PTEN基因抗白血病作用及其机制的研究
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摘要
背景:与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensin hemology deleted on chromosome ten gene,PTEN)是磷酸酶家族中发现的首个抑癌基因,PTEN蛋白具有蛋白磷酸酶和脂质磷酸酶双重活性,其脂质磷酸酶活性可以将3,4,5三磷酸磷脂酰肌醇(PIP3)3位上的磷酸基团去掉,使PIP3转化为4,5二磷酸磷脂酰肌醇(PIP2),此功能在于调控细胞的增殖与凋亡。其蛋白磷酸酶活性可以抑制丝裂原活化蛋白激酶(MAPK)、黏着斑激酶(FAK)的磷酸化,调节细胞的迁移及黏附。PTEN通过上述多种信号传导通路影响细胞生长、分化、黏附及细胞周期进程,抑制肿瘤细胞增殖、侵袭、转移,并促进肿瘤细胞凋亡。研究发现在多种实体瘤中存在PTEN基因的突变、等位基因的缺失或低表达,从而影响其肿瘤抑制功能,并与部分肿瘤的预后不良密切相关。近年来PTEN在造血系统肿瘤,如白血病发病中的作用逐渐受到重视,研究表明PTEN基因在白血病中存在不同程度的缺失、低表达、甲基化,而突变罕见。造血细胞PTEN基因突变或缺失的小鼠最初可以出现类似于人类骨髓增殖性疾病(MPD)表现,随后转化为急性髓系或淋巴细胞白血病,把PTEN基因突变或缺失的小鼠造血祖细胞移植到SCID鼠后,受体鼠亦发生白血病,并伴有白血病细胞髓外浸润。同时PTEN基因的表达水平对区分白血病干细胞和正常造血干细胞发挥了关键作用。国内外研究表明PTEN基因的异常可能参与了白血病的发病、多药耐药、血管新生及髓外浸润。
PTEN基因抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的作用在多种实体肿瘤细胞中已经得到证实,而在造血系统肿瘤如白血病中的作用及其机制尚不完全清楚。
K562细胞系为人慢性粒细胞白血病急性红白血病变细胞系,具有较高的PI3K/Akt活性,同时低表达PTEN基因及蛋白,为我们将外源性野生型PTEN基因进行转染提供了条件。因此我们将腺病毒介导的PTEN基因转染人白血病原代细胞及K562细胞系,观察野生型PTEN对白血病细胞增殖、凋亡、侵袭、细胞周期进程、多药耐药及血管新生的影响,并检测其可能的作用机制,为基因治疗白血病提供理论依据。研究分以下三部分:
第一部分腺病毒介导的野生型PTEN基因对人慢性粒细胞白血病细胞系K562及原代白血病细胞增殖、凋亡、细胞周期的影响
目的探讨肿瘤抑制基因PTEN对人原代白血病细胞及K562细胞系增殖、凋亡及细胞周期的影响,并对其可能的作用机制进行初步探讨。
方法将携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad- PTEN-GFP)或空载体腺病毒(Ad-GFP)转染人原代白血病细胞及K562细胞系。MTT检测细胞生长曲线;流式细胞仪检测腺病毒转染细胞的转染效率、细胞凋亡率和细胞周期分布;光镜下检测细胞形态变化,DNA ladder,Hoechst33342荧光染色检测、细胞集落形成等实验检测细胞增殖及凋亡。荧光定量PCR(FQ-PCR)检测PTEN、Bcl-2、Bcl-xL、Bax、Survivin、Xiap、Smac、Caspase-3、-7、-9、CyclinD1、CyclinD2、CDK4、P27kip1 mRNA水平变化,Western Blot检测PTEN、Bcl-2、Akt、p-Akt水平变化。Caspase活性检测试剂盒检测Caspase-3/7、Caspase-9蛋白活性。
结果
1当MOI为200时,转染第三天用流式细胞仪检测腺病毒转染K562细胞效率最高为81.2±5.4%。通过计数绿色荧光细胞数量检测转染原代细胞效率较低,选取了5例原代白血病细胞,平均转染效率为7.32±6.41%。
2当MOI为200时在转染第3天PTEN mRNA及蛋白表达水平达到最高。转染后d3,Ad-PTEN-GFP组PTEN表达mRNA水平(23.58±4.52)及蛋白表达水平(0.912±0.102)高于Ad-GFP组的mRNA(0.650±0.516)及蛋白(0.117±0.028)表达水平和未转染对照组的mRNA(0.575±0.226)及蛋白(0.086±0.021)表达水平,均P<0.01。
3 Ad-PTEN-GFP以感染复数为200转染细胞后第5天与空载体腺病毒(Ad-GFP)相比,Ad-PTEN-GFP转染人原代白血病细胞后,细胞增殖受抑,对转染的5例原代细胞增殖抑制率为33.8±10.4%。Ad-PTEN-GFP以感染复数为200转染K562细胞后第5天,细胞最大生长抑制率为38.67%,明显高于Ad-GFP组的抑制率5.34%。Ad-PTEN-GFP组细胞凋亡率为22.4±2.11%,明显高于Ad-GFP组的2.8±0.67%和未转染组的0.2±0.02%,均P<0.05。
4腺病毒转染K562细胞5天后,未转染组及Ad-GFP转染组的细胞呈圆形或椭圆形,体积较大,染色质疏松;转染Ad-PTEN-GFP组部分细胞体积缩小,染色质高度浓缩边集,核碎裂核溶解。Hoechst33342荧光染色显示正常细胞核Hoechst着色的形态呈圆形,淡蓝色;而凋亡细胞的核由于浓集而呈亮蓝色。转染Ad-PTEN-GFP组细胞凋亡细胞比例(35.6±2.2%)明显高于未转染组(3.1%±1.13%)和转染Ad-GFP组(6.2%±0.87%),均P<0.01。
5以MOI=200腺病毒转染K562细胞5天后,Ad-PTEN-GFP组DNA琼脂糖凝胶电泳出现大小为180至200bp及其倍数的梯状条带,片断之间呈一定间隔,这是DNA在核小体间成倍断裂的结果,Ad-GFP组及未转染组细胞未出现DNA降解现象。
6细胞周期显示以MOI=200 Ad-PTEN-GFP转染K562细胞7天,G0/G1期细胞比例由54.9±3.61%增加至78.5±5.17%(P<0.05),G2/M期细胞比例由30.2±2.42%降至13.6±2.01%,(P<0.05),处于G0/G1期细胞比例逐渐增加,处于G2/M期细胞比例逐渐减少,细胞周期阻滞在G0/G1。
7当MOI为200时,K562细胞转染Ad-PTEN-GFP第三天Western blot检测Akt及p-Akt结果显示,总Akt表达水平无明显变化,而p-Akt表达水平在未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别为(0.587±0.078;0.547±0.061;0.021±0.011),转染PTEN 3d后p-Akt表达水平明显减低(P<0.01)。
8以MOI=200转染K562细胞系3天后,Ad-PTEN-GFP组细胞内Caspase-9活性(0.786±0.032)、Caspase-3/7活性(1.362±0.039),均高于Ad-GFP组的Caspase-9(0.591±0.024)和Caspase-3/7(1.103±0.024)活性,(P<0.05)。
9以MOI=200转染K562细胞系3天后,Ad-PTEN-GFP组细胞内Survivin(0.0700±0.0059)、Xiap(0.00889±0.0006)、Smac (0.0600±0.0039) mRNA表达水平均明显低于Ad-GPF组细胞内Survivin(0.437±0.079)、Xiap(0.0661±0.0072)、Smac(0.158±0.0078)和未转染组K562细胞内Survivin(0.453±0.081)、Xiap(0.070±0.0079)、Smac(0.177±0.0085) mRNA表达水平,转染Ad-PTEN-GFP后与转染Ad-GFP相比Survivin mRNA表达水平降低6.14倍、Xiap降低7.44倍,而Smac mRNA降低2.95倍。
10以MOI=200转染K562细胞系3天后,未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组凋亡相关基因的表达水平分别为,Bcl-2 mRNA:0.732±0.078、0.723±0.148、0.195±0.043;Bcl-xL mRNA:0.0181±0.0009、0.0176±0.0011、0.0047±0.0008;Bax mRNA :0.376±0.031、0.395±0.036、0.912±0.117。结果显示未转染组、Ad-GFP组与转染Ad-PTEN-GFP组各基因的表达水平有明显差异,均P<0.01。转染野生型PTEN后Bcl-2与Bcl-xL抗凋亡基因表达减低,而促凋亡基因Bax mRNA表达升高,Bcl-2蛋白表达水平下调,分别为(1.016±0.147;0.996±0.166;0.582±0.067)。Bcl-2/Bax mRNA表达水平由Ad-GFP组1.83降至Ad-PTEN-GFP组0.214,降低8.55倍。
11以MOI=200转染K562细胞系3天后,细胞周期相关基因的检测结果显示,未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别如下:CyclinD1 mRNA (0.711±0.174;0.698±0.162;0.243±0.102)、CyclinD2 mRNA (0.123±0.0196 ; 0.106±0.0178 ; 0.0441±0.0144)、CDK4 mRNA (0.859±0.165;0.876±0.142;0.272±0.092)、P27kip1 mRNA (0.0721±0.0102;0.0683±0.00883;0.470±0.172)。与未转染组和转染Ad-GFP组比较,转染Ad-PTEN-GFP后CyclinD1、CyclinD2、CDK4 mRNA表达水平明显下调(均P<0.05),而P27kip1 mRNA表达水平升高(各组P<0.05)
结论高表达PTEN基因能够明显抑制原代白血病细胞和K562细胞增殖并诱导凋亡,可能与PTEN抑制PI3K/Akt信号通路,并调控多种凋亡相关分子家族基因如Bcl-2家族、Caspase家族和IAP家族密切相关,并通过调控细胞周期相关分子表达使细胞周期阻滞在G0/G1期。
第二部分腺病毒介导的野生型PTEN基因对人慢性粒细胞白血病阿霉素耐药细胞系K562/ADM多药耐药逆转的研究
目的探讨野生型抑癌基因PTEN对人慢性粒细胞白血病阿霉素耐药细胞系K562/ADM增殖、凋亡的影响,及其与多种化疗药物联合应用的协同作用进行研究,并对其逆转多药耐药的分子作用机制进行探讨。
方法用携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad- PTEN-GFP)及空载体腺病毒(Ad-GFP)转染对阿霉素耐药的慢性粒细胞性白血病细胞株K562/ADM细胞系,和或联合应用阿霉素(ADM)、阿糖胞苷(Ara-c)、三氧化二砷(As2O3)等不同类型化疗药物,观察PTEN基因与化疗药物的协同作用。MTT检测细胞生长曲线,计算不同药物的IC50及联合作用的IC50,计算耐药逆转倍数;流式细胞仪检测腺病毒转染K562细胞的转染效率、细胞凋亡率;荧光定量PCR(FQ-PCR)检测PTEN、NF-κB、I-κB、P53、MDR1、MRP1及凋亡相关基因Bcl-2、Bcl-xL及Bax水平变化,Western Blot检测PTEN、Akt、p-Akt、P65蛋白水平变化。
结果
1当MOI为200时,转染第3天流式细胞仪检测腺病毒转染K562/ADM细胞效率最高,为82.2±5.8%。
2当MOI为200时在转染第3天,K562/ADM细胞中的PTEN mRNA及蛋白表达水平达到最高。转染后d3,Ad-PTEN-GFP组PTEN mRNA表达水平(25.43±3.77)及蛋白表达水平(1.240±0.219)高于Ad-GFP组PTEN mRNA(0.473±0.117)及PTEN蛋白(0.105±0.014)表达水平和未转染对照组PTEN mRNA(0.477±0.071)及PTEN蛋白(0.073±0.011)表达水平(P<0.01)。而K562及K562/ADM细胞中的PTEN mRNA及蛋白表达水平无显著差异(P>0.05)。
3以MOI为200转染K562/ADM细胞,第5天Ad-PTEN-GFP组增殖抑制率为32.26%,明显高于Ad-GFP组(3.31%),有明显统计学差异(P<0.01),与亲代K562细胞比较,PTEN基因对K562/ADM细胞的抑制率稍低,但两者差异无统计学意义(P>0.05)。
4流式细胞凋亡检测显示以MOI为200转染K562/ADM细胞后,Ad-PTEN-GFP联合10mg/L阿霉素(ADM)或5μmol阿糖胞苷(Ara-c)或1μmol/L三氧化二砷(As2O3)干预组细胞凋亡率分别为:17.8±2.3、28.6±2.9、61.7±3.2,明显高于转染Ad-GFP后联合各化疗药物组的凋亡率:2.3±0.7、15.2±1.8、20.4±2.1,三者均有显著性差异(P<0.01)。
5以MOI为200转染K562/ADM细胞后联合或单独应用ADM、Ara-c、As2O3等化疗药物72h后PTEN基因对不同化疗药物的耐药逆转倍数分别为ADM 3.8倍、Ara-c 2.64倍、As2O3 2.65倍。
6当MOI为200时,转染Ad-PTEN-GFP第三天Western blot检测Akt及p-Akt结果显示,总Akt表达水平无明显变化,而p-Akt表达水平在未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别为:0.633±0.065、0.646±0.066、0.022±0.007;转染PTEN 3d后K562/ADM细胞p-Akt表达水平明显减低(P<0.01)。
7以MOI=200转染K562/ADM细胞后3天,实时荧光定量PCR法(FQ-PCR)结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组,NF-κB mRNA与β-actin mRNA相对表达水平分别为16.08±2.85;15.20±2.08;1.17±0.06(P<0.05),转染PTEN基因后P65蛋白表达水平亦明显减低。I-κB相对表达水平分别为0.21±0.08;0.18±0.07;0.23±0.11,无明显差异(P>0.05)。P53 mRNA与β-actin mRNA的相对表达水平分别为2.95±0.32;3.07±0.75;5.93±1.51,(P<0.05)。转染野生型PTEN基因后NF-κB mRNA及p65蛋白表达水平明显减低,p65蛋白活性呈时间依赖性减低。I-κB mRNA表达水平无明显改变,P53 mRNA表达水平轻度升高。
8以MOI=200转染K562/ADM细胞3天后,FQ- PCR结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组中MDR1 mRNA与β-actin mRNA相对表达水平分别为0.25±0.02;0.24±0.03;0.13±0.01(P<0.05)。MRP mRNA表达水平分别为0.44±0.07;0.42±0.07;0.47±0.09(P>0.05)。表明转染PTEN基因后MDR1 mRNA表达水平减低,而MPR mRNA表达水平无明显改变。
9以MOI=200转染K562/ADM细胞3天后,FQ- PCR结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组中Bcl-2 mRNA表达水平分别为0.040±0.013;0.036±0.010;0.011±0.004(P<0.05)。Bcl-xL mRNA表达水平分别为:0.0071±0.0003;0.0076±0.0003;0.0037±0.0001(P<0.05)。Bax mRNA表达水平分别为:0.129±0.028;0.141±0.018;0.432±0.071(P<0.05)。结果表明转染野生型PTEN基因后Bcl-2 mRNA、Bcl-xL
mRNA表达水平明显减低,而Bax mRNA表达水平增加。结论野生型PTEN基因可能通过调控PI3K/Akt信号传导通路、抑制K562/ADM细胞增殖及诱导凋亡,并通过下调NF-κB、MDR1、Bcl-2及上调P53、Bax等多种信号分子,逆转K562/ADM细胞的多药耐药。
第三部分PTEN基因对白血病细胞VEGF调控的研究及其临床意义
目的探讨肿瘤抑制基因PTEN及血管内皮生长因子(VEGF)及其受体VEGFR1(FLT1)在白血病中的表达和临床意义;研究野生型PTEN基因的抗血管新生作用。方法(1)应用实时荧光定量PCR(FQ-PCR)检测50例初治急性髓系白血病(AML),10例缓解期AML,10例慢性粒细胞白血病慢性期(CML-CP)、10例CML急变期(CML-BC)患者和10例健康人骨髓单个核细胞(MMNC)中PTEN、VEGF、VEGFR1 mRNA的表达。(2)将携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP)转染K562细胞系,观察不同感染复数(MOI)条件下PTEN基因对VEGF及FLT1表达的影响。(3)通过MTT及流式细胞术检测PTEN基因对脐静脉内皮细胞系ECV304增殖、凋亡的影响。(4)鸡胚绒毛尿囊膜(CAM)体内血管生长实验方法检测PTEN基因对血管生成的影响。
结果
1 PTEN、VEGF、FLT1 mRNA在髓系白血病中的表达水平及相互关系。
1.1 PTEN mRNA在急性髓系白血病(AML)初治患者中表达水平(0.051±0.059)明显低于AML缓解组(1.015±0.411)和正常对照组NC(1.059±0.595),(P<0.01);CML-BC患者中PTEN mRNA表达水平(0.022±0.021)低于CML-CP患者(1.134±1.124)及NC(P<0.01)。
1.2 VEGF mRNA在AML初治患者中表达水平(0.442±0.192)明显高于AML缓解组(0.075±0.042)和NC(0.059±0.039),(P<0.01);CML-BC患者(0.512±0.322)VEGF mRNA表达水平高于CML-CP(0.219±0.131)及NC(P<0.01)。
1.3 FLT1 mRNA在AML初治患者中表达水平(1.569±0.694)明显高于AML缓解组(0.375±0.241)和NC(0.402±0.238),(P<0.01);CML-BC患者中FLT1 mRNA表达水平(1.579±0.623)高于CML-CP(0.598±0.311)及NC(P<0.01)。
1.4 Pearson直线相关法分析显示,AML患者中PTEN mRNA与VEGF mRNA呈负相关(r=-0.631,P<0.05),与FLT1 mRNA表达同样呈负相关(r=-0.522,P<0.05)。
2 PTEN基因对K562细胞系的侵袭性及p-Akt、VEGF及FLT1调控的研究
2.1当MOI为200时,转染第三天流式细胞仪检测腺病毒转染K562细胞效率最高为81.2±5.4%,符合基因治疗对载体的要求。
2.2当MOI为50、100、200、400转染K562细胞系后,转染72h内随着PTEN mRNA表达水平升高,VEGF、FLT1 mRNA表达水平逐渐降低,呈负相关趋势,相关系数分别为:r=-0.903,P<0.05;r=-0.900,P<0.05;VEGF蛋白表达水平同样降低。当MOI=200时PTEN mRNA表达水平增加到41.01倍,VEGF、FLT1 mRNA表达水平下调分别为4.80倍和4.68倍,ELISA方法显示VEGF蛋白表达水平下调2.21倍。
2.3当MOI为200时,转染Ad-PTEN-GFP第三天Western blot检测总Akt及p-Akt结果显示,总Akt表达水平无明显变化,而p-Akt表达水平在未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别为0.587±0.078、0.547±0.061、0.021±0.011,转染PTEN 3d后p-Akt表达水平明显减低(P<0.01)。
2.4 Transwell侵袭实验显示在MOI=200时,Ad-PTEN-GFP转染K562细胞48h后,400倍显微镜下计数10个视野内黏附于下室面有荧光的细胞数量,取平均值计数Ad-GFP组、Ad-PTEN-GFP组的细胞数分别为50±11和24±6(P<0.01),PTEN基因转染后明显抑制K562细胞侵袭性。
3 PTEN基因对人脐静脉内皮细胞系ECV304增殖及凋亡的影响。
3.1以MOI为100转染ECV304细胞,MTT检测细胞增殖,结果显示第5天Ad-PTEN-GFP组最大增殖抑制率为50.38±4.15%,明显高于Ad-GFP组4.28±0.73,(P<0.01)。
3.2流式细胞凋亡检测显示以MOI为100转染ECV304细胞5天后,Ad-PTEN-GFP组细胞凋亡率为18.56±2.11,明显高于Ad-GFP组2.86±1.01及未转染组1.34±0.32,(P<0.01)。
4鸡胚尿囊膜血管生成实验结果显示生理盐水组及Ad-GFP处理组鸡胚尿囊膜血管生长良好,呈树枝状,分支适中,Ad-PTEN-GP干预组出现比较明显的血管稀疏区及苍白的无血管区,血管数目明显减少。Ad-GFP组、Ad-PTEN-GFP组血管指数分别为92.2±4.37%与47.6±3.37%,两者比较差异有显著性(P<0.05)。
结论:
1.在AML细胞中PTEN基因低表达,并且与VEGF和FLT1基因呈负相关。
2. PTEN基因通过抑制PI3K/Akt通路,下调VEGF及FLT1表达,抑制白血病细胞侵袭及血管新生。
Background: Phosphatase and tensin hemology deleted on chromosome ten gene (PTEN),is a novel tumor suppressor gene in phosphatase family. PTEN protein has protein phosphatase and lipid phosphatase dual activity. PTEN is a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3). By converting PIP3 into phosphatidylinositol 4,5-bisphosphate (PIP2), PTEN negatively regulates the PI3 kinase-Akt signaling pathway. Because of this function, PTEN can inhibit proliferation, induce apoptosis and regulate cell cycle progression. The protein phosphatase activity of PTEN can regulate cellular processes such as migration and focal adhesion, by targetting mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK) pathway and inhibiting phosphorylation of several proteins. Briefly PTEN can regulate the growth, differentiation, adhesion and cell cycle of normal cells; supress the proliferation, invasion and metastasis of tumor cells; induce apoptosis of tumor cells.
Previous studies found that genetic mutation/deletion or lower expression of PTEN gene is so common in many types of human solid cancers, indicating that PTEN is one of the most frequently candidate of tumor suppressors. The above mentioned abnormality of PTEN may decrease its tumor suppression function that is closely corelated with the poor prognosis of human malignant diseases.
In the past decade, the function of PTEN gene in the genesis of hematopoietic malignancies such as leukemia has been generally focused. Studies showed that PTEN gene frequently deleted or methylated, expressed at low level but seldom mutated in leukemia. Mice with PTEN-mutant or PTEN- deletion had an increased proliferation of myeloid and T-lymphoid lineages and developed myeloproliferative disorder (MPD), and then transformed to acute myeloid/lymphoid leukaemia in the late stage of MPD. If the hemetopoietic stem cells from these mice were transplanted to SCID mice, the recipient mice also developed leukemia and with extramedullary infiltrations of leukemia cells. Meanwhile, PTEN gene played an important role in distinguishing leukemia stem cell and haemopoietic stem cell. Resent study indicated that the abnormality of PTEN gene probably involved in leukemia pathogenesis, multidrug resistance, angiogenesis and extramedullary infiltrations.
In many solid tumors, the ability of PTEN gene in inhibiting tumor cells proliferation and promoting apoptosis was confirmed already by many studies. However, the effect and its mechanism of PTEN gene in the genesis of hematopoietic malignancies such as leukemia were still not clear.
K562 cells, a leukemia cell line established from a patient with chronic myelocytic leukemia (CML) in blast crisis of erythroleukemia, have high activity of PI3K/Akt pathway and low-expression levels of PTEN gene and protein. In the present study, we transfected fresh human leukemia cells or K562 cell line cells with wild-type PTEN gene mediated by adenovirus vector. The effects and the machinesim of wild-type PTEN gene on leukemia cells proliferation, apoptosis, invasion, cell cycle progression, multidrug resistance, and angiogenesis were studied. The result of this study may provid an important theoretical basis for leukemia therapy with PTEN gene. This paper consists of the following three parts:
Part one: Effects of wild-type PTEN gene on proliferation, apoptosis and cell cycle progression of K562 cells and primary leukemia cell
Objective To investigate in vitro the effects of wild-type tumor-suppressing gene PTEN on proliferation, apoptosis, cell cycle of human primary leukemia cells and human chronic myeloid leukemia (CML) cell line K562 cells and to explore its possible machinesims.
Method The recombinant adenovirus vector-PTEN (Ad-PTEN) gene containing green fluorescent protein (Ad-PTEN-GFP) or the empty adenovirus vector containing green fluorescent protein (Ad-GFP) was transfected into K562 cells or primary leukemia cells from CML patients in blast crisis. The growth inhibition rate of leukemia cells was observed by MTT assay; the transfection efficiency, the apoptosis rate and cell cycle distribution were assessed by flow cytometry (FCM). We also obsereved apoptosis by morphological characteristics under optical microscope; DNA ladder, Hoechst 33342 fluorescent staining and cell colony fomation methods were also used to detect proliferation and apoptosis of K562 cells. The mRNA expression levels of PTEN, and the members of apoptosis family Bcl-2, Bcl-xL, Bax, Caspase-3, Caspase-7, Caspase-9, Survivin, Xiap, Smac, and cell cycle related molecular CyclinD1, CyclinD2, CDK4, and P27kip1 were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); The expression levels of PTEN, Bcl-2, Akt and p-Akt protein were detected by western blotting; Caspase-3/7, Caspase-9 protein activity were detected by caspase activity measuring kits.
Results
1 The transfection efficiency was about 81.2±5.4% in K562 cell line and 7.32±6.41% in five sample of primary leukemia cells after the cells were transfected with Ad-PTEN-GFP or Ad-GFP for 3 days, detected by flow cytometry at the condition of multiple of infection (MOI)=200.
2 The expression of PTEN mRNA and protein increased to the highest level at the third day of transfection, with MOI=200. In Ad-PTEN-GFP group, PTEN mRNA expression level was (23.58±4.52) and protein level was (0.912±0.102) that was higher than that in Ad-GPF group (PTEN mRNA was 0.650±0.516 and protein was 0.117±0.028) and that in untransfected group (PTEN mRNA was 0.575±0.226 and protein was 0.086±0.021), P<0.01.
3 Compared with empty vector adenovirus (Ad-GFP) group, the proliferation inhibiting rate of five primary leukemia cells transfected with Ad-PTEN-GFP was 33.8±10.4% at the fifth day. The maximum proliferation inhibiting rate was 38.67% in K562 cells transfected with Ad-PTEN-GFP for 5 days, which was higher than the 5.34% in Ad-GPF groups. The apoptosis rate was higher in Ad-PTEN-GFP group (22.4±2.11%) than that in Ad-GFP group (2.8±0.67%) and untransfected group (0.2±0.02%), P<0.05.
4 Cell morphology examination result showed that in the untransfected group and in Ad-GFP group, K562 cells were plump, the cytoplasm was abundant, the chromatin was delicate and loose, but in Ad-PTEN-GFP group the cells shrank and displayed the morphological sign of apoptosis: chromatin condensation, marginalization, nuclear pyknosis and fragmentation, the appearance of apoptotic bodies. Hoechst33342 fluorescent staining showed that the nucleus of the non-apoptosis cells were round with light blue clour, but the nucleus of apoptosis cell were sapphirine clour because of the nuclear pyknosis. The apoptosis rate was 35.6±2.2% in Ad-PTEN-GFP and 3.1%±1.13% in untransfected group, 6.2%±0.87% in Ad-GFP group at the 5th day of transfection。
5 After transfection with PTEN gene, 180bp to 200bp DNA bands appeared on agarose gel electrophoresis, among DNA extracted from the transfected K562 cells. The cells in Ad-GFP group and untransfected group did not show the phenomenon of DNA degradation at the 5th day of transfection with MOI=200.
6 After transfection with Ad-PTEN-GFP at MOI=200 for seven days, cell cycle distribution showed the ratio of G0/G1 phase cells increased from 54.9±3.61% to 78.5±5.17% (P<0.05), the ratio of G2/M phase decreased form 30.2±2.42% to 13.6±2.01% (P<0.05), and the cell cycle was arresd at G0/G1 phase.
7 After transfection with Ad-PTEN-GFP at MOI=200 for three days, western blot results showed the Akt protein expression level had no change but p-Akt expression level (0.021±0.011) decreased, compared with that in Ad-GFP group (0.587±0.078) and that in untransfected group (0.547±0.061), the difference was statisticly significant, P<0.01.
8 After transfection with Ad-PTEN-GFP at MOI=200 for 3 days, the activitay of caspase-9 (0.786±0.032), caspase-3/7 (1.362±0.039) was up-regulated compared with the activity of caspase-9 (0.591±0.024) and Caspase-3/7 (1.103±0.024) in Ad-GFP group, and the difference was significant (P<0.05).
9 After K562 cells had been transfected Ad-PTEN-GFP at MOI=200 for the third days, the expression level of survivin mRNA (0.0700±0.0059), xiap mRNA (0.00889±0.0006), smac mRNA (0.0600±0.0039) was lower than the expression level of survivin (0.437±0.079), xiap (0.0661±0.0072), smac (0.158±0.0078) in Ad-GPF group and the expression level of Survivin (0.453±0.081), xiap (0.070±0.0079), smac (0.177±0.0085) in untransfected group. Compared with the expresson level of the apoptosis related genes in Ad-GFP group, the expression levels of survivin, xiap, smac mRNA in the K562 cells transfected with Ad-PTEN-GFP decreased 6.14 fold, 7.44 fold and 2.95 fold, respectively.
10 After K562 cells transfected with Ad-PTEN-GFP at MOI=200 for 3 days, the Bcl-2 mRNA expression levels in untransfected group, Ad-GFP group and Ad-PTEN-GFP group were 0.732±0.078, 0.723±0.148, 0.195±0.043 respectively; the Bcl-xL mRNA expression levels were 0.0181±0.0009, 0.0176±0.0011, 0.0047±0.0008; the Bax mRNA expression levels were 0.376±0.031, 0.395±0.036, 0.912±0.117, respectively. This result showed that compared with that in the untransfected group and Ad-GFP group, the expression levels of Bcl-2, Bcl-xL mRNA and Bcl-2 protein were down-regulated, but Bax mRNA was up-regulated in Ad-PTEN-GFP group. The ratio of Bcl-2 to Bax mRNA was 1.83 in Ad-GFP group to 0.214 inAd-PTEN-GFP group, and decreased 8.55 fold.
11 After K562 cells transfected with Ad-PTEN-GFP at MOI=200 for 3 days, the expression levels of cell cycle related moleculars in untransfected group, Ad-GFP group and Ad-PTEN-GFP group changed as following: CyclinD1 mRNA (0.711±0.174; 0.698±0.162; 0.243±0.102), CyclinD2 mRNA (0.123±0.0196; 0.106±0.0178; 0.0441±0.0144), CDK4 mRNA (0.859±0.165; 0.876±0.142; 0.272±0.092), P27kip1 mRNA (0.0721±0.0102; 0.0683±0.00883; 0.470±0.172). Compared with the levels in untransfected group and Ad-GFP group, in Ad-PTEN-GFP group the expression levels of CyclinD1 mRNA, CyclinD2 and CDK4 mRNA expression levels decreased, but P27kip1mRNA increased and the difference was statistically significant (P<0.05).
Conclusions
The over-expression of wild-type PTEN gene inhibited proliferation and induced apoptosis of the primary leukemia cells and K562 cell line cells, probably via inhibition PI3K/Akt pathway and regulating the expression levels of many apoptosis related genes, such as Bcl-2 family, Caspase family and IAP family. Cell cycle distribution examination results showed the cell cycle arresd at G0/G1 phase through regulating the cell cycle related gene expression.
Part two: Mechanism of PTEN gene on multidrug resistance reversal in K562/ADM cells
Objective To explorer in vitro the effects of wild-type tumor-suppressing gene PTEN on proliferation, apoptosis of Adriamycin resistant human chronic myeloid leukemia (CML) cell line K562/ADM cells and the possible molecular machinesim of multidrug resistance reversal by investigating the synergistic effect of PTEN with different chemotherapeutic drugs.
Method The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562/ADM cells, then, the cells were treated with Adriamycin (ADM) or cytarabine (Ara-c) or arsenic trioxide (As2O3) to investigate the synergistic effect of chemotherapeutics with PTEN gene. The growth inhibition rate of K562/ADM cells was measured by MTT assay after the cells were transfected with PTEN gene and combined with or without chemotherapeutic drugs; the drug resistance reversal index was measured by the IC50 of different chemotherapeutic drugs combined with or without PTEN gene transfection; the transfection efficiency and the apoptosis rate were assessed by flow cytometry (FCM). The mRNA expression levels of PTEN, NF-κB, I-Κb, P53, MDR1, MRP1 and apoptosis related gene Bcl-2, Bcl-xL and Bax were detected by FQ-PCR. The protein expression levels of PTEN, Akt, p-Akt and P65 were detected by western blot.
Results
1 The transfection efficiency was about 82.2±5.8% in K562/ADM cell line cells after the cells transfected with Ad-GFP or Ad-PTEN-GFP on the third day detected by flow cytometry at the condition of multiple of infection (MOI)=200.
2 With MOI=200, at the third day of transfection, the expression level of PTEN mRNA and protein increased to the highest level. In Ad-PTEN-GFP group, PTEN mRNA expression level was (25.43±3.77) and protein level was (1.240±0.219), which were higher than that in Ad-GPF group (PTEN mRNA was 0.473±0.117 and protein was 0.105±0.014) and untransfected group (PTEN mRNA was 0.477±0.071 and protein was 0.073±0.011), P<0.01. PTEN mRNA and protein expression levels in K562/ADM cells had no statistical significance difference when compared with that of K562 cells, P>0.05.
3 Compared with empty vector adenovirus (Ad-GFP) group, the proliferation inhibiting rate was 32.26% in Ad-PTEN-GFP transfected K562/ADM cells at the fifth day, that was higher than the inhibition rate in Ad-GPF group (3.31%), P<0.01; and was lower than the inhibition rate in parental K562 cells, but the difference had no statistical significance, P>0.05.
4 The apoptosis rate was detected by FCM. After transfection with Ad-PTEN-GFP or Ad-GFP at MOI=200, the apoptosis rate in Ad-PTEN-GFP group combined with 10mg/L ADM or 5μmol Ara-c or 1μmol/L As2O3 were 17.8±2.3, 28.6±2.9, 61.7±3.2 respectively which were higher than that in Ad-GFP combined with chemotherapeutic drugs (2.3±0.7, 15.2±1.8, 20.4±2.1 respectively), P<0.01.
5 After transfection with Ad-PTEN-GFP and combined with the treatment of different chemotherapeutic drugs such as ADM, Ara-c, As2O3 for 72h, the drug resistance reversal index was 3.8 in ADM, 2.64 in Ara-c, 2.65 in As2O3 respectively.
6 After transfection with Ad-PTEN-GFP for 3 days, western blot examination results showed the Akt expression levels had no change but Ser473 p-Akt expression levels (0.022±0.007) decreased when compared with that in Ad-GFP group (0.646±0.066) and untransfected group (0.633±0.065), P<0.01.
7 After K562/ADM cells transfected with Ad-PTEN-GFP or Ad-GFP at MOI=200 for 3 days, the FQ-PCR results showed that in untransfected group, Ad-GFP group, and Ad-PTEN-GFP group, the mRNA expression level of NF-κB (P65) was 16.08±2.85, 15.20±2.08, 1.17±0.06, respectively, P<0.05, and P65 protein expression level was down-regulated after transfection with PTEN gene; The I-κB mRNA expression levels in untransfected group, Ad-GFP group and Ad-PTEN-GFP group were 0.21±0.08, 0.18±0.07, 0.23±0.11, the difference had no statistical significance (P>0.05). The P53 mRNA expression levels in untransfected group (2.95±0.32), Ad-GFP group (3.07±0.75) were lower than that in Ad-PTEN-GFP group(5.93±1.51),P<0.05.
8 After K562/ADM cells transfected Ad-PTEN-GFP at MOI=200 for 3 days, in untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the MDR1 mRNA expression levels were 0.25±0.02, 0.24±0.03, 0.13±0.01 (P<0.05) respectively; The MRP mRNA expression levels were 0.44±0.07, 0.42±0.07, 0.47±0.09 (P>0.05). The results showed that after transfection with PTEN gene, the MDR1 mRNA was down-regulated and MRP mRNA had no significant change.
9 After K562/ADM cells transfected Ad-PTEN-GFP at MOI=200 for 3 days, in untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the Bcl-2 mRNA expression levels were 0.040±0.013, 0.036±0.010, 0.011±0.004, respectively (P<0.05). Bcl-xL mRNA expression levels were 0.0071±0.0003, 0.0076±0.0003, 0.0037±0.0001, respectively (P<0.05). Bax mRNA expression levels were 0.129±0.028, 0.141±0.018, 0.432±0.071 (P<0.05). The results showed the Bcl-2 and Bcl-xL mRNA expression levels were down-regulated and Bax mRNA up-regulated after transfection PTEN gene,
Conclusions
Wild-type PTEN gene inhibited proliferation and induced apoptosis and enhanced the drug sensitivity or reversed drug resistance via inhibiting PI3K/Akt pathway and its downstream partener of the cell signaling transduction pathway in K562/ADM cells, such as down-regulated NF-κB, MDR1, Bcl-2 and up-regulated p53 and Bax.
Part three:Regulatory mechanism of PTEN gene on VEGF in leukemia cells and its clinical significance
Objective To investigate the clinical significance of wild-type PTEN gene with VEGF and VEGFR1 (FLT1) mRNA expression in myeloid leukemia; and explore the anti-angiogenesis effects of wild-type PTEN gene via regulating VEGF and FLT1 expresson.
Method (1) The expression of PTEN, VEGF and FLT1 mRNA were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) in 50 de novo acute myeloid leukemia (AML) patients, 10 AML patients in complete remission (CR), 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal controls.(2)The recombinated adenovirus containing green fluorescent protein (GFP) and PTEN gene(Ad-PTEN-GFP)or empty vector (Ad-GFP)was transfected into K562 cells. Then the VEGF, FLT1 and PTEN mRNA expression levels and the correlation between the expression of these genes were measured. (3) Human umbilical vein endothelial cell (HUVEC) line ECV304 cells were also transfected with or without PTEN gene; the proliferation inhibition rate and apoptosis rate were measured by MTT and flow cytometry (FCM). (4)Chick chorioallantoic membrane (CAM) test was used to testify the effect of PTEN gene on angiogenesis.
Results
1 The expression levels of PTEN, VEGF, FLT1 mRNA in myeloid leukemia and its relationship.
1.1 PTEN mRNA expression levels in the newly diagnosed acute myeloid leukemia (AML) patients (expression level were 0.051±0.059) were higher than that in AML-CR patients (1.015±0.411) and NC group (1.059±0.595), (P<0.05). In CML-BC patients, the PTEN mRNA expression levels (0.022±0.021) were lower than that in CML-CP patients (1.134±1.124) and in NC, (P<0.01).
1.2 VEGF mRNA expression levels in the newly diagnosed AML patients (expression levels were 0.442±0.192) were higher than those in AML-CR patients (0.075±0.042) and NC group(0.059±0.039), (P<0.01). In CML-BC patients (0.512±0.322) the VEGF mRNA expression levels were higher than CML-CP patients (0.219±0.131) and NC, (P<0.01).
1.3 VEGFR1 (FLT1) mRNA expression levels in the newly diagnosed AML patients (expression levels were 1.569±0.694) were higher than those in AML-CR patients (0.375±0.241) and NC group(0.402±0.238), (P<0.01). In CML-BC patients (1.579±0.623) the VEGF mRNA expression levels were higher than those in CML-CP patients (0.598±0.311) and NC, (P<0.01).
1.4 Pearson correlation analysis showed PTEN mRNA expression levels had negative correlation with VEGF and FLT1 mRNA expression levels, and the correlation coefficient were r=-0.631, (P<0.05) and r=-0.522, (P<0.05) respectively.
2 The effect of wild-type PTEN gene transfection on invasion activity of K562 cells and the regulation of p-Akt, VEGF and VEGFR1 (FLT1)
2.1 The Ad-GFP or Ad-PTEN-GFP transfection efficiency was about 81.2±5.4% in K562 cell line on the third day detected by flow cytometry with multiple of infection (MOI) of 200.
2.2When K562 cells were transfected with Ad-GFP or Ad-PTEN-GFP for 72 hours at different MOI (MOI=50, 100, 200 and 400), PTEN mRNA expression level increased while the VEGF and FLT1 mRNA expression levels decreased. The expression levels of PTEN mRNA and VEGF mRNA or FLT1 mRNA showed negative correlation, and the correlation coefficient were r=-0.903, (P<0.05) and r=-0.900, (P<0.05) respectively. VEGF proteins were also decreased. After transfection with Ad-GFP or Ad-PTEN-GFP for 3 days, compared with Ad-GFP group, in Ad-PTEN-GFP group, PTEN mRNA expression levels increased 41.01 fold but VEGF and FLT1 mRNA expression levels decreased 4.80 and 4.68 fold. The expression level of VEGF protein decreased 2.21 fold detected by ELISA method.
2.3 After transfection with Ad-PTEN-GFP at MOI=200 for 3 days, Akt expression levels in K562 cells had no change, but p-Akt expression level (0.021±0.011) decreased compared with that in Ad-GFP group (0.587±0.078) and in untransfected group (0.547±0.061), P<0.01.
2.4 After transfection with Ad- GFP or Ad-PTEN-GFP at MOI=200 for 48 hours, transwell invasion test results showed that the number of fluorescent staining cells, that were adhered to the bottom of the upper chamber of transwell, was 50±11 in Ad-GFP group, which was higher than the number of cells in Ad-PTEN-GPF group (24±6). These results indicated that the invasion or migration ability of K562 cells was suppressed when the cells were transfected with Ad-PTEN-GFP.
3 The anti-proliferation and apoptosis induction effect of PTEN gene on human umbilical vein endothelial cell (HUVEC) line ECV304 cells.
3.1 After ECV304 cells were transfected with Ad-PTEN-GFP at MOI=100, the maximum proliferation inhibition rate was 50.38±4.15% at the fifth day of transfection, which was higher than that in Ad-GFP group (4.28±0.73), P<0.01.
3.2 The apoptosis rate in Ad-PTEN-GFP group was 18.56±2.11, which was higher than that in Ad-GFP group (1.34±0.32), P<0.01, after ECV304 cells transfected at MOI=100 for 5 days.
4 PTEN gene significantly suppressed blood vessels’formation in chick embryo choriallantoic membrane (CAM). In control group and Ad-GFP group, blood vessels surrounding gelatin sponge grew well, it was tree-like and well-branched. While avascular area surrounding gelatin sponge could be observed in Ad-PTEN-GFP group, its blood vessels were less than control group. In Ad-GFP and Ad-PTEN-GFP group, the blood vessel index was 92.2±4.37% and 47.6±3.37% respectively P<0.05.
Conclusions
1 PTEN gene expressed at low level in the newly diagnosed AML patients and its expression level was negatively correlated with the expression level of VEGF and FLT1 gene.
2 PTEN gene inhibited the invasion ability of K562 cells and had anti-angiogenesis effect by inhibiting PI3K/Akt signaling transduction pathway and down-regulated the expression of VEGF and FLT1 genes. PTEN gene may be a candidate of effective anti-leukemia gene.
PTEN基因抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的作用在多种实体肿瘤细胞中已经得到证实,而在造血系统肿瘤如白血病中的作用及其机制尚不完全清楚。
K562细胞系为人慢性粒细胞白血病急性红白血病变细胞系,具有较高的PI3K/Akt活性,同时低表达PTEN基因及蛋白,为我们将外源性野生型PTEN基因进行转染提供了条件。因此我们将腺病毒介导的PTEN基因转染人白血病原代细胞及K562细胞系,观察野生型PTEN对白血病细胞增殖、凋亡、侵袭、细胞周期进程、多药耐药及血管新生的影响,并检测其可能的作用机制,为基因治疗白血病提供理论依据。研究分以下三部分:
第一部分腺病毒介导的野生型PTEN基因对人慢性粒细胞白血病细胞系K562及原代白血病细胞增殖、凋亡、细胞周期的影响
目的探讨肿瘤抑制基因PTEN对人原代白血病细胞及K562细胞系增殖、凋亡及细胞周期的影响,并对其可能的作用机制进行初步探讨。
方法将携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad- PTEN-GFP)或空载体腺病毒(Ad-GFP)转染人原代白血病细胞及K562细胞系。MTT检测细胞生长曲线;流式细胞仪检测腺病毒转染细胞的转染效率、细胞凋亡率和细胞周期分布;光镜下检测细胞形态变化,DNA ladder,Hoechst33342荧光染色检测、细胞集落形成等实验检测细胞增殖及凋亡。荧光定量PCR(FQ-PCR)检测PTEN、Bcl-2、Bcl-xL、Bax、Survivin、Xiap、Smac、Caspase-3、-7、-9、CyclinD1、CyclinD2、CDK4、P27kip1 mRNA水平变化,Western Blot检测PTEN、Bcl-2、Akt、p-Akt水平变化。Caspase活性检测试剂盒检测Caspase-3/7、Caspase-9蛋白活性。
结果
1当MOI为200时,转染第三天用流式细胞仪检测腺病毒转染K562细胞效率最高为81.2±5.4%。通过计数绿色荧光细胞数量检测转染原代细胞效率较低,选取了5例原代白血病细胞,平均转染效率为7.32±6.41%。
2当MOI为200时在转染第3天PTEN mRNA及蛋白表达水平达到最高。转染后d3,Ad-PTEN-GFP组PTEN表达mRNA水平(23.58±4.52)及蛋白表达水平(0.912±0.102)高于Ad-GFP组的mRNA(0.650±0.516)及蛋白(0.117±0.028)表达水平和未转染对照组的mRNA(0.575±0.226)及蛋白(0.086±0.021)表达水平,均P<0.01。
3 Ad-PTEN-GFP以感染复数为200转染细胞后第5天与空载体腺病毒(Ad-GFP)相比,Ad-PTEN-GFP转染人原代白血病细胞后,细胞增殖受抑,对转染的5例原代细胞增殖抑制率为33.8±10.4%。Ad-PTEN-GFP以感染复数为200转染K562细胞后第5天,细胞最大生长抑制率为38.67%,明显高于Ad-GFP组的抑制率5.34%。Ad-PTEN-GFP组细胞凋亡率为22.4±2.11%,明显高于Ad-GFP组的2.8±0.67%和未转染组的0.2±0.02%,均P<0.05。
4腺病毒转染K562细胞5天后,未转染组及Ad-GFP转染组的细胞呈圆形或椭圆形,体积较大,染色质疏松;转染Ad-PTEN-GFP组部分细胞体积缩小,染色质高度浓缩边集,核碎裂核溶解。Hoechst33342荧光染色显示正常细胞核Hoechst着色的形态呈圆形,淡蓝色;而凋亡细胞的核由于浓集而呈亮蓝色。转染Ad-PTEN-GFP组细胞凋亡细胞比例(35.6±2.2%)明显高于未转染组(3.1%±1.13%)和转染Ad-GFP组(6.2%±0.87%),均P<0.01。
5以MOI=200腺病毒转染K562细胞5天后,Ad-PTEN-GFP组DNA琼脂糖凝胶电泳出现大小为180至200bp及其倍数的梯状条带,片断之间呈一定间隔,这是DNA在核小体间成倍断裂的结果,Ad-GFP组及未转染组细胞未出现DNA降解现象。
6细胞周期显示以MOI=200 Ad-PTEN-GFP转染K562细胞7天,G0/G1期细胞比例由54.9±3.61%增加至78.5±5.17%(P<0.05),G2/M期细胞比例由30.2±2.42%降至13.6±2.01%,(P<0.05),处于G0/G1期细胞比例逐渐增加,处于G2/M期细胞比例逐渐减少,细胞周期阻滞在G0/G1。
7当MOI为200时,K562细胞转染Ad-PTEN-GFP第三天Western blot检测Akt及p-Akt结果显示,总Akt表达水平无明显变化,而p-Akt表达水平在未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别为(0.587±0.078;0.547±0.061;0.021±0.011),转染PTEN 3d后p-Akt表达水平明显减低(P<0.01)。
8以MOI=200转染K562细胞系3天后,Ad-PTEN-GFP组细胞内Caspase-9活性(0.786±0.032)、Caspase-3/7活性(1.362±0.039),均高于Ad-GFP组的Caspase-9(0.591±0.024)和Caspase-3/7(1.103±0.024)活性,(P<0.05)。
9以MOI=200转染K562细胞系3天后,Ad-PTEN-GFP组细胞内Survivin(0.0700±0.0059)、Xiap(0.00889±0.0006)、Smac (0.0600±0.0039) mRNA表达水平均明显低于Ad-GPF组细胞内Survivin(0.437±0.079)、Xiap(0.0661±0.0072)、Smac(0.158±0.0078)和未转染组K562细胞内Survivin(0.453±0.081)、Xiap(0.070±0.0079)、Smac(0.177±0.0085) mRNA表达水平,转染Ad-PTEN-GFP后与转染Ad-GFP相比Survivin mRNA表达水平降低6.14倍、Xiap降低7.44倍,而Smac mRNA降低2.95倍。
10以MOI=200转染K562细胞系3天后,未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组凋亡相关基因的表达水平分别为,Bcl-2 mRNA:0.732±0.078、0.723±0.148、0.195±0.043;Bcl-xL mRNA:0.0181±0.0009、0.0176±0.0011、0.0047±0.0008;Bax mRNA :0.376±0.031、0.395±0.036、0.912±0.117。结果显示未转染组、Ad-GFP组与转染Ad-PTEN-GFP组各基因的表达水平有明显差异,均P<0.01。转染野生型PTEN后Bcl-2与Bcl-xL抗凋亡基因表达减低,而促凋亡基因Bax mRNA表达升高,Bcl-2蛋白表达水平下调,分别为(1.016±0.147;0.996±0.166;0.582±0.067)。Bcl-2/Bax mRNA表达水平由Ad-GFP组1.83降至Ad-PTEN-GFP组0.214,降低8.55倍。
11以MOI=200转染K562细胞系3天后,细胞周期相关基因的检测结果显示,未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别如下:CyclinD1 mRNA (0.711±0.174;0.698±0.162;0.243±0.102)、CyclinD2 mRNA (0.123±0.0196 ; 0.106±0.0178 ; 0.0441±0.0144)、CDK4 mRNA (0.859±0.165;0.876±0.142;0.272±0.092)、P27kip1 mRNA (0.0721±0.0102;0.0683±0.00883;0.470±0.172)。与未转染组和转染Ad-GFP组比较,转染Ad-PTEN-GFP后CyclinD1、CyclinD2、CDK4 mRNA表达水平明显下调(均P<0.05),而P27kip1 mRNA表达水平升高(各组P<0.05)
结论高表达PTEN基因能够明显抑制原代白血病细胞和K562细胞增殖并诱导凋亡,可能与PTEN抑制PI3K/Akt信号通路,并调控多种凋亡相关分子家族基因如Bcl-2家族、Caspase家族和IAP家族密切相关,并通过调控细胞周期相关分子表达使细胞周期阻滞在G0/G1期。
第二部分腺病毒介导的野生型PTEN基因对人慢性粒细胞白血病阿霉素耐药细胞系K562/ADM多药耐药逆转的研究
目的探讨野生型抑癌基因PTEN对人慢性粒细胞白血病阿霉素耐药细胞系K562/ADM增殖、凋亡的影响,及其与多种化疗药物联合应用的协同作用进行研究,并对其逆转多药耐药的分子作用机制进行探讨。
方法用携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad- PTEN-GFP)及空载体腺病毒(Ad-GFP)转染对阿霉素耐药的慢性粒细胞性白血病细胞株K562/ADM细胞系,和或联合应用阿霉素(ADM)、阿糖胞苷(Ara-c)、三氧化二砷(As2O3)等不同类型化疗药物,观察PTEN基因与化疗药物的协同作用。MTT检测细胞生长曲线,计算不同药物的IC50及联合作用的IC50,计算耐药逆转倍数;流式细胞仪检测腺病毒转染K562细胞的转染效率、细胞凋亡率;荧光定量PCR(FQ-PCR)检测PTEN、NF-κB、I-κB、P53、MDR1、MRP1及凋亡相关基因Bcl-2、Bcl-xL及Bax水平变化,Western Blot检测PTEN、Akt、p-Akt、P65蛋白水平变化。
结果
1当MOI为200时,转染第3天流式细胞仪检测腺病毒转染K562/ADM细胞效率最高,为82.2±5.8%。
2当MOI为200时在转染第3天,K562/ADM细胞中的PTEN mRNA及蛋白表达水平达到最高。转染后d3,Ad-PTEN-GFP组PTEN mRNA表达水平(25.43±3.77)及蛋白表达水平(1.240±0.219)高于Ad-GFP组PTEN mRNA(0.473±0.117)及PTEN蛋白(0.105±0.014)表达水平和未转染对照组PTEN mRNA(0.477±0.071)及PTEN蛋白(0.073±0.011)表达水平(P<0.01)。而K562及K562/ADM细胞中的PTEN mRNA及蛋白表达水平无显著差异(P>0.05)。
3以MOI为200转染K562/ADM细胞,第5天Ad-PTEN-GFP组增殖抑制率为32.26%,明显高于Ad-GFP组(3.31%),有明显统计学差异(P<0.01),与亲代K562细胞比较,PTEN基因对K562/ADM细胞的抑制率稍低,但两者差异无统计学意义(P>0.05)。
4流式细胞凋亡检测显示以MOI为200转染K562/ADM细胞后,Ad-PTEN-GFP联合10mg/L阿霉素(ADM)或5μmol阿糖胞苷(Ara-c)或1μmol/L三氧化二砷(As2O3)干预组细胞凋亡率分别为:17.8±2.3、28.6±2.9、61.7±3.2,明显高于转染Ad-GFP后联合各化疗药物组的凋亡率:2.3±0.7、15.2±1.8、20.4±2.1,三者均有显著性差异(P<0.01)。
5以MOI为200转染K562/ADM细胞后联合或单独应用ADM、Ara-c、As2O3等化疗药物72h后PTEN基因对不同化疗药物的耐药逆转倍数分别为ADM 3.8倍、Ara-c 2.64倍、As2O3 2.65倍。
6当MOI为200时,转染Ad-PTEN-GFP第三天Western blot检测Akt及p-Akt结果显示,总Akt表达水平无明显变化,而p-Akt表达水平在未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别为:0.633±0.065、0.646±0.066、0.022±0.007;转染PTEN 3d后K562/ADM细胞p-Akt表达水平明显减低(P<0.01)。
7以MOI=200转染K562/ADM细胞后3天,实时荧光定量PCR法(FQ-PCR)结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组,NF-κB mRNA与β-actin mRNA相对表达水平分别为16.08±2.85;15.20±2.08;1.17±0.06(P<0.05),转染PTEN基因后P65蛋白表达水平亦明显减低。I-κB相对表达水平分别为0.21±0.08;0.18±0.07;0.23±0.11,无明显差异(P>0.05)。P53 mRNA与β-actin mRNA的相对表达水平分别为2.95±0.32;3.07±0.75;5.93±1.51,(P<0.05)。转染野生型PTEN基因后NF-κB mRNA及p65蛋白表达水平明显减低,p65蛋白活性呈时间依赖性减低。I-κB mRNA表达水平无明显改变,P53 mRNA表达水平轻度升高。
8以MOI=200转染K562/ADM细胞3天后,FQ- PCR结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组中MDR1 mRNA与β-actin mRNA相对表达水平分别为0.25±0.02;0.24±0.03;0.13±0.01(P<0.05)。MRP mRNA表达水平分别为0.44±0.07;0.42±0.07;0.47±0.09(P>0.05)。表明转染PTEN基因后MDR1 mRNA表达水平减低,而MPR mRNA表达水平无明显改变。
9以MOI=200转染K562/ADM细胞3天后,FQ- PCR结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组中Bcl-2 mRNA表达水平分别为0.040±0.013;0.036±0.010;0.011±0.004(P<0.05)。Bcl-xL mRNA表达水平分别为:0.0071±0.0003;0.0076±0.0003;0.0037±0.0001(P<0.05)。Bax mRNA表达水平分别为:0.129±0.028;0.141±0.018;0.432±0.071(P<0.05)。结果表明转染野生型PTEN基因后Bcl-2 mRNA、Bcl-xL
mRNA表达水平明显减低,而Bax mRNA表达水平增加。结论野生型PTEN基因可能通过调控PI3K/Akt信号传导通路、抑制K562/ADM细胞增殖及诱导凋亡,并通过下调NF-κB、MDR1、Bcl-2及上调P53、Bax等多种信号分子,逆转K562/ADM细胞的多药耐药。
第三部分PTEN基因对白血病细胞VEGF调控的研究及其临床意义
目的探讨肿瘤抑制基因PTEN及血管内皮生长因子(VEGF)及其受体VEGFR1(FLT1)在白血病中的表达和临床意义;研究野生型PTEN基因的抗血管新生作用。方法(1)应用实时荧光定量PCR(FQ-PCR)检测50例初治急性髓系白血病(AML),10例缓解期AML,10例慢性粒细胞白血病慢性期(CML-CP)、10例CML急变期(CML-BC)患者和10例健康人骨髓单个核细胞(MMNC)中PTEN、VEGF、VEGFR1 mRNA的表达。(2)将携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP)转染K562细胞系,观察不同感染复数(MOI)条件下PTEN基因对VEGF及FLT1表达的影响。(3)通过MTT及流式细胞术检测PTEN基因对脐静脉内皮细胞系ECV304增殖、凋亡的影响。(4)鸡胚绒毛尿囊膜(CAM)体内血管生长实验方法检测PTEN基因对血管生成的影响。
结果
1 PTEN、VEGF、FLT1 mRNA在髓系白血病中的表达水平及相互关系。
1.1 PTEN mRNA在急性髓系白血病(AML)初治患者中表达水平(0.051±0.059)明显低于AML缓解组(1.015±0.411)和正常对照组NC(1.059±0.595),(P<0.01);CML-BC患者中PTEN mRNA表达水平(0.022±0.021)低于CML-CP患者(1.134±1.124)及NC(P<0.01)。
1.2 VEGF mRNA在AML初治患者中表达水平(0.442±0.192)明显高于AML缓解组(0.075±0.042)和NC(0.059±0.039),(P<0.01);CML-BC患者(0.512±0.322)VEGF mRNA表达水平高于CML-CP(0.219±0.131)及NC(P<0.01)。
1.3 FLT1 mRNA在AML初治患者中表达水平(1.569±0.694)明显高于AML缓解组(0.375±0.241)和NC(0.402±0.238),(P<0.01);CML-BC患者中FLT1 mRNA表达水平(1.579±0.623)高于CML-CP(0.598±0.311)及NC(P<0.01)。
1.4 Pearson直线相关法分析显示,AML患者中PTEN mRNA与VEGF mRNA呈负相关(r=-0.631,P<0.05),与FLT1 mRNA表达同样呈负相关(r=-0.522,P<0.05)。
2 PTEN基因对K562细胞系的侵袭性及p-Akt、VEGF及FLT1调控的研究
2.1当MOI为200时,转染第三天流式细胞仪检测腺病毒转染K562细胞效率最高为81.2±5.4%,符合基因治疗对载体的要求。
2.2当MOI为50、100、200、400转染K562细胞系后,转染72h内随着PTEN mRNA表达水平升高,VEGF、FLT1 mRNA表达水平逐渐降低,呈负相关趋势,相关系数分别为:r=-0.903,P<0.05;r=-0.900,P<0.05;VEGF蛋白表达水平同样降低。当MOI=200时PTEN mRNA表达水平增加到41.01倍,VEGF、FLT1 mRNA表达水平下调分别为4.80倍和4.68倍,ELISA方法显示VEGF蛋白表达水平下调2.21倍。
2.3当MOI为200时,转染Ad-PTEN-GFP第三天Western blot检测总Akt及p-Akt结果显示,总Akt表达水平无明显变化,而p-Akt表达水平在未转染组、转染Ad-GFP组、转染Ad-PTEN-GFP组分别为0.587±0.078、0.547±0.061、0.021±0.011,转染PTEN 3d后p-Akt表达水平明显减低(P<0.01)。
2.4 Transwell侵袭实验显示在MOI=200时,Ad-PTEN-GFP转染K562细胞48h后,400倍显微镜下计数10个视野内黏附于下室面有荧光的细胞数量,取平均值计数Ad-GFP组、Ad-PTEN-GFP组的细胞数分别为50±11和24±6(P<0.01),PTEN基因转染后明显抑制K562细胞侵袭性。
3 PTEN基因对人脐静脉内皮细胞系ECV304增殖及凋亡的影响。
3.1以MOI为100转染ECV304细胞,MTT检测细胞增殖,结果显示第5天Ad-PTEN-GFP组最大增殖抑制率为50.38±4.15%,明显高于Ad-GFP组4.28±0.73,(P<0.01)。
3.2流式细胞凋亡检测显示以MOI为100转染ECV304细胞5天后,Ad-PTEN-GFP组细胞凋亡率为18.56±2.11,明显高于Ad-GFP组2.86±1.01及未转染组1.34±0.32,(P<0.01)。
4鸡胚尿囊膜血管生成实验结果显示生理盐水组及Ad-GFP处理组鸡胚尿囊膜血管生长良好,呈树枝状,分支适中,Ad-PTEN-GP干预组出现比较明显的血管稀疏区及苍白的无血管区,血管数目明显减少。Ad-GFP组、Ad-PTEN-GFP组血管指数分别为92.2±4.37%与47.6±3.37%,两者比较差异有显著性(P<0.05)。
结论:
1.在AML细胞中PTEN基因低表达,并且与VEGF和FLT1基因呈负相关。
2. PTEN基因通过抑制PI3K/Akt通路,下调VEGF及FLT1表达,抑制白血病细胞侵袭及血管新生。
Background: Phosphatase and tensin hemology deleted on chromosome ten gene (PTEN),is a novel tumor suppressor gene in phosphatase family. PTEN protein has protein phosphatase and lipid phosphatase dual activity. PTEN is a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3). By converting PIP3 into phosphatidylinositol 4,5-bisphosphate (PIP2), PTEN negatively regulates the PI3 kinase-Akt signaling pathway. Because of this function, PTEN can inhibit proliferation, induce apoptosis and regulate cell cycle progression. The protein phosphatase activity of PTEN can regulate cellular processes such as migration and focal adhesion, by targetting mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK) pathway and inhibiting phosphorylation of several proteins. Briefly PTEN can regulate the growth, differentiation, adhesion and cell cycle of normal cells; supress the proliferation, invasion and metastasis of tumor cells; induce apoptosis of tumor cells.
Previous studies found that genetic mutation/deletion or lower expression of PTEN gene is so common in many types of human solid cancers, indicating that PTEN is one of the most frequently candidate of tumor suppressors. The above mentioned abnormality of PTEN may decrease its tumor suppression function that is closely corelated with the poor prognosis of human malignant diseases.
In the past decade, the function of PTEN gene in the genesis of hematopoietic malignancies such as leukemia has been generally focused. Studies showed that PTEN gene frequently deleted or methylated, expressed at low level but seldom mutated in leukemia. Mice with PTEN-mutant or PTEN- deletion had an increased proliferation of myeloid and T-lymphoid lineages and developed myeloproliferative disorder (MPD), and then transformed to acute myeloid/lymphoid leukaemia in the late stage of MPD. If the hemetopoietic stem cells from these mice were transplanted to SCID mice, the recipient mice also developed leukemia and with extramedullary infiltrations of leukemia cells. Meanwhile, PTEN gene played an important role in distinguishing leukemia stem cell and haemopoietic stem cell. Resent study indicated that the abnormality of PTEN gene probably involved in leukemia pathogenesis, multidrug resistance, angiogenesis and extramedullary infiltrations.
In many solid tumors, the ability of PTEN gene in inhibiting tumor cells proliferation and promoting apoptosis was confirmed already by many studies. However, the effect and its mechanism of PTEN gene in the genesis of hematopoietic malignancies such as leukemia were still not clear.
K562 cells, a leukemia cell line established from a patient with chronic myelocytic leukemia (CML) in blast crisis of erythroleukemia, have high activity of PI3K/Akt pathway and low-expression levels of PTEN gene and protein. In the present study, we transfected fresh human leukemia cells or K562 cell line cells with wild-type PTEN gene mediated by adenovirus vector. The effects and the machinesim of wild-type PTEN gene on leukemia cells proliferation, apoptosis, invasion, cell cycle progression, multidrug resistance, and angiogenesis were studied. The result of this study may provid an important theoretical basis for leukemia therapy with PTEN gene. This paper consists of the following three parts:
Part one: Effects of wild-type PTEN gene on proliferation, apoptosis and cell cycle progression of K562 cells and primary leukemia cell
Objective To investigate in vitro the effects of wild-type tumor-suppressing gene PTEN on proliferation, apoptosis, cell cycle of human primary leukemia cells and human chronic myeloid leukemia (CML) cell line K562 cells and to explore its possible machinesims.
Method The recombinant adenovirus vector-PTEN (Ad-PTEN) gene containing green fluorescent protein (Ad-PTEN-GFP) or the empty adenovirus vector containing green fluorescent protein (Ad-GFP) was transfected into K562 cells or primary leukemia cells from CML patients in blast crisis. The growth inhibition rate of leukemia cells was observed by MTT assay; the transfection efficiency, the apoptosis rate and cell cycle distribution were assessed by flow cytometry (FCM). We also obsereved apoptosis by morphological characteristics under optical microscope; DNA ladder, Hoechst 33342 fluorescent staining and cell colony fomation methods were also used to detect proliferation and apoptosis of K562 cells. The mRNA expression levels of PTEN, and the members of apoptosis family Bcl-2, Bcl-xL, Bax, Caspase-3, Caspase-7, Caspase-9, Survivin, Xiap, Smac, and cell cycle related molecular CyclinD1, CyclinD2, CDK4, and P27kip1 were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); The expression levels of PTEN, Bcl-2, Akt and p-Akt protein were detected by western blotting; Caspase-3/7, Caspase-9 protein activity were detected by caspase activity measuring kits.
Results
1 The transfection efficiency was about 81.2±5.4% in K562 cell line and 7.32±6.41% in five sample of primary leukemia cells after the cells were transfected with Ad-PTEN-GFP or Ad-GFP for 3 days, detected by flow cytometry at the condition of multiple of infection (MOI)=200.
2 The expression of PTEN mRNA and protein increased to the highest level at the third day of transfection, with MOI=200. In Ad-PTEN-GFP group, PTEN mRNA expression level was (23.58±4.52) and protein level was (0.912±0.102) that was higher than that in Ad-GPF group (PTEN mRNA was 0.650±0.516 and protein was 0.117±0.028) and that in untransfected group (PTEN mRNA was 0.575±0.226 and protein was 0.086±0.021), P<0.01.
3 Compared with empty vector adenovirus (Ad-GFP) group, the proliferation inhibiting rate of five primary leukemia cells transfected with Ad-PTEN-GFP was 33.8±10.4% at the fifth day. The maximum proliferation inhibiting rate was 38.67% in K562 cells transfected with Ad-PTEN-GFP for 5 days, which was higher than the 5.34% in Ad-GPF groups. The apoptosis rate was higher in Ad-PTEN-GFP group (22.4±2.11%) than that in Ad-GFP group (2.8±0.67%) and untransfected group (0.2±0.02%), P<0.05.
4 Cell morphology examination result showed that in the untransfected group and in Ad-GFP group, K562 cells were plump, the cytoplasm was abundant, the chromatin was delicate and loose, but in Ad-PTEN-GFP group the cells shrank and displayed the morphological sign of apoptosis: chromatin condensation, marginalization, nuclear pyknosis and fragmentation, the appearance of apoptotic bodies. Hoechst33342 fluorescent staining showed that the nucleus of the non-apoptosis cells were round with light blue clour, but the nucleus of apoptosis cell were sapphirine clour because of the nuclear pyknosis. The apoptosis rate was 35.6±2.2% in Ad-PTEN-GFP and 3.1%±1.13% in untransfected group, 6.2%±0.87% in Ad-GFP group at the 5th day of transfection。
5 After transfection with PTEN gene, 180bp to 200bp DNA bands appeared on agarose gel electrophoresis, among DNA extracted from the transfected K562 cells. The cells in Ad-GFP group and untransfected group did not show the phenomenon of DNA degradation at the 5th day of transfection with MOI=200.
6 After transfection with Ad-PTEN-GFP at MOI=200 for seven days, cell cycle distribution showed the ratio of G0/G1 phase cells increased from 54.9±3.61% to 78.5±5.17% (P<0.05), the ratio of G2/M phase decreased form 30.2±2.42% to 13.6±2.01% (P<0.05), and the cell cycle was arresd at G0/G1 phase.
7 After transfection with Ad-PTEN-GFP at MOI=200 for three days, western blot results showed the Akt protein expression level had no change but p-Akt expression level (0.021±0.011) decreased, compared with that in Ad-GFP group (0.587±0.078) and that in untransfected group (0.547±0.061), the difference was statisticly significant, P<0.01.
8 After transfection with Ad-PTEN-GFP at MOI=200 for 3 days, the activitay of caspase-9 (0.786±0.032), caspase-3/7 (1.362±0.039) was up-regulated compared with the activity of caspase-9 (0.591±0.024) and Caspase-3/7 (1.103±0.024) in Ad-GFP group, and the difference was significant (P<0.05).
9 After K562 cells had been transfected Ad-PTEN-GFP at MOI=200 for the third days, the expression level of survivin mRNA (0.0700±0.0059), xiap mRNA (0.00889±0.0006), smac mRNA (0.0600±0.0039) was lower than the expression level of survivin (0.437±0.079), xiap (0.0661±0.0072), smac (0.158±0.0078) in Ad-GPF group and the expression level of Survivin (0.453±0.081), xiap (0.070±0.0079), smac (0.177±0.0085) in untransfected group. Compared with the expresson level of the apoptosis related genes in Ad-GFP group, the expression levels of survivin, xiap, smac mRNA in the K562 cells transfected with Ad-PTEN-GFP decreased 6.14 fold, 7.44 fold and 2.95 fold, respectively.
10 After K562 cells transfected with Ad-PTEN-GFP at MOI=200 for 3 days, the Bcl-2 mRNA expression levels in untransfected group, Ad-GFP group and Ad-PTEN-GFP group were 0.732±0.078, 0.723±0.148, 0.195±0.043 respectively; the Bcl-xL mRNA expression levels were 0.0181±0.0009, 0.0176±0.0011, 0.0047±0.0008; the Bax mRNA expression levels were 0.376±0.031, 0.395±0.036, 0.912±0.117, respectively. This result showed that compared with that in the untransfected group and Ad-GFP group, the expression levels of Bcl-2, Bcl-xL mRNA and Bcl-2 protein were down-regulated, but Bax mRNA was up-regulated in Ad-PTEN-GFP group. The ratio of Bcl-2 to Bax mRNA was 1.83 in Ad-GFP group to 0.214 inAd-PTEN-GFP group, and decreased 8.55 fold.
11 After K562 cells transfected with Ad-PTEN-GFP at MOI=200 for 3 days, the expression levels of cell cycle related moleculars in untransfected group, Ad-GFP group and Ad-PTEN-GFP group changed as following: CyclinD1 mRNA (0.711±0.174; 0.698±0.162; 0.243±0.102), CyclinD2 mRNA (0.123±0.0196; 0.106±0.0178; 0.0441±0.0144), CDK4 mRNA (0.859±0.165; 0.876±0.142; 0.272±0.092), P27kip1 mRNA (0.0721±0.0102; 0.0683±0.00883; 0.470±0.172). Compared with the levels in untransfected group and Ad-GFP group, in Ad-PTEN-GFP group the expression levels of CyclinD1 mRNA, CyclinD2 and CDK4 mRNA expression levels decreased, but P27kip1mRNA increased and the difference was statistically significant (P<0.05).
Conclusions
The over-expression of wild-type PTEN gene inhibited proliferation and induced apoptosis of the primary leukemia cells and K562 cell line cells, probably via inhibition PI3K/Akt pathway and regulating the expression levels of many apoptosis related genes, such as Bcl-2 family, Caspase family and IAP family. Cell cycle distribution examination results showed the cell cycle arresd at G0/G1 phase through regulating the cell cycle related gene expression.
Part two: Mechanism of PTEN gene on multidrug resistance reversal in K562/ADM cells
Objective To explorer in vitro the effects of wild-type tumor-suppressing gene PTEN on proliferation, apoptosis of Adriamycin resistant human chronic myeloid leukemia (CML) cell line K562/ADM cells and the possible molecular machinesim of multidrug resistance reversal by investigating the synergistic effect of PTEN with different chemotherapeutic drugs.
Method The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562/ADM cells, then, the cells were treated with Adriamycin (ADM) or cytarabine (Ara-c) or arsenic trioxide (As2O3) to investigate the synergistic effect of chemotherapeutics with PTEN gene. The growth inhibition rate of K562/ADM cells was measured by MTT assay after the cells were transfected with PTEN gene and combined with or without chemotherapeutic drugs; the drug resistance reversal index was measured by the IC50 of different chemotherapeutic drugs combined with or without PTEN gene transfection; the transfection efficiency and the apoptosis rate were assessed by flow cytometry (FCM). The mRNA expression levels of PTEN, NF-κB, I-Κb, P53, MDR1, MRP1 and apoptosis related gene Bcl-2, Bcl-xL and Bax were detected by FQ-PCR. The protein expression levels of PTEN, Akt, p-Akt and P65 were detected by western blot.
Results
1 The transfection efficiency was about 82.2±5.8% in K562/ADM cell line cells after the cells transfected with Ad-GFP or Ad-PTEN-GFP on the third day detected by flow cytometry at the condition of multiple of infection (MOI)=200.
2 With MOI=200, at the third day of transfection, the expression level of PTEN mRNA and protein increased to the highest level. In Ad-PTEN-GFP group, PTEN mRNA expression level was (25.43±3.77) and protein level was (1.240±0.219), which were higher than that in Ad-GPF group (PTEN mRNA was 0.473±0.117 and protein was 0.105±0.014) and untransfected group (PTEN mRNA was 0.477±0.071 and protein was 0.073±0.011), P<0.01. PTEN mRNA and protein expression levels in K562/ADM cells had no statistical significance difference when compared with that of K562 cells, P>0.05.
3 Compared with empty vector adenovirus (Ad-GFP) group, the proliferation inhibiting rate was 32.26% in Ad-PTEN-GFP transfected K562/ADM cells at the fifth day, that was higher than the inhibition rate in Ad-GPF group (3.31%), P<0.01; and was lower than the inhibition rate in parental K562 cells, but the difference had no statistical significance, P>0.05.
4 The apoptosis rate was detected by FCM. After transfection with Ad-PTEN-GFP or Ad-GFP at MOI=200, the apoptosis rate in Ad-PTEN-GFP group combined with 10mg/L ADM or 5μmol Ara-c or 1μmol/L As2O3 were 17.8±2.3, 28.6±2.9, 61.7±3.2 respectively which were higher than that in Ad-GFP combined with chemotherapeutic drugs (2.3±0.7, 15.2±1.8, 20.4±2.1 respectively), P<0.01.
5 After transfection with Ad-PTEN-GFP and combined with the treatment of different chemotherapeutic drugs such as ADM, Ara-c, As2O3 for 72h, the drug resistance reversal index was 3.8 in ADM, 2.64 in Ara-c, 2.65 in As2O3 respectively.
6 After transfection with Ad-PTEN-GFP for 3 days, western blot examination results showed the Akt expression levels had no change but Ser473 p-Akt expression levels (0.022±0.007) decreased when compared with that in Ad-GFP group (0.646±0.066) and untransfected group (0.633±0.065), P<0.01.
7 After K562/ADM cells transfected with Ad-PTEN-GFP or Ad-GFP at MOI=200 for 3 days, the FQ-PCR results showed that in untransfected group, Ad-GFP group, and Ad-PTEN-GFP group, the mRNA expression level of NF-κB (P65) was 16.08±2.85, 15.20±2.08, 1.17±0.06, respectively, P<0.05, and P65 protein expression level was down-regulated after transfection with PTEN gene; The I-κB mRNA expression levels in untransfected group, Ad-GFP group and Ad-PTEN-GFP group were 0.21±0.08, 0.18±0.07, 0.23±0.11, the difference had no statistical significance (P>0.05). The P53 mRNA expression levels in untransfected group (2.95±0.32), Ad-GFP group (3.07±0.75) were lower than that in Ad-PTEN-GFP group(5.93±1.51),P<0.05.
8 After K562/ADM cells transfected Ad-PTEN-GFP at MOI=200 for 3 days, in untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the MDR1 mRNA expression levels were 0.25±0.02, 0.24±0.03, 0.13±0.01 (P<0.05) respectively; The MRP mRNA expression levels were 0.44±0.07, 0.42±0.07, 0.47±0.09 (P>0.05). The results showed that after transfection with PTEN gene, the MDR1 mRNA was down-regulated and MRP mRNA had no significant change.
9 After K562/ADM cells transfected Ad-PTEN-GFP at MOI=200 for 3 days, in untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the Bcl-2 mRNA expression levels were 0.040±0.013, 0.036±0.010, 0.011±0.004, respectively (P<0.05). Bcl-xL mRNA expression levels were 0.0071±0.0003, 0.0076±0.0003, 0.0037±0.0001, respectively (P<0.05). Bax mRNA expression levels were 0.129±0.028, 0.141±0.018, 0.432±0.071 (P<0.05). The results showed the Bcl-2 and Bcl-xL mRNA expression levels were down-regulated and Bax mRNA up-regulated after transfection PTEN gene,
Conclusions
Wild-type PTEN gene inhibited proliferation and induced apoptosis and enhanced the drug sensitivity or reversed drug resistance via inhibiting PI3K/Akt pathway and its downstream partener of the cell signaling transduction pathway in K562/ADM cells, such as down-regulated NF-κB, MDR1, Bcl-2 and up-regulated p53 and Bax.
Part three:Regulatory mechanism of PTEN gene on VEGF in leukemia cells and its clinical significance
Objective To investigate the clinical significance of wild-type PTEN gene with VEGF and VEGFR1 (FLT1) mRNA expression in myeloid leukemia; and explore the anti-angiogenesis effects of wild-type PTEN gene via regulating VEGF and FLT1 expresson.
Method (1) The expression of PTEN, VEGF and FLT1 mRNA were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) in 50 de novo acute myeloid leukemia (AML) patients, 10 AML patients in complete remission (CR), 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal controls.(2)The recombinated adenovirus containing green fluorescent protein (GFP) and PTEN gene(Ad-PTEN-GFP)or empty vector (Ad-GFP)was transfected into K562 cells. Then the VEGF, FLT1 and PTEN mRNA expression levels and the correlation between the expression of these genes were measured. (3) Human umbilical vein endothelial cell (HUVEC) line ECV304 cells were also transfected with or without PTEN gene; the proliferation inhibition rate and apoptosis rate were measured by MTT and flow cytometry (FCM). (4)Chick chorioallantoic membrane (CAM) test was used to testify the effect of PTEN gene on angiogenesis.
Results
1 The expression levels of PTEN, VEGF, FLT1 mRNA in myeloid leukemia and its relationship.
1.1 PTEN mRNA expression levels in the newly diagnosed acute myeloid leukemia (AML) patients (expression level were 0.051±0.059) were higher than that in AML-CR patients (1.015±0.411) and NC group (1.059±0.595), (P<0.05). In CML-BC patients, the PTEN mRNA expression levels (0.022±0.021) were lower than that in CML-CP patients (1.134±1.124) and in NC, (P<0.01).
1.2 VEGF mRNA expression levels in the newly diagnosed AML patients (expression levels were 0.442±0.192) were higher than those in AML-CR patients (0.075±0.042) and NC group(0.059±0.039), (P<0.01). In CML-BC patients (0.512±0.322) the VEGF mRNA expression levels were higher than CML-CP patients (0.219±0.131) and NC, (P<0.01).
1.3 VEGFR1 (FLT1) mRNA expression levels in the newly diagnosed AML patients (expression levels were 1.569±0.694) were higher than those in AML-CR patients (0.375±0.241) and NC group(0.402±0.238), (P<0.01). In CML-BC patients (1.579±0.623) the VEGF mRNA expression levels were higher than those in CML-CP patients (0.598±0.311) and NC, (P<0.01).
1.4 Pearson correlation analysis showed PTEN mRNA expression levels had negative correlation with VEGF and FLT1 mRNA expression levels, and the correlation coefficient were r=-0.631, (P<0.05) and r=-0.522, (P<0.05) respectively.
2 The effect of wild-type PTEN gene transfection on invasion activity of K562 cells and the regulation of p-Akt, VEGF and VEGFR1 (FLT1)
2.1 The Ad-GFP or Ad-PTEN-GFP transfection efficiency was about 81.2±5.4% in K562 cell line on the third day detected by flow cytometry with multiple of infection (MOI) of 200.
2.2When K562 cells were transfected with Ad-GFP or Ad-PTEN-GFP for 72 hours at different MOI (MOI=50, 100, 200 and 400), PTEN mRNA expression level increased while the VEGF and FLT1 mRNA expression levels decreased. The expression levels of PTEN mRNA and VEGF mRNA or FLT1 mRNA showed negative correlation, and the correlation coefficient were r=-0.903, (P<0.05) and r=-0.900, (P<0.05) respectively. VEGF proteins were also decreased. After transfection with Ad-GFP or Ad-PTEN-GFP for 3 days, compared with Ad-GFP group, in Ad-PTEN-GFP group, PTEN mRNA expression levels increased 41.01 fold but VEGF and FLT1 mRNA expression levels decreased 4.80 and 4.68 fold. The expression level of VEGF protein decreased 2.21 fold detected by ELISA method.
2.3 After transfection with Ad-PTEN-GFP at MOI=200 for 3 days, Akt expression levels in K562 cells had no change, but p-Akt expression level (0.021±0.011) decreased compared with that in Ad-GFP group (0.587±0.078) and in untransfected group (0.547±0.061), P<0.01.
2.4 After transfection with Ad- GFP or Ad-PTEN-GFP at MOI=200 for 48 hours, transwell invasion test results showed that the number of fluorescent staining cells, that were adhered to the bottom of the upper chamber of transwell, was 50±11 in Ad-GFP group, which was higher than the number of cells in Ad-PTEN-GPF group (24±6). These results indicated that the invasion or migration ability of K562 cells was suppressed when the cells were transfected with Ad-PTEN-GFP.
3 The anti-proliferation and apoptosis induction effect of PTEN gene on human umbilical vein endothelial cell (HUVEC) line ECV304 cells.
3.1 After ECV304 cells were transfected with Ad-PTEN-GFP at MOI=100, the maximum proliferation inhibition rate was 50.38±4.15% at the fifth day of transfection, which was higher than that in Ad-GFP group (4.28±0.73), P<0.01.
3.2 The apoptosis rate in Ad-PTEN-GFP group was 18.56±2.11, which was higher than that in Ad-GFP group (1.34±0.32), P<0.01, after ECV304 cells transfected at MOI=100 for 5 days.
4 PTEN gene significantly suppressed blood vessels’formation in chick embryo choriallantoic membrane (CAM). In control group and Ad-GFP group, blood vessels surrounding gelatin sponge grew well, it was tree-like and well-branched. While avascular area surrounding gelatin sponge could be observed in Ad-PTEN-GFP group, its blood vessels were less than control group. In Ad-GFP and Ad-PTEN-GFP group, the blood vessel index was 92.2±4.37% and 47.6±3.37% respectively P<0.05.
Conclusions
1 PTEN gene expressed at low level in the newly diagnosed AML patients and its expression level was negatively correlated with the expression level of VEGF and FLT1 gene.
2 PTEN gene inhibited the invasion ability of K562 cells and had anti-angiogenesis effect by inhibiting PI3K/Akt signaling transduction pathway and down-regulated the expression of VEGF and FLT1 genes. PTEN gene may be a candidate of effective anti-leukemia gene.
引文
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