Shen Hui-Ling, Chen Zi-Xing, Wang Wei, Cen Jian-Nong, Hu Shao-Yan, Zhao Ye
Jiangsu Institute of Hematology, The First Hospital Affiliated to Suzhou University, Suzhou 215006, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2005 Dec;13(6):989-95.
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.
为研究外源性WT1基因亚型对白血病细胞系NB4凋亡的潜在影响及其可能的分子机制，采用电穿孔法分别将含全长人WT1亚型（WTA：-17aa/-KTS）cDNA的真核表达重组载体（pCB6(+)/WTA）和空载载体导入白血病细胞系NB4。通过RT-PCR和Western blot检测细胞中的WTA mRNA和蛋白。采用流式细胞术和琼脂糖凝胶电泳检测Annexin V结合情况，以验证外源性WTA是否能诱导NB4细胞凋亡。将重组载体导入NB4细胞后，通过半定量RT-PCR检测p21、p53、bcl-2、bcl-XL和c-myc基因的表达。结果显示，在0.8 μmol/L三氧化二砷作用48小时后，NB4/WTA细胞呈现凋亡的形态学特征，凝胶电泳显示明显的DNA梯状条带，Annexin V标记的凋亡率升高。RT-PCR显示NB4/WTA细胞中p21和c-myc基因表达增加，bcl-2表达降低，p53、bcl-XL表达相对恒定。结论是，外源性WTA基因的导入和表达可通过下调Bcl-2基因表达、上调p21和c-myc基因表达导致NB4/WTA细胞凋亡。
