Angiogenesis And Anti-angiogenic Gene Therapy In Leukemia And Solid Tumors

Objective: To explore the gene coexpression pattern of VEGF and its receptors (Flt-1 and KDR) in variety of human malignant cell lines. Methods: After total RNA was isolated from thirty tumor cell lines and four endothelial cells, gene expression of VEGF and its receptors was measured by semi-quantitative reverse transcriptase polymerase chain reaction using the transcriptinal level of the house-keeping gene for internal calibration. Results: VEGF transcript was detected in majority of tumor cell lines (29/30) and in all endothelial cell lines at moderate or high levels, while the expression of VEGF was low in human umbilical vein endothelial cell (HUVEC). Moreover, Flt-1 gene expression was found in 50% of hematopoietic malignancies (6/12), 28% of solid tumors (5/18), and endothelial cells (EA.hy926 and HUVEC). In contrast, the expression of KDR gene was found in 2 (16.7%) hematopoietic cell lines and 6 (33.3%) solid tumor lines. In endothelial cells, KDR gene expression was detectable in HMEC-1 and HUVEC only. Importantly, ECV304 cells express neither Flt-1 nor KDR gene. Conclusion: Overexpression of VEGF gene in all tumor cell lines tested provides a potential biological marker for malignancies and the coexpression of VEGF with its receptors suggesting an autocrine pathway in tumor cells.2 Quantitative analysis of the expression of vascular endothelial growth factor gene during all-trans retinoic acid-induced differentiation of NB4 leukemia cellsObjective: To assess the regulation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 acute promyelocytic leukemia cell line. Methods: Using gene recombinant technologies, a competitor DNA fragment, known as VEGF gene mimic, was constructed, and a competitive quantitative RT-PCR (cQRT-PCR) method to monitor the alternation of VEGF gene expression levels after ATRA treatment was developed. For construction of standard curve from which the amount of target cDNA was derived, serial dilutions of the target were coamplified with constant amount of mimic using VEGF gene-specific primers, and the intensity of bands corresponding to the target and mimic weremeasured. The expression of surface antigen CD11b and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. Results: It was showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression, with a detectable range from lxlO4 to 2xlO5 molecules. The number of VEGF gene transcripts, detected by means of cQRT-PCR assay, was 42.3x105, 12.6xl05, 3.6x105, and less than l.0x105 per microgram of NB4 total cellular RNA at 0, 12, 24, or 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the upregulation of CD11b expression and an increased NBT reduction rate. Conclusion: ATRA efficiently represses VEGF expression and should exert an antileukemic effect, apart from induction of differentiation, via inhibition of angiogenesis.