Lemur tyrosine kinase 2 silencing inhibits the proliferation of gastric cancer cells by regulating GSK-3ß phosphorylation and ß-catenin nuclear translocation.

Previous studies on the mechanism of proliferation and cell cycle progression of gastric cancer cells have shown promising perspectives for the prevention and treatment of gastric cancer. The aim of the present study was to investigate the role of lemur tyrosine kinase 2 (LMTK2) in gastric cancer cell proliferation and cell cycle progression, as well as in tumor-bearing nude mouse models. The expression levels of LMTK2 were determined in gastric cancer cell lines. In addition, the effects of LMTK2 silencing or overexpression on cell proliferation were measured using Cell Counting Kit-8, BrdU and colony formation assays. Cell cycle progression was analyzed using flow cytometry and western blotting. The expression levels of proteins associated with the ß-catenin pathway were assessed using western blot analysis. A tumor-bearing nude mouse model was established by injecting gastric cancer cells, and the effect of LMTK2 knockdown or overexpression on tumor growth was examined. The expression levels of LMTK2 were found to be upregulated in all gastric cancer cell lines. Moreover, LMTK2 knockdown inhibited cell proliferation, colony formation and cell cycle progression. LMTK2 knockdown also inhibited the activation of GSK-3ß/ß-catenin signaling, as evidenced by reduced GSK-3ß phosphorylation and nuclear ß-catenin levels. LMTK2 knockdown also suppressed tumor growth, whereas overexpression accelerated this process. In conclusion, LMTK2 silencing can inhibit the proliferation of gastric cancer cells in vitro and tumor growth in vivo by regulating GSK-3ß phosphorylation and ß-catenin nuclear translocation.

Selective leukemia cell death by activation of the double-stranded RNA-dependent protein kinase PKR.

Deregulated activity of the BCR-ABL tyrosine kinase encoded by the Bcr-Abl oncogene represents an important therapeutic target for all the chronic myelogenous leukemia (CML) phases. In this study, we sought to identify targeted PKR activation by Bcr-Abl AS RNA, an anti-sense RNA complementary to the unique mRNA fragments flanking the fusion point of Bcr-Abl, which can be used as an effective anti-leukemia strategy in K562 cells. Moreover, we observed expression of Bcr-Abl AS RNA in K562 cells which resulted in selective apoptosis induction through specific activation of PKR, leading to phosphorylation of eIF2α, global inhibition of protein synthesis, caspase-8 activation and BAX up-regulation. The targeted PKR activation and induced apoptosis were reversed by the PKR inhibitor 2-aminopurine. Taken together, our results indicate that targeted PKR activation led to selective apoptosis induction in K562 cells, which correlated with caspase-8 activity and enhanced expression of BAX.

[Targeted blockage of RNA binding protein E2 by decoy RNA induces the granulocytic differentiation of K562 cells].

OBJECTIVE: To use a decoy RNA targeted blockage of the RNA binding protein E2 (hnRNP E2) resulting in the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene's abnormal translation and investigate its effect on the granulocytic differentiation of K562 cells and the probable molecular mechanism. METHODS: The hnRNP E2 decoy RNA expression plasmid was constructed and transfected into K562 cells with cationic liposome, and stable expression cells were obtained by G418 selection. The changes of C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR) gene expression were detected by RT-PCR and Western blot. The morphologic changes were observed after Wright-Giemsa staining. The expression of granulocytic differentiation antigens CD13 and CD15 was studied by immunocytochemistry. RESULTS: The stably expressed pG cells were obtained. Its C/EBP alpha mRNA level remained unchanged, while 42kD-C/EBP alpha protein expression was increased by (49.7 +/- 5.5)% (P < 0.05); and G-CSFR mRNA was increased by (42.1 +/- 3.6)% (P < .05), and its protein was increased by (37.4 +/- 6.2)% (P < 0.05) compared to that in the K562 control cells. The characteristics of polymorphonuclear neutrophils appeared in pG cells and CD13 and CD15 positive cell ratios were (18.7 +/- 2.5)% and (26.3 +/- 2.9)% respectively. CONCLUSIONS: HnRNP E2 decoy RNA can induce granulocytic differentiation of K562 cells, and G-CSF promotes this effect. The mechanisms may be that decoy RNA specifically blocks hnRNP E2, hence regulates the translation of C/ EBP alpha mRNA, restores the expression of 42kD-C/EBP alpha, and then up-regulates the expression of G-CSFR gene.

[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.

[Effect and possible mechanism of HnRNP E2 decoy RNA on proliferation of K562 leukemia cells].

BACKGROUND & OBJECTIVE: The abnormal expression of poly(rC)-binding protein E2 (hnRNP E2) induced by BCR/ABL plays an important role in blast crisis of chronic myeloid leukemia (CML). The present study was to investigate the effect of hnRNP E2 decoy RNA on the cell proliferation in K562 leukemia cells, and further elucidate the possible underlying mechanisms. METHODS: Decoy hnRNP E2 plasmid was constructed and transfected into K562 cells using cationic liposome. Stably transfected cells were selected with G418. The cell proliferation rate was determined by cell growth curve using trypan blue staining, and the cell cycle was analyzed by flow cytometry. The changes of CCAAT/enhancer-binding protein alpha (C/EBP alpha) and c-Myc gene expression were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: The proliferation rate of stably transfected K562 cells was inhibited by (62.73+/-12.92)%. The cell cycle was arrested at S phase [stably transfected K562 cell group: (55.59+/-4.67)%, control group: (44.70+/-4.21)%, P<0.05]; C/EBPalpha mRNA level remained unchanged. However the 42 ku-C/EBPalpha protein expression was elevated by (49.72+/-5.58)%; c-Myc mRNA and protein expression was inhibited by (58.27+/-7.23)% and (57.26+/-6.52)%, respectively. CONCLUSION: HnRNP E2 decoy RNA could inhibit the proliferation of K562 cells, and this may be caused by the blockage of the binding between hnRNP E2 and C/EBPalpha mRNA and subsequent elevation of 42 ku-C/EBPalpha by decoy RNA.