[The effect of on VEGF-C cDNA transfection on NB4 cell proliferation, differentiation and resistance to apoptosis].
OBJECTIVE
To explore the biological effect on NB4 cells proliferation, all-trans retinoic acid (ATRA) inducing differentiation and resistance to apoptosis by vascular endothelial growth factor (VEGF)-C cDNA transfection.


METHODS
The recombinant eukaryotic expression plasmid pcDNA3.1-VEGF-C and the vacant pcDNA3.1 vector were introduced separately into NB4 cells by lipofectamine mediation. The positive clones were screened by G418 and identified by reverse transcriptase-PCR (RT-PCR) and Western blotting. The proliferation capacity of NB4/VEGF-C cells was analysed by MTT assay and colony forming assay in vitro. After NB4/VEGF-C cells were induced by ATRA, the expression level of C/EBPalpha gene, CD11b on cells surface and morphological alteration were analysed by real-time quantitative PCR (RQ-PCR), flow cytometry (FCM), and Wright-Giemsa staining, respectively. FCM Annexin V-FITC/PI dual labeling technique was performed to investigate the etoposide (Vp16) induced NB4/VEGF-C cells apoptosis and bcl-2 gene expression level in these cells was analysed by RQ-PCR. The NB4/pcDNA3.1 cells was used as control in the above experiments.


RESULTS
A stable NB4 cell line that secrets VEGF-C and its control lines were established. The proliferation capacity of the former was stronger than that of the latter. The expression level of C/EBPalpha gene of NB4/VEGF-C cells on ATRA treatment was only 1/32 that of NB4/pcDNA3.1 cells. The CD11b level and the degree of differentiation of NB4/VEGF-C were weaker than that of NB4/pcDNA3.1 cells. The percentage of apoptotic NB4/VEGF-C cells induced by Vp16 [(7.20 +/- 2.52)%] was significantly lower than that of NB4/pcDNA3.1 cells [(16.07 +/- 3.58)%] (P = 0.005), but the bcl-2 gene expression level of NB4/VEGF-C cells is 2.28-fold that of NB4/pcDNA3.1 cells.


CONCLUSIONS
The VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of leukemic cells by autocrine pathway and inhibit the cell differentiation mediated by ATRA and chemotherapy-induced apoptosis. VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.
Ding
Kai-yang
KY
Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Bai
Xia
X
Dai
Lan
L
Dong
Ning-zheng
NZ
Ruan
Chang-geng
CG
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism; D039481:CD11b Antigen / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D039481:CD11b Antigen; D018076:DNA, Complementary; C509295:ITGAM protein, human; D042582:Vascular Endothelial Growth Factor C; D014212:Tretinoin