Background & objective: The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor, and is highly expressed in leukemia cells and negatively correlated with the prognosis of leukemia. WT1 mRNA undergoes alternative splicing at 2 sites resulting in 4 isoforms. The mRNA splice isoforms are constantly expressed in all tissues expressing WT1 with fixed proportions. The functional significance of WT1 isoforms expression in leukemia is unclear. This study was designed to explore the potential effects of exogenous WT1 gene isoforms on proliferation of leukemia cell line NB4 and its possible molecular mechanisms.
Methods: The recombinant eukaryotic expression vector pCB6+/WTA containing full-length human WT1 isoform WTA (-17AA/-KTS) cDNA, and pCB6+ vector with neomycin resistance gene were transfected into NB4 cells by electroperforation. The positive cell clones (NB4/WTA) were selected with G418. The transfected cells were monocloned by limited dilution. The expressing of WTA mRNA and protein in transfected cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The proliferating ability of leukemia cells was measured by trypan blue exclusion assay, MTT assay, colony formation assay and cell cycle analysis. The expression of p53, p21, Cyclin E, Cyclin D1 and Cyclin A1 was detected by semi-quantitative RT-PCR.
Results: WT1 gene isoforms were transfected into NB4 cells, and the cell line stably expressing exogenous WT1 gene isoforms was established successfully. The monoclonal cells overexpressing WT1 were obtained. The growth of NB4/WTA cells was slower than that of untransfected NB4 cells and NB4 cells transfected with pCB6+ vector (NB4/CMV): the growth of NB4/WTA cells reached plate phase [(78.7+/-18.0 x 10(4)/ml] at the 3rd day, while that of control cells reached plate phase [(146.0+/-21.0) x 10(4)/ml] at the 4th day. The inhibitory rates of NB4/WTA cells at different time points were significantly higher than those of control NB4 cells (P<0.005). The colony forming efficiency of NB4/WTA cells decreased markedly and became much lower when exposed to 0.2 micromol/L As2O3. The proportion of S phase cells was increased in NB4/WTA group. The expression of p21 and Cyclin A1 was higher in NB4/WTA cells than in control cells.
Conclusions: Exogenous WTA gene could inhibit the proliferation and colony forming ability of leukemia cells through up-regulating the expression of p21 and Cyclin A1 and arresting cell cycle at S phase.