RESUMO
Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destruction. However, how pathogens respond to irradiation stress has not yet been fully elucidated. Here, we observed that Pseudomonas aeruginosa PAO1 could release nucleic acids into the extracellular environment under X-ray irradiation. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), X-ray irradiation was observed to induce outer membrane vesicle (OMV) formation in P. aeruginosa PAO1. The size distribution of the OMVs of the irradiated PAO1 was similar to that of the OMVs of the non-irradiated PAO1 according to nanoparticle tracking analysis (NTA). The pyocin-related proteins are involved in OMV production in P. aeruginosa PAO1 under X-ray irradiation conditions, and that this is regulated by the key SOS gene recA. The OMV production was significantly impaired in the irradiated PAO1 Δlys mutant, suggesting that Lys endolysin is associated with OMV production in P. aeruginosa PAO1 upon irradiation stress. Meanwhile, no significant difference in OMV production was observed between PAO1 lacking the pqsR, lasR, or rhlR genes and the parent strain, demonstrating that the irradiation-induced OMV biosynthesis of P. aeruginosa was independent of the Pseudomonas quinolone signal (PQS).
RESUMO
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.
Assuntos
Morte Celular , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , eIF-2 Quinase/metabolismo , Apoptose , Caspases/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo , eIF-2 Quinase/genéticaRESUMO
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.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , RNA/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica , Humanos , Células K562 , TraduçãoRESUMO
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.
Assuntos
Ciclo Celular , Proliferação de Células , Ativação Enzimática , Poli I-C/farmacologia , eIF-2 Quinase/metabolismo , 2-Aminopurina/farmacologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Vetores Genéticos , Humanos , Células K562 , RNA Antissenso/farmacologia , RNA de Cadeia Dupla/farmacologia , RNA Mensageiro/metabolismo , Retroviridae/genética , TransfecçãoRESUMO
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.