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


CONCLUSIONS
Targeted activation of PKR could inhibit the proliferation of K562 cells through inhibiting protein synthesis, and arresting progression of cell cycle.
Zhao
Shi-Qiao
SQ
Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing, 400016, P. R. China.
Feng
Wen-Li
WL
Zeng
Jian-Ming
JM
Bai
Wei-Jun
WJ
Wang
Xiao-Zhong
XZ
Wen
Jian-Ping
JP
Cao
Wei-Xi
WX
Luo
Yun-Ping
YP
Huang
Zong-Gan
ZG
D015075:2-Aminopurine / Q000494:pharmacology; D002453:Cell Cycle; D049109:Cell Proliferation; D004789:Enzyme Activation; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D011070:Poly I-C / Q000494:pharmacology*; D016372:RNA, Antisense / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D019892:eIF-2 Kinase / Q000378:metabolism*
D016372:RNA, Antisense; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D015075:2-Aminopurine; D016044:Fusion Proteins, bcr-abl; D019892:eIF-2 Kinase; D011070:Poly I-C