Effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib.</p><p><b>METHODS</b>Tumor suppressor gene p14(ARF) was transduced into K562 (K562-p14(ARF)) and 4 blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector with cells transduced by empty vector as control. Fluorescence microscopy and flow cytometry were applied to measure transduction efficiency, and Western blotting assay was used to detect p14(ARF) protein of K562 cells. WST-8 method was used to determine cell growth inhibition rate of K562 cells transduced by the target gene under different concentrations of imatinib (0, 0.015, 0.062, 0.125, 0.25, 0.5, 1.0, 2.0 μmol/L). Cell apoptosis and leukemic cellular colony-forming ability were detected by Annexin V-FITC/PI dyeing using flow cytometry (FCM) and semi-solid culture method respectively.</p><p><b>RESULTS</b>Fluorescence microscopy and FCM showed that transduction efficiency (GFP positive cells) of K562-p14(ARF), K562-VSV and CML-BC1 cells were close to 100%, and CML-BC 2-4 cells were 80% to 90% on average. Results of Western blotting showed that the levels of ARF protein expression of K562 cells transduced by p14(ARF) were significantly higher than of untransduced cells; the apoptosis rate of K562-p14(ARF) was 20%; the mean apoptosis rate of 4 primary leukemic cells transduced by the p14(ARF) [(71.1±22.4)%] was significantly higher than of control group [(12.4±6.2)%] (P<0.05). Imatinib significantly inhibited the proliferation of K562-p14(ARF) cells in a dose-dependent manner. The mean leukemic cellular colony-forming unit of 4 primary leukemic cells transduced by the p14(ARF) (41.5±13.2) was significantly lower than of the control group (88.5±7.9) (P<0.05).</p><p><b>CONCLUSION</b>Increased p14(ARF) gene expression could induce apoptosis of CML cells; Moreover, it could enhance inhibitory effect on cell proliferation when combined with imatinib.</p>

Influence of gene transduction mediated by lentivirus vector on gene expression of human CD34+ cord blood cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the influence of gene transduction mediated by lentivirus vector on human CD34+ cord blood cell (CBCs) gene expression.</p><p><b>METHODS</b>CD34+ cells were isolated and transduced with the third-generation self-inactivating ( SIN) lentiviral vector carrying green fluorescent protein (GFP). The total RNA from transduced cells was extracted and the differences of genotypes between the transduced and non-transduced CD34+ cells were determined with cDNA microarray analysis.</p><p><b>RESULTS</b>In 23000 genes two were upregulated and six downregulated. These changes were not confirmed by semi-quantitative RT-PCR method.</p><p><b>CONCLUSIONS</b>Lentiviral vector used in this study do not influence significantly on the gene expression of CD34+ CBCs, and the vector system may be a useful and safe one in clinical gene therapy.</p>