[Experimental study on apoptosis of leukemia cell line NB4 transfected with WT1 gene]

In order to study the potential effects of exogenous WT1 gene isoform on apoptosis in leukemia cell line NB4 and its possible molecular mechanisms, the eukaryotic expression recombinant vector (pCB6(+)/WTA) containing full-length human WT1 isoform (WTA: -17aa/-KTS) cDNA and the vacant vector-alone were introduced into the leukemia cell line NB4 respectively by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Binding of Annexin V were tested by flow cytometry and agarose gel electrophoresis to verify whether exogenous WTA could induce apoptosis of NB4 cells. Expressions of p21, p53, bcl-2, bcl-XL and c-myc genes were determined by semi-quantitative RT-PCR after introducing recombinant vectors into the NB4 cells. The results showed that in exposure to As(2)O(3) at 0.8 micromol/L for 48 hours, the NB4/WTA cells exhibited the morphological hallmarks of apoptosis, the marked DNA ladder shown by gel electrophoresis, and the enhanced apoptosis rate marked by Annexin V. RT-PCR showed an increase in p21 and c-myc genes expression, a decrease in bcl-2 and a relative constant expression of p53, bcl-XL in NB4/WTA cells. It is concluded that the introduction and expression of exogenous WTA gene can lead to apoptosis of NB4/WTA cells by down-regulating the Bcl-2 gene expression and up-regulating the p21 and c-myc genes expression.