[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.
Shen
Hui-Ling
HL
Jiangsu Institute of Hematology, The First Hospital Affiliated to Suzhou University, Suzhou 215006, China.
Chen
Zi-Xing
ZX
Wang
Wei
W
Cen
Jian-Nong
JN
Hu
Shao-Yan
SY
Zhao
Ye
Y
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D025721:WT1 Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051020:bcl-X Protein / Q000235:genetics
C494811:BCL2L1 protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C489427:MYC protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D025721:WT1 Proteins; D051020:bcl-X Protein