Study On The Biological Functions Of WT1 Gene Isoforms In Leukemia Cells

Objective: To establish leukemia cell lines which stably expressed exogenous WT1 gene by electroporation-mediated transfection of 4 full-length WT1 isoforms cDNA clones into the leukemia cell line NB4 using optimized parameters. The potential effects of exogenous WT1 gene isoforms on the cell biologic behaviors including proliferation, apoptosis and differentiation in leukemia cell line NB4 and its possible molecular mechanisms were then studied.Methods: The GFP-containing plasmid was transferred into leukemia cell lines K562 and NB4 in suspension by electroporation using differently experimental parameters such as the voltage, the electric capacity, the cell growth condition and the operation temperature. The transfection efficiencies were evaluated by flow cytometry (FCM) and fluorescent microscopy. The eukaryotic expression recombinant vectors (pCB6+/WTl) containing 4 clones of full-length human WT1 isoforms (WTA:-17aa/-KTS WTB:+17aa/-KTS WTC:-17aa/+KTS WTD:+17aa/+KTS) cDNA were introduced into the leukemia cell line NB4 by electroporation .The positive cell clones(NB4/WTl) were selected with G418. The integration of WT1 gene isoforms in NB4 cells were confirmed by PCR. The WT1 mRNA and protein in cells were detected by RT-PCR and western blotting. The transfected cells were subcloned by limited dilution. The proliferation ability of leukemia cells was measured by trypan blue exclusion assay, MTT assay , colony forming assay and cell cycles analysis. Cell morphological analysis, NBT reduction and the expression of CD11b antigen in NB4 cells were examined to access the cell differentiation. Binding of AnnexinV tested by flow cytometry and agarose gel electrophoresis were performed to verify whether exogenous WTA could induce apoptosis in NB4 cells. Expressions of PML/RAR RbAP46 P21 P53 Bc1-2, Bel-XL and C-myc genes were determined by semi-quantitative RT-PCR after recombinant vectors were introduced into the NB4 cells. Furthermore, cDNA microarray was used to explore the alteration of gene expression profiles when increasing WTA expression in leukemia cells.Results: (1)The maximal electroporation transfection rate was achieved 40.6% and 32.9% for these two cell lines, respectively, under the condition of voltage 250V or 350V ,electric capacity 950F. The transfection efficiencies were closely related to the vigorous state of the cells. The operation temperature and the concentration of serum in buffer have no influence on the transfection efficiencies. (2)WT1 gene isoforms have been transferred into NB4 cells and the cell lines stably overexpressing exogenous WT1 gene isoforms were established successfully. WT1 mRNAand protein expression in the G418-selected cells increased remarkably when compared with the control cells transfected with the plasmid pCB6+. The monoclonal cell strains that overexpressed WT1 mRNA and protein were thus obtained. (3)The cells with overexpression of WTA had a significant decrease in proliferation rate measured by growth curves of the cells, and the colony forming ability was lower compared with NB4 and NB4 cells transfected with pCB6+ vector. The growth of cells transfected with WTA gene was rapid in 3 days, reaching (78.7 18.0) 104/ml, in comparison with the 146.0 21.0 104/ml for the control cells on the 4th day. The colony forming efficiency of NB4/WTA cells decreased markedly and became much lower when exposed to As2O3 at 0.2M. The cell cycle analysis found that NB4/ WTA cells arrested in S stage increased.(4) NB4/WTA cells treated with ATRA 0.5M for 2 days were induce to partially differentiate. Compared with NB4/WTA cells, NB4 and pCB6+ vector transfected NB4 cells had a higher morphological differentiation rate and higher expression level of CD11b antigen by flow cytometry in presence of ATRA (0.5M) for 48 hours . The percentage of NBT reduction in NB4/WTA cells was lower than that of control groups in absence of drugs. The differences in NBT reduction rate between NB4/WTA and control cells treated with ATRA 0.5M gradually increased over three days. (5)Exposure to As2O3 at...