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Objective: To analyze alterations in the gene expression profiles of Velcade-treated K562 cells using bioinformatics methods., Methods: The total RNAs of Velcade-treated and control K562 cells were amplified and labeled with fluorescent dyes. The labeled RNAs were hybridized to Agilent Human 1A Microarray, and the raw expression data were processed with Agilent Feature Extraction Software. GeneSifter and GATHER were used for data analysis of the differentially expressed genes to perform gene ontology classification, KEGG pathway analysis, functional protein association network construction and literature mining., Results: Totally 228 differentially expressed genes were identified in the Velcade-treated K562 cells. including 84 up-regulated and 144 down-regulated genes. Chymase 1 gene had the greatest down-regulation by 10.80 folds (log ratio), and interferon alpha-21 gene was also down-regulated by 2.31 folds. Gene ontology classification suggested enhanced aging and leukocyte activity. KEGG pathway analysis showed significant impact of Velcade on JAK-STAT signaling pathway, cytotoxicity mediated by natural killer cells, and antigen processing and presentation pathways. Protein-protein interaction analysis revealed that ubiquitin-dependent protein catabolism, antigen presentation and immune response, as well as JAK-STAT signaling pathway were the major elements of the protein network. Literature mining showed that the differentially expressed genes were strongly associated with terms such as leukemia, apoptosis, cell cycle, proteasome, inhibitor, aging and IkappaB, etc., Conclusions: Velcade may inhibit the cell survival pathways such as NF-kappaB and JAK-STAT signaling pathways to enhance the cytotoxicity and inducing tumor cell apoptosis. Velcade might also be involved in antigen processing and presentation, immune response and inflammation. Chymase 1 gene is probably the key target of Velcade.

Objective: To study the effect of antizyme 1 (ZA1) gene transfection on the cell proliferation, cell cycle and apoptosis of human neuroblastoma SH-SY5Y cells in vitro., Methods: The recombinant eukaryotic expression vector pAZ1m was constructed by cloning mutant AZ1 gene into the vector pEGFP-N1, and subsequently transfected in SH-SY5Y cells. The transfected cell proliferation was examined using MTT assay, and the changes in cell cycle and apoptosis were assayed using flow cytometry analysis. RT-PCR was performed to measure cyclin D1 and caspase-3 mRNA expressions, Western blotting carried out to examine cyclin D1 protein expression, and the changes in caspase-3 activity were detected using a caspase-3 detection kit., Results: AZ1 gene transfection significantly inhibited the proliferation of SH-SY5Y cells, causing cell cycle arrest at G0/G1 stage and down-regulated cyclin D1 and up-regulated caspase-3 expressions. Obviously increased caspase-3 activity was also observed in the transfected cells., Conclusion: Exogenous AZ1 gene transfection can inhibit the proliferation and cause cell cycle arrest of SH-SY5Y cells by down-regulating cyclin D1 expression. Up-regulated caspase-3 expression resulting from AZ1 gene transfection may induce apoptosis of the neuroblastoma cells.

Objective: To study the effect of the S-bioallethrin on human lymphocytes by microarray technique., Methods: The changes of normal human lymphocytes treated with S-bioallethrin were examined with light microscope, flow cytometry, electron microscope, DNA ladder and microarray techniques., Results: Morphological study showed that the lymphocytes underwent apoptosis after S-bioallethrin exposure, which as further confirmed by the expression changes of 346 genes., Conclusion: S-bioallethrin can induce apoptosis of normal human lymphocytes and changes in their gene expression profiles.

Objective: To investigate the mechanisms for the cytopathic effect (CPE) of human cytomegalovirus (HCMV) in ECV304 endothelial-like cells., Methods: PCR and indirect immunofluorescence were used to detect HCMV infection by examining immediate-early (IE) gene and protein expression of the virus in ECV304 cells. Phase-contrast and electron microscopies were performed to observe the morphological changes of the infected and uninfected cells, and DNA ladder analysis and flow cytometry were carried out to study HCMV-induced cell apoptosis., Results: In HCMV-infected ECV304 cells, cytopathic effects were first observed at approximately 72 h post-infection. The cells with CPE changes exhibited detachment from the monolayer, cell rounding and shrinkage. The expression of the IE gene was detected. Chromatin condensation and nuclear fragmentation along with dramatic changes of the mitochondria were observed by electron microscopy at 96 h post-infection. Cellular DNA fragmentation was observed in the infected cells, which had cells apoptotic rates of 4.1% and 45.7% at 96 h and 144 h post-infection, respectively., Conclusion: HCMV can induce apoptosis of ECV304 endothelial-like cells.

Objective: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells., Methods: siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting., Results: Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate., Conclusion: RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.

Objective: To study the feasibility of using MTT assay to detect cell apoptosis., Methods: K562 cells were grown in RPMI 1640 medium supplemented with 10% calf serum and 4 micromol/L arsenic trioxide. Apoptosis was induced in the cultured cells by As2O3, and the cells were detected with optical microscope, DNA gel electrophoresis and MTT staining respectively., Result: MTT staining could also accurately detect cell apoptosis, by which the apoptotic cells were easily distinguished from normal cells and dead cells., Conclusion: MTT staining is simple, convenient and practical for detecting cell apoptosis.