靶向激活蛋白激酶PKR对白血病K562细胞增殖的影响及机制

背景与目的:由t(9;22)(q34;q11)导致的bcr-abl融合基因在慢性粒细胞白血病(chronic myeloid leukemia,CML)发病中起着重要的作用.本研究运用CML特异的bcr/abl融合基因的mRNA与外源重组反义RNA形成双链RNA (double strand RNA,dsRNA)能激活双链RNA依赖性蛋白激酶(double-stranded RNA-dependent protein kinase,PKR)的策略,研究其对白血病K562细胞增殖的影响及可能的机制.方法:将dsRNA类似物聚肌苷酸-聚胞啶酸(Polyriboinosinic Polyribocytidylic Acid,polyIC)、含ber/abl融合基因序列40bp的逆转录病毒载体RV-40AS、RV-40AS 2-氨基嘌呤(2-aminopurine,2AP)和含绿色荧光蛋白(green fluorescent protein,GFP)的逆转录病毒载体RV-GFP作用于K562细胞,并以ECV304细胞作对照细胞株.通过细胞计数、MTT法和半固体集落形成实验检测其对细胞生长增殖的影响,用流式细胞仪检测处理前后细胞周期的变化,用Western blot法检测细胞内PKR、磷酸化PKR(phosphated PKR,p-PKR)、真核翻译启始因子2α(eukaryotic initiation factor-2α,eIF2α)、磷酸化eIF2α(phosphated eIF2α,p-eIF2α)蛋白表达的变化,用3H-亮氨酸掺入实验检测细胞总蛋白合成水平的变化.结果:polyIC对K562细胞和ECV304细胞生长和增殖均具有非特异性抑制作用.而RV-40AS仅对K562细胞具有特异性的抑制效应.PKR抑制剂能阻断RV-40AS对K562 细胞的抑制效应.polyIC和RV-40AS作用K562细胞24 h 后,S期细胞减少[polyIC组(37.26±2.35)%,未处理组(58.53±5.42)%,P＜0.05;RV-40AS组(31.48±3.65)%,未处理组(58.53±5.42)%,P＜0.05],G0/G1期细胞增多[pdylC组(50.97±2.18)%,未处理组(36.44±4.20)%,P＜0.05;RV-40AS组(57.47±3.61)%, 未处理组(36.44±4.20)%,P＜0.05].polyIC处理K562细胞组、ECV304细胞组以及RV-40AS处理K562细胞组的p-PKR和p-eIF2α蛋白表达显著上调,且总蛋白合成水平下降[RV-40AS处理的K562细胞组(3.5±1.9)cpm/ng,未处理组(26.8±2.6)cpm/ng,P＜0.05].结论:外源重组反义RNA与bcr/abl融合基因的mRNA形成的dsRNA可通过激活PKR而抑制K562细胞的生长增殖,其机制是通过活化的PKR使蛋白合成启始因子eIF2a磷酸化,从而阻断细胞内蛋白合成的启动,及阻止细胞周期进程来实现.

Effect of targeted activation of protein kinase PKR on proliferation of leukemia cell line K562 and its mechanism

BACKGROUND & OBJECTIVE: The bcr-abl fusion gene induced by reciprocal translocation of t(9; 22)(q34; q11) plays an important role in pathogenesis of chronic myeloid leukemia (CML). Using the strategy of activating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) by the dsRNA formed between the CML-specific bcr/abl fusion gene mRNA and the exogenous recombinant antisense RNA, this study was to investigate the effect of the activated PKR on the proliferation of leukemia cell line K562, and explore its possible mechanisms. METHODS: dsRNA analogue polyriboinosinic polyribocytidylic acid (PolyIC), retroviral vector containing 40 bp of bcr/abl fusion gene sequence (RV-40AS), RV-40AS and 2-aminopurine (2-AP), and retroviral vector containing green fluorescent protein sequence (RV-GFP) were transfected or infected into K562 cells respectively; ECV304 cells were used as control. Cell proliferation was determined by cell counting, MTT assay, and semisolid clone formation experiment. Cell cycle was analyzed by flow cytometry (FCM). The expression of PKR, phosphated PKR (p-PKR), eukaryotic initiation factor-2alpha (eIF2alpha), and phosphated eIF2alpha (p-eIF2alpha) was detected by Western blot. Total protein synthesis was studied by 3H-leucine incorporation. RESULTS: polyIC inhibited the proliferation of K562 cells and ECV304 cells unspecifically, while RV-40AS only inhibited the proliferation of K562 cells specifically. 2-AP blocked the inhibitory effect of RV-40AS on the proliferation of K562 cells. The S phase proportion was significantly lower in polyIC-and RV-40AS-treated K562 cells than in untreated cells [(37.26+/-2.35)% and (31.48+/-3.65)% vs. (58.53+/-5.42)%, P<0.05], while the G0/G1 phase proportion was significantly higher in polyIC-and RV-40AS-treated cells than in untreated cells [(50.97+/-2.18)% and (57.47+/-3.61)% vs. (36.44+/-4.20)%, P<0.05]. The expression of p-PKR and p-eIF2alpha in polyIC-and RV-40AS-treated K562 cells and polyIC-treated ECV304 cells was obviously up-regulated. The total protein synthesis level was significantly lower in RV-40AS-treated K562 cells than in untreated K562 cells [(3.5+/-1.9) cpm/ng vs. (26.8+/-2.6) cpm/ng, P<0.05]. CONCLUSION: Targeted activation of PKR could inhibit the proliferation of K562 cells through inhibiting protein synthesis, and arresting progression of cell cycle.