| A study of multidrug resistance andit's modulation in leukemiaObjectives: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 (AUCROC) 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 (IC50) 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.AUCROC: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)×108copy/μg RNAto (3.9±2.37)×107copy/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)×105 copy/μgRNA to (5.4±2.45)×104 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.
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