[Experimental study on the role of VEGF autocrine loop in K562 leukemia cells].
OBJECTIVE
lo investigate the effects of anti-VEGF antibody and anti-VEGF hairpin ribozyme gene on the proliferation, apoptosis and related gene expression of the leukemia K562 cells and the possible molecular mechanisms.


METHODS
K562 cells were cultured in different concentrations of anti-VEGF antibody. The recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation. Cell proliferative capacity was determined by MTT, colony formation assay and cells cycles analysis. Cell apoptosis was assayed by DNA ladder and Annexin V -FITC/PI flow cytometry.


RESULTS
The anti-VEGF antibody was able to inhibit growth and induce apoptosis of K562 cells in a dose-dependent manner. Exposure to anti-VEGF antibody at 0. 165 microg/ml for 72 hours, the cells exhibited typical DNA ladders. The apoptosis rate peaked at antibody concentration of 0. 825 microg/ml. RT-PCR showed a decrease of MRP and TOPO II expression but a relative constant expression of GST. The introduction of exogenous anti-VEGF ribozyme gene resulted in a decrease of the proliferative capacity and colony forming efficiency from (30.5 +/- 3.3) % in control group to (16.3 +/- 2.8) % in K562/RZ group, higher G1 and lower S ratio in cell cycle distribution in comparison with the control groups. Typical DNA fragmentation and higher Annexin V + ratio occurred in K562/RZ cells after treated with 0.5 micromot/L of As2O3 for 3 days, the apoptosis rate increased from 13.4% in control group to 31. 5% in As2O3 treated group.


CONCLUSIONS
Anti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPO II genes of K562 cells in vitro. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of the cells by delaying the progression G1 into S phase in cell cycles and induce cell apoptosis by down-regulating VEGF gene expression.
Xu
Wen-Lin
WL
Department of Hematology, The Affiliated People's Hospital of Jiangsu University, Zhenjiang 212002, China.
Shen
Hui-Ling
HL
Yuan
Wei
W
Jiang
Yun-Wei
YW
Wu
Zhao-Yang
ZY
Fei
Xia
X
Tang
Hua-Rong
HR
D000906:Antibodies / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D008081:Liposomes; D016337:RNA, Catalytic / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D008081:Liposomes; D016337:RNA, Catalytic; D042461:Vascular Endothelial Growth Factor A; C418481:hairpin ribozyme