The effect of on VEGF-C cDNA transfection on NB4 cell proliferation, differentiation and resistance to apoptosis.
- Author:
Kai-yang DING
1
;
Xia BAI
;
Lan DAI
;
Ning-zheng DONG
;
Chang-geng RUAN
Author Information
- Publication Type:Journal Article
- MeSH: Apoptosis; drug effects; genetics; physiology; Blotting, Western; CCAAT-Enhancer-Binding Protein-alpha; genetics; metabolism; CD11b Antigen; genetics; metabolism; Cell Differentiation; genetics; physiology; Cell Line, Tumor; Cell Proliferation; DNA, Complementary; genetics; Drug Resistance, Neoplasm; Flow Cytometry; Genetic Vectors; Humans; Leukemia, Promyelocytic, Acute; genetics; metabolism; pathology; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tretinoin; pharmacology; Vascular Endothelial Growth Factor C; genetics; metabolism; physiology
- From: Chinese Journal of Hematology 2006;27(4):244-248
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSA 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.
CONCLUSIONThe 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.
