Effect of wild-type p53 gene on the number and proteins of centrosome in leukemic K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.</p><p><b>METHODS</b>The recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.</p><p><b>RESULTS</b>Infection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.</p><p><b>CONCLUSION</b>There is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.</p>

Targeted blockage of RNA binding protein E2 by decoy RNA induces the granulocytic differentiation of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Targeted blockage of STAT5 by a decoy oligodeoxynucleotide inhibits the growth and proliferation of K562 cells / 中华血液学杂志

<p><b>OBJECTIVES</b>To investigate targeted blockage of BCR/ABL oncoprotein mediated cell transformation by STAT5 decoy oligodeoxynucleotide (ODN), its effect on the growth and proliferation inhibition of K562 cells and the related molecular mechanisms.</p><p><b>METHODS</b>STAT5 decoy ODN, designed and synthesized in vitro, was transfected into K562 cells by cationic lipid. The cell growth curve and colony formation assay were used to reflect the growth and proliferation capacity of K562 cells, RT-PCR to detect the expression of three genes downstream STAT5.</p><p><b>RESULTS</b>Confocal microscopy demonstrated that STAT5 decoy ODN was successfully transfected into K562 cells (95.2% positive cells). STAT5 decoy ODN inhibited the growth of K562 cells (inhibition rate 77.7%) and their colony formation capacity (Decoy ODN treated group 8.3% vs control group 35.7%, P < 0.05) after the treatment with STAT5 decoy ODN, the expressions of c-myc, bcl-X(L), cyclin D1 mRNA were down-regulated by 15.4%, 30.8%, 29.1%, respectively in the K562 cells.</p><p><b>CONCLUSIONS</b>STAT5 decoy ODN inhibits the growth and proliferation of K562 cells. The mechanisms may be that decoy ODN blocks the transcriptional activation potent of STAT5 and down-regulates the expression of these tumor related genes downstream STAT5.</p>

VEGF antisense oligonucleotide inhibits the expression of vascular endothelial growth factor in human leukemic cell lines / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of vascular endothelial growth factor (VEGF) antisense phosphorothioated oligodeoxynucleotide (AS-ODN) on the expression of VEGF in human leukemic cell lines (HL-60 and K562 cells).</p><p><b>METHODS</b>The levels of VEGF mRNA and protein in leukemic cells incubated with VEGF AS-ODN were measured by RT-PCR, immunohistochemistry assay and ELISA. MTT test was used to examine the influence of the culture supernatant (CS) of VEGF AS-ODN treated leukemic cells on the proliferation of human umbilical vein endothelial cells (ECV304).</p><p><b>RESULTS</b>After leukemic cells were treated with different concentrations (2.5 approximately 15.0 micro mol/L) of VEGF AS-ODN for 24 h, VEGF mRNA level in the cells decreased remarkably in a concentration dependent manner, no change was found in the VEGF missense ODN treated cells (MS-ODN). When the leukemic cells were treated with 5 micro mol/L VEGF AS-ODN for 24 h, VEGF protein level decreased greatly both in the cells and in the CS; and the proliferation stimulating effect of the treated CS on the ECV304 cells reduced. Meanwhile, there was no obvious change in VEGF protein and its effect in the VEGF MS-ODN treated group.</p><p><b>CONCLUSION</b>VEGF AS-ODN could inhibit VEGF expression in human leukemic cell lines in vitro.</p>

Study on the transcriptional activation of MTS1 gene beta promoter / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of MTS1 gene beta promoter transcriptional activation in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and identify the fragment with transcriptional activation.</p><p><b>METHODS</b>Seven pGL3 recombinant plasmids with the same 3'-end transcriptional start site but the different 5'sequences were constructed by gene recombinant technique and transfected into Jurkat cell line which is biallelic deletion of MTS1 gene by transient transfection. Luciferase report gene was detected to observe beta promoter transcriptional activation.</p><p><b>RESULTS</b>Seven pGL3 recombinant plasmids containing different fragments of beta promoter were obtained, all of them showed transcriptional activation in Jurkat cell line. Among them, the 0.38 kb fragment cut by SacII-SacI is fundamental in transcription.</p><p><b>CONCLUSION</b>MTS1 gene beta promoter can be activated in Jurkat cell line.</p>

Quantification of bcr/abl mRNA in patients with chronic myeloid leukemia by using real-time quantitative fluorescence PCR with self-quenched primer / 中华检验医学杂志

Objective To establish a quantitative RT-PCR method with self-quenched fluorogenic probe for detection of bcr/abl mRNA in patients with chronic myeloid leukemia for providing a useful tool for diagnosis of CML,evaluation of therapeutic effect and monitoring of minimal residual disease(MRD). Methods bcr/abl gene from cultured K562 cells was amplified by conventional RT-PCR.The standard quantitative plasmid was constructed by A-T clone method.The self-quenched fluorogenic quantitative RT- PCR method(FQ-RT-PCR)for determination of bcr/abl mRNA was established successfully using the ABI PRISM 7000 PCR Detector.The linear range,sensitivity,stability,and repetitiveness of the method were determined.The marrow samples from 25 CML patients and 3 ALL patients were assessed.Results The sensitivity of the FQ-RT-PCR was 10 copies/?l recombined plasmid,and bcr/abl mRNA can be detected from 1 K562 cell in 10~5 normal cells.The linear range was 10~2-10~9 copies/?l recombined plasmid.The coefficient variation(CV)value was 2.1% in intra-assay and 6.1% in inter-assay.The median ber/abl mRNA expression level was 4.50?10~4 copies/?g RNA [(0.45-89.00)?10~4],5.45?10~4 copies/?g RNA [(2.95-19.30)?10~4 ],13.00?10~4 copies/?g RNA [(4.10-89.00)?10~4] and 2.35?10~4 copies/?g RNA [(0.45-5.12)?10~4] in 25 CML patients,11 patients in the incipient chronic phase,6 patients in blastic crisis,8 patients in chronic period after treatment,respectively.The bcr/abl mRNA level in blastic crisis was significantly higher than that in chronic phase(q= 3.41,P