、mdr1和MRP诊断临床耐药价值较高；其联合试验可以提高灵敏度和特异度。
3．siRNA可有效、特异地逆转K562／A02 mdr1和GSTπ的多药耐药性。
A study of multidrug resistance andit's modulation in leukemia
Objectives:
1.To investigate whether Pgp,mdrl,MRP and TopoⅡare theprognostic factors of clinical resistance in acute leukemiapatients.
2.To determine the diagnostic value of Pgp,mdrl,MRP and TopoⅡof clinical resistance in acute leukemia patients.
3.To investigate the effectiveness and the specificity of hairpinsmall interference RNA(siRNA) on mdrl and GSTπexpression inmultidrug resistance human leukemia cell line K562/A02.
Methods:
1.45 cases were sequentially recruited.Flow cytometry usingmonoclonal antibody UIC2 was used to detect the expression ofPgp.RT-PCT was used to detect the expression of mdrl,MRP andTopoⅡ.Univariate and multivariate logistic regression wereused to analyse the relationship between the four factors andthe prognosis of acute leukemia.
2.According to the golden standard of clinical resistance,AreaUnder Receiver-Operating Characteristic Curve (AUC~(ROC)) was used to assess the accuracy of the four indicators and to determinethe cut-off point.
3.The Hairpin siRNA were synthesized according to the sequencetargeting mdrl and GSTπcorresponded to the coding region 79-99and 308-327,and cloned to pSilencer2.1-U6 neo,the clonedproducts were pSilence-mdrl and pSilence-GSTπ,transfected intoK562/A02.Expression of mdrl and GSTπmRNA were assayed by SYBRGreen I real-time PCR.Western blot analysis andimmunofluorescence test were used to detect the effectivenessand the specificity of the gene silence.50% inhibitionconcentration (IC_(50)) of doxorubicin(ADM) on K562/A02 wasdetermined by MTT method.
Results:
1.The expressions of Pgp,mdrl and MRP were significantly higherin resistant group than that in sensitive group (P＜0.01).Theexpression of TopoⅡwas lower in resistant group,but thedifference was not statistically significant.Logisticregression of univariate analysis showed that theover-expression of Pgp,mdrl and MRP,the lower expression ofTopoⅡand age over 55 were the prognostic factors of clinicalresistance in acute leukemia.Sex,initial white blood cellcount (WBC),bone marrow (BM) blast percentage and FAB subtypeswere not significant for the clinical resistance.Multivariateanalysis adjusted by above factors showed that theover-expression of Pgp,mdrl and MRP,the low expression of TopoⅡand age over 55 were still highly significant for clinicalresistance.Pgp:RR=14.87,P=0.003;mdrl:RR=19.98,P=0.003; MRP:RR=16.53,P=0.006;TopoⅡ:RR=0.23,P=0.046;Age:RR=10.87,P=0.013.
2.AUC~(ROC):Pgp 0.842±0.06,mdrl 0.729±0.079,MRP 0.739±0.077,TopoⅡ0.52±0.087.The cut-off point of Pgp,mdrl,MRP and TopoⅡwere 10%,0.8,1.0 and 0.9,respectively.The sensitivity andspecificity were Pgp 74% and 82%,mdrl 65% and 86%,MRP 61%and 86%,TopoⅡ65% and 41%.The parallel and serial testsof Pgp and mdrl;Pgp and MRP;Pgp,mdrl and MRP could increasethe sensitivity and the specificity up to 91%.
3.After transfected with pSilence-mdrl,the expression of mdrlmRNA in K562/A02 was reduced 71.5% compared to the mocktransfection,from (2.8±1.65)×10~8copy/μg RNAto (3.9±2.37)×10~7copy/μg RNA (P＜0.01);While the expression of GSTπmRNA inK562/A02 transfected with pSilence GSTπwas reduced 39.8%compared to the mock transfection,from (2.3±1.14)×10~5 copy/μgRNA to (5.4±2.45)×10~4 copy/μg RNA (P＜0.01).
4.Western blot results showed that mdrl and GSTπtargeted siRNAinhibited the expression of Pgp and GSTπprotein,with no effectonα-tubulin expression in comparison with mock treatment;lamin A/C siRNA decreased lamin A/C protein expression but hadno effect on the expression of Pgp and GSTπprotein.Immunofluorescence test also showed siRNA significantlyinhibited the expression of Pgp and GSTπprotein compared to themock treatment.
5.The resistance index after transfection was decreased to 8 and10 from 23 compared to the mock transfection,P＜0.01.
Conclusions:
1.The over-expression of Pgp,mdrl and MRP,and the lowerexpression of TopoⅡwere the unfavorable prognostic factorsof clinical resistance in acute leukemia patients.
2.The accuracy of Pgp,mdrl and MRP for diagnosing the clinicalresistance were high.The multiple tests could increase thesensitivity and the specificity.
3.The hairpin siRNA could effectively and specifically modulate themultidrug resistance in K562/A02 cell line.
引文
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10.Friche E,Danks MK,Schmidt CA,et al.Decreased DNA topoisomerase Ⅱ in daunorubicin-resistant ehrlich ascites cells.Cancer Res,1991,51:4213-4218.
11.Cheng SH,Lam W,Lee AS,et al.Low-level doxorubicin resistance in benzo[a]pyrene-treated KB-3-1 cells is associated with increased LRP expression and altered subcellular drug distribution.Toxicol Appl Pharmacol,2000,164:134-142.
12.Raderer M,Scheithauer W.Clinical trials of agents that reverse multidrug resistance.A literature review.Cancer,1993,72:3553-3563.
13．蔡宇，孙桂芝．中药逆转肿瘤化疗多药耐药性(MDR)体外研究概述．中国实验方剂杂志，2001，7：60-61．
14.Cucco C,Calabretta B.In vitro and vivo reversal of multidrug resistance in a human leukemia-resistant cell line by mdrl antisense oligodeoxynucleotides.Cancer Res,1996,56:4332-4337.
15.Ren Y,Wei D,Zhan X.Inhibition of P-glycoprotein and increasing of drug-sensitivity of a human carcinoma cell line(KB-A-1)by an antisense oligodeoxynucleotide-doxorubicin conjugate in vitro.Biotechnol Appl Biochem,2005,41:137-143.
16．曹江，胡汛，潘锵荣，等．MDR1反义RNA表达质粒的构建及其在多药耐药细胞抗药机理研究中的应用．中华医学杂志，1995，25：557—560．
17.Stein U,Walther W,Shoemaker RH.Modulation of mdrl expression by cytokines in human colon carcinoma cells:an approach for reversal of multidrug resistance.Br J Cancer.1996,74:1384-1391.
18.Kobayashi H,Takemura Y,Wang FS,et al.Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells:Screening for effective target sites.Int J Cancer,1999,81:944-950.
19.Fire A,Xu S,Montgomery M,et al.Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature,1998,391:806-811.
20.Wu H,Hait WN.,Yang JM.Small interfering RNA-induced suppression of MDR1(P-Glycoprotein)restores sensitivity to multidrug-resistant cancer cells.Cancer Res,2003 ,63:1515-1519.
21.Sui G,Soohoo C,Affar EB,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.PNAS,2002,99:5515-5520.
第一部分
1.Arceci RJ.Clinical significance of P-glycoprotein in multidrug resistance malignancies.Blood,1993,81:2215-2222.
2.Fletcher RH,Fletcher SW,Wanger EH.Clinical Epidemiology—the essentials.2ed,Baltimore,Williams & Wilkins.1988,42-75.
3. Lee GR, Foerster J, Luken J, et al. Wintrobe' s Clinical Hematology. 10~(th) ed. Vol 2 Baltimore: Williams & Wilkins 1999, 2209-2233.
4. Hiddemann W, Buchner T. Treatment strategies in acute myeloid leukemia (AML). Blut, 1990, 60: 163-171.
5. Zwig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem, 1993, 39: 561-577.
6. Broxterman HJ, Sonneveld P, van Putten WJ, et al. P-glycoprotein in primary acute myeloid leukemia and treatment outcome of idarubicin/cytosine arabinoside-based induction therapy. Leukemia, 2000, 14: 1018-1024.
7. Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science, 1992, 258: 1650-1654.
8. Deffie AM, McPherson JP, Gupta RS, et al. Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia, Chinese hamster ovary, and human HeLa cells, with emphasis on the role of DNA topoisomerase II. Biochem Cell Biol, 1992, 70: 354-364.
9. FricheE, Danks MK, Schmidt CA, et al. Decreased DNA topoisomerase II in daunorubicin-resistant ehrlich ascites cells. Cancer Res, 1991, 51: 4213-4218.
10. Nooter K, Burger H, Stoter G. Multidrug resistance-associated pretein (MRP) in haematological malignancies. Leukemia Lymphoma, 1995, 20: 381-387.
11.Zhou DC,Zittoun R,Marie JP.Expression of multidrug resistance-associated protein(MRP)and multidrug resistance(mdrl)genes in acute myeloid leukemia.Leukemia,1995,9:1661-1666.
12.Slapak CA,Mizunuma N,Kufe DW.Expression of the multidrug resistance associated protein and P-glycoprotein in doxorubicin-selected humanmyeloid leukemia cells.Blood,1994,84:3113-3121.
第二部分
1.Arceci RJ.Clinical significance of P-glycoprotein in multidrug resistance malignancies.Blood,1993,81:2215-2222.
2.Fire A,Xu S,Montgomery M,et al.Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature 1998,391:806-811.
3.Wu H,Hait WH,Yang JM.Small interfering RNA-induced suppression of mdrl(P-glycoprotein)restores sensitivity to multidrug resistance cance cells.Cancer Res,2003,63:1515-1519.
4.Elbashir SM,Harborth J,Weber K,et al.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods,2002,26:199-213.
5．方鹏骞．半数效量．见：刘筱娴．医学统计学．北京：科学出版社，2000，156-165．
6．高船舟，曲淑贤，吕广艳，等．P-170糖蛋白在多药耐药(MDR)K562／ADM细胞中的定位研究大连医科大学学报，2002，24：91-93．
7.McClean S,Hill BT.An overview of membrane,cytosolic and nuclear proteins associated with the expression of resistance to multiple drugs in vitro.Biochem Biophys Acta,1992,1114:107-127.
8.Sui G,Soohoo C,Affar EB,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.Proc Natl Acad Sci USA,2002,99:5515-5520.
9.Siraj AK,Ozbek U,Sazawal S,et al.Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia.Clin Cancer Res.2002,8:3832-3840.
10．彭智，肖志坚，王一，等．siRNA逆转K562／A02细胞多药耐药的研究．中华血液学杂志，2004，25：5-7．
11.Lewis DL,Hagstrom JE,Loomis AG,et al.Efficient delivery of siRNA for inhibition by small interfering RNA.Blood,2003,101:1566-1569.
12.McCaffrey AP,Meuse L,Pham TTT,et al.RNA interference in adult mice.Nature,2002,418:38-39.
1.Arceci RJ.Clinical significance of P-glycoprotein in multidrug resistance malignancies.Blood,1993,81:2215-2222.
2.Roninson IB,Chin JE,Choi KG.et al.Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells.Proc Natl Acad Sci U S A,1986,83:4538-4542.
3.Thiebaut F,Tsuruo T,Hamada H,et al.Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA, 1987, 84: 7735-7738.
4. Mannervik B, Awasthi YC, Board PG, et al. Nomenclature for human glutathione transferases. Biochem J, 1992, 282: 305-306.
5. Puchalski RB, Fahl WE. Expression of recombinant glutathon S-transferase π Ya or Y_(b1) confers resistane to alkylating agents. Proc Natl Acad Sci USA, 1990, 87: 2443-2447.
6. Manoharan TH, Welch PJ, Guilich AM, et al. Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance. Mol pharmacol, 1991, 39: 461-467.
7. Leyland-Jones BR, Townsend AJ, Tu CPD, et al. Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human class glutathione S-transferase gene. Cancer Res, 1991, 51: 587-594.
8. Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a novel transporter gene in a multidrug resistant human lung cancer cell line. Science, 1992, 258: 1650-1654.
9. Keppler D, Konig J, Buchler M. The canalicular multidrug resistance protein, cMRP/MRP2, a novel conjugate export pump expressed in the apical membrane of hepatocytes. Adv Enzyme Regul,1997, 37: 321-333.
10. Kool M, de Haas M, Scheffer GL, et al. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res, 1997, 57: 3537-3547.
11.Hopper E,Belinsky MG,Zeng H,et al.Analysis of the structure and expression pattern of MRP7(ABCC10)),a new member of the MRP sub-family.Cancer Lett,2001,162:181-191.
12.Deffie AM,McPherson JP,Gupta RS,et al.Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia,Chinese hamster ovary,and human HeLa cells,with emphasis on the role of DNA topoisomerase Ⅱ.Biochem Cell Biol,1992,70:354-364.
13.Friche E,Danks MK,Schmidt CA,et al.Decreased DNA topoisomerase Ⅱ in daunorubicin-resistant ehrlich ascites cells.Cancer Res,1991,51:4213-4218.
14.O'Brian CA,Ward NE,Stewart JR,et al.Prospects for targeting protein kinase C isozymes in the therapy of drug-resistant cancer--an evolving story.Cancer Metastasis Rev,2001,20:95-100.
15.Raderer M,Scheithauer W.Clinical trials of agents that reverse multidrug resistance.Cancer,1993,72:3553-2563.
16.Pea F,Damiani D,Michieli M ,et al.Multidrug resistance modulation in vivo:the effect of cyclosporin A alone or with dexverapamil on idarubicin pharmacokinetics in acute leukemia.Eur J Clin Pharmacol.1999,55:361-368.
17．蔡宇，孙桂芝．中药逆转肿瘤化疗多药耐药性(MDR)体外研究概述．中国实验方剂杂志，2001，7：60-61．
18.Barker RH Jr,Metelev V,Coakley A,et al.Plasmodium falciparum:effect of chemical structure on efficacy and specificity of antisense oligonucleotides against malaria in vitro.Exp Parasitol,1998,88:51-59.
19.RenY,Wei D,ZhanX.Inhibition of P-glycoproteinandincreasing of drug-sensitivity of a human carcinoma cell line(KB-A-1)by an antisense oligodeoxynucleotide-doxorubicin conjugate in vitro.Biotechnol Appl Biochem,2005,41:137-143.
20．李惠芳，孙桂香，卢薇薇，等．多药耐药基因反义寡核苷酸逆转肿瘤细胞耐药的初步研究．中华血液学杂志，1997，18：76—78．
21.Cucco C,Calabretta B.In vitro and vivo reversal of multidrug resistance in a human leukemia-resistant cell line by MDR1 antisense oligodeoxynucleotides.Cancer Res,1996,56:4332-4337.
22.Tomizawa J,Itoh T.Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript.Proc Natl Acad Sci U S A,1981,78:6096-6100.
23.Xiao B,Shao YQ,You H,etal.Transductin of Fas gene or Bcl-2 antisense RNA sensitizes cultured drug resistant gastric cancer cells to chemotherapeutic drugs.World J Gastroenterol,1998,4:421-425.
24.Zhang YM,Zhao YQ,Pan YL,et al.Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells.World J Gastronenterol,2003,9:894-498.
25.Arienti F,Gambacorti-Passerini C,Borin L,et al.Increased susceptibility to lymphokine activated killer(LAK)lysis of relapsing vs.newly diagnosed acute leukemic cells without changes in drug resistance or in the expression of adhesion molecules.Ann Oncol,1992,3:155-162.
26. Weitzmann MN, Savage N. Cyclic adenosine 3', 5' -monophosphate, a second messenger in interleukin-1 mediated K562 cytostasis. Biochem Biophys Res Commun, 1993, 190: 564-570.
27. Lebrun JJ, Vale WW. Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type Ⅰ and type Ⅱ activin receptors and human erythroid differentiation. Mol Cell Biol, 1997, 17: 1682-1691.
28. el-Sonbaty SS, Watanabe M, Hochito K, et al. Exogenously expressed granulocyte colony-stimulating factor (G-CSF) receptor on K562 cells can transduce G-CSF-triggered growth and differentiation signals. Int J Hematol, 1995, 61: 61-68.
29. KiehntopfM, BrachMA, Licht T, etal. Ribozyme-mediated cleavage of the MDR-1 transcript restores chemosensitivity in previously resistant cancer cells. EMBO J, 1994, 13: 4645-4652.
30. Kobayashi H, Takemura Y, Wang FS, et al. Retrovirus-mediated transfer of anti-MDRl hammerhead ribozymes into multidrug-resistant human leukemia cells: Screening for effective target sites. Int J Cancer, 1999, 81: 944-950.
31. Fire A, Xu S, Montgomery M, et al. Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans. Nature 1998, 391: 806-811.
32. Masiero M, Nardo G, Indraccolo S, Favaro E. RNA interference: implications for cancer treatment. Mol Aspects Med 2007; 28(1) :143 - 166.
33. Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference. Oncogene, 2002, 21:5716-5724.
34.Wu H,Hait WN,Yang JM.Small interfering RNA-induced suppression of MDR1(P-Glycoprotein)restores sensitivity to multidrug resistant cancer cells.Cancer Res,2003,63:1515-1519.
35．彭智，肖志坚，王一，等．siRNA逆转K562／A02细胞多药耐药性的研究．中华血液学杂志，2004，25(1)：5-7．
36.Lage H.MDR1/P-glycoprotein(ABCB1)as target for RNA interference-mediated reversal of multidrug resistance.Curr Drug Targets 2006;7(7):813-821
37.Shao SL,Sun YY,Li XY et al.The reversion effectof the RNAi-silencing mdrl gene on multidrug resistance of the leukemia cell HT9.Cell Bio Int,2008,32:893-898.
38.Brummelia TR,Bernards R,Agami R,et al.A system for stable expression of short interfering RNAs in mammalian cells.Science 2002,296:550-556.
39.Scherr M,Morgan MA,Eder M.Gene silencing mediated by small interfering RNAs in mammalian cells.Curr Med Chem,2003,10:245-256.
40.Sui G,Soohoo C,Affarel B,etal.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.Proc Natl Acad Sci,2002,99:5515-5520.
41.Lewis DL,Hagstrom JE,Loomis AG,et al.Efficient delivery of siRNA for inhibition of gene expression in postnatal mice.Nature Genetics,2002,32:107-108.
42.McCaffrey AP,Meuse L,Pham TTT,et al.RNA interference in adult mice. Nature, 2002, 418: 38-39.
43. Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens. Drug Discov Today 2006; 11 (21-22):975-982.
2. Arceci RJ. Clinical significance of P-glycoprotein in multidrug resistance malignancies. Blood, 1993, 81: 2215-2222.
3. Musto P, Melillo L, Lombardi G. et al. High risk of early resistant relapse for leukemic patients with presence of multidrug resistance associated P-glycoprotein positive cells in complete remission. Br J Haematol, 1981, 77: 50-53.
4. Zhou DC, Marie JP, Suberville AM, et al. Relevance of mdrl gene expression in acute myeloid leukemia and comparison of different diagnostic methods. Leukemia, 1992, 6: 879-885.
5. Herwei jer H, Sonneveld P, Boas F, et al. Expression of mdrl and mdr3 multidrug-resistance genes in human acute and chronic leukemias in association with stimulation of drug accumulation by cyclosporine. J Natl Cancer Inst, 1990, 82: 1133-1140.
6. McClean S, Hill BT. An overview of membrane, cytosolic and nuclear proteins associated with the expression of resistance to multiple drugs in vitro. Biochem Biophys Acta, 1992, 1114: 107-127.
7. Hida T, Kuwabara M, Ariyoshi Y, et al. Serum glutathione s-transfterase-π level as a tumor marker for non-small cell lung cncer. Cancer, 1994, 73: 1377-1382.
8. Cole SPC, Bhardwaj G, GerlachJH, et al. Overexpression of a novel transporter gene in a multidrug resistant human lung cancer cell line. Science, 1992, 258: 1650-1654.
9.Deffie AM,McPherson JP,Gupta RS,et al.Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia,Chinese hamster ovary,and human HeLa cells,with emphasis on the role of DNA topoisomerase Ⅱ.Biochem Cell Biol,1992,70:354-364.
10.Friche E,Danks MK,Schmidt CA,et al.Decreased DNA topoisomerase Ⅱ in daunorubicin-resistant ehrlich ascites cells.Cancer Res,1991,51:4213-4218.
11.Cheng SH,Lam W,Lee AS,et al.Low-level doxorubicin resistance in benzo[a]pyrene-treated KB-3-1 cells is associated with increased LRP expression and altered subcellular drug distribution.Toxicol Appl Pharmacol,2000,164:134-142.
12.Raderer M,Scheithauer W.Clinical trials of agents that reverse multidrug resistance.A literature review.Cancer,1993,72:3553-3563.
13．蔡宇，孙桂芝．中药逆转肿瘤化疗多药耐药性(MDR)体外研究概述．中国实验方剂杂志，2001，7：60-61．
14.Cucco C,Calabretta B.In vitro and vivo reversal of multidrug resistance in a human leukemia-resistant cell line by mdrl antisense oligodeoxynucleotides.Cancer Res,1996,56:4332-4337.
15.Ren Y,Wei D,Zhan X.Inhibition of P-glycoprotein and increasing of drug-sensitivity of a human carcinoma cell line(KB-A-1)by an antisense oligodeoxynucleotide-doxorubicin conjugate in vitro.Biotechnol Appl Biochem,2005,41:137-143.
16．曹江，胡汛，潘锵荣，等．MDR1反义RNA表达质粒的构建及其在多药耐药细胞抗药机理研究中的应用．中华医学杂志，1995，25：557—560．
17.Stein U,Walther W,Shoemaker RH.Modulation of mdrl expression by cytokines in human colon carcinoma cells:an approach for reversal of multidrug resistance.Br J Cancer.1996,74:1384-1391.
18.Kobayashi H,Takemura Y,Wang FS,et al.Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells:Screening for effective target sites.Int J Cancer,1999,81:944-950.
19.Fire A,Xu S,Montgomery M,et al.Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature,1998,391:806-811.
20.Wu H,Hait WN.,Yang JM.Small interfering RNA-induced suppression of MDR1(P-Glycoprotein)restores sensitivity to multidrug-resistant cancer cells.Cancer Res,2003 ,63:1515-1519.
21.Sui G,Soohoo C,Affar EB,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.PNAS,2002,99:5515-5520.
第一部分
1.Arceci RJ.Clinical significance of P-glycoprotein in multidrug resistance malignancies.Blood,1993,81:2215-2222.
2.Fletcher RH,Fletcher SW,Wanger EH.Clinical Epidemiology—the essentials.2ed,Baltimore,Williams & Wilkins.1988,42-75.
3. Lee GR, Foerster J, Luken J, et al. Wintrobe' s Clinical Hematology. 10~(th) ed. Vol 2 Baltimore: Williams & Wilkins 1999, 2209-2233.
4. Hiddemann W, Buchner T. Treatment strategies in acute myeloid leukemia (AML). Blut, 1990, 60: 163-171.
5. Zwig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem, 1993, 39: 561-577.
6. Broxterman HJ, Sonneveld P, van Putten WJ, et al. P-glycoprotein in primary acute myeloid leukemia and treatment outcome of idarubicin/cytosine arabinoside-based induction therapy. Leukemia, 2000, 14: 1018-1024.
7. Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science, 1992, 258: 1650-1654.
8. Deffie AM, McPherson JP, Gupta RS, et al. Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia, Chinese hamster ovary, and human HeLa cells, with emphasis on the role of DNA topoisomerase II. Biochem Cell Biol, 1992, 70: 354-364.
9. FricheE, Danks MK, Schmidt CA, et al. Decreased DNA topoisomerase II in daunorubicin-resistant ehrlich ascites cells. Cancer Res, 1991, 51: 4213-4218.
10. Nooter K, Burger H, Stoter G. Multidrug resistance-associated pretein (MRP) in haematological malignancies. Leukemia Lymphoma, 1995, 20: 381-387.
11.Zhou DC,Zittoun R,Marie JP.Expression of multidrug resistance-associated protein(MRP)and multidrug resistance(mdrl)genes in acute myeloid leukemia.Leukemia,1995,9:1661-1666.
12.Slapak CA,Mizunuma N,Kufe DW.Expression of the multidrug resistance associated protein and P-glycoprotein in doxorubicin-selected humanmyeloid leukemia cells.Blood,1994,84:3113-3121.
第二部分
1.Arceci RJ.Clinical significance of P-glycoprotein in multidrug resistance malignancies.Blood,1993,81:2215-2222.
2.Fire A,Xu S,Montgomery M,et al.Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature 1998,391:806-811.
3.Wu H,Hait WH,Yang JM.Small interfering RNA-induced suppression of mdrl(P-glycoprotein)restores sensitivity to multidrug resistance cance cells.Cancer Res,2003,63:1515-1519.
4.Elbashir SM,Harborth J,Weber K,et al.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods,2002,26:199-213.
5．方鹏骞．半数效量．见：刘筱娴．医学统计学．北京：科学出版社，2000，156-165．
6．高船舟，曲淑贤，吕广艳，等．P-170糖蛋白在多药耐药(MDR)K562／ADM细胞中的定位研究大连医科大学学报，2002，24：91-93．
7.McClean S,Hill BT.An overview of membrane,cytosolic and nuclear proteins associated with the expression of resistance to multiple drugs in vitro.Biochem Biophys Acta,1992,1114:107-127.
8.Sui G,Soohoo C,Affar EB,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.Proc Natl Acad Sci USA,2002,99:5515-5520.
9.Siraj AK,Ozbek U,Sazawal S,et al.Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia.Clin Cancer Res.2002,8:3832-3840.
10．彭智，肖志坚，王一，等．siRNA逆转K562／A02细胞多药耐药的研究．中华血液学杂志，2004，25：5-7．
11.Lewis DL,Hagstrom JE,Loomis AG,et al.Efficient delivery of siRNA for inhibition by small interfering RNA.Blood,2003,101:1566-1569.
12.McCaffrey AP,Meuse L,Pham TTT,et al.RNA interference in adult mice.Nature,2002,418:38-39.
1.Arceci RJ.Clinical significance of P-glycoprotein in multidrug resistance malignancies.Blood,1993,81:2215-2222.
2.Roninson IB,Chin JE,Choi KG.et al.Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells.Proc Natl Acad Sci U S A,1986,83:4538-4542.
3.Thiebaut F,Tsuruo T,Hamada H,et al.Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA, 1987, 84: 7735-7738.
4. Mannervik B, Awasthi YC, Board PG, et al. Nomenclature for human glutathione transferases. Biochem J, 1992, 282: 305-306.
5. Puchalski RB, Fahl WE. Expression of recombinant glutathon S-transferase π Ya or Y_(b1) confers resistane to alkylating agents. Proc Natl Acad Sci USA, 1990, 87: 2443-2447.
6. Manoharan TH, Welch PJ, Guilich AM, et al. Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance. Mol pharmacol, 1991, 39: 461-467.
7. Leyland-Jones BR, Townsend AJ, Tu CPD, et al. Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human class glutathione S-transferase gene. Cancer Res, 1991, 51: 587-594.
8. Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a novel transporter gene in a multidrug resistant human lung cancer cell line. Science, 1992, 258: 1650-1654.
9. Keppler D, Konig J, Buchler M. The canalicular multidrug resistance protein, cMRP/MRP2, a novel conjugate export pump expressed in the apical membrane of hepatocytes. Adv Enzyme Regul,1997, 37: 321-333.
10. Kool M, de Haas M, Scheffer GL, et al. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res, 1997, 57: 3537-3547.
11.Hopper E,Belinsky MG,Zeng H,et al.Analysis of the structure and expression pattern of MRP7(ABCC10)),a new member of the MRP sub-family.Cancer Lett,2001,162:181-191.
12.Deffie AM,McPherson JP,Gupta RS,et al.Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia,Chinese hamster ovary,and human HeLa cells,with emphasis on the role of DNA topoisomerase Ⅱ.Biochem Cell Biol,1992,70:354-364.
13.Friche E,Danks MK,Schmidt CA,et al.Decreased DNA topoisomerase Ⅱ in daunorubicin-resistant ehrlich ascites cells.Cancer Res,1991,51:4213-4218.
14.O'Brian CA,Ward NE,Stewart JR,et al.Prospects for targeting protein kinase C isozymes in the therapy of drug-resistant cancer--an evolving story.Cancer Metastasis Rev,2001,20:95-100.
15.Raderer M,Scheithauer W.Clinical trials of agents that reverse multidrug resistance.Cancer,1993,72:3553-2563.
16.Pea F,Damiani D,Michieli M ,et al.Multidrug resistance modulation in vivo:the effect of cyclosporin A alone or with dexverapamil on idarubicin pharmacokinetics in acute leukemia.Eur J Clin Pharmacol.1999,55:361-368.
17．蔡宇，孙桂芝．中药逆转肿瘤化疗多药耐药性(MDR)体外研究概述．中国实验方剂杂志，2001，7：60-61．
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