The complexes were separated on a 5% polyacrylamide gel and exposed to an X-ray film for autoradiography. The sequence of the probe used here is 5′-CAG GGC TGG GGA TTC CCC ATC TCC ACA GTT TCA CTT-3′. Approximately 20,000 cpm of 32P-labeled DNA probe for NF-κB (p65) were added and reaction binding proceeded for 15 minutes. Aliquots of nuclear extracts were preincubated with 1 mg poly(deoxyinosinic-deoxycytidylic acid) in binding buffer for 10 minutes at room temperature. Nuclear extracts were prepared from HDMEC-LSXN, HDMEC-Bcl-2, or HDMEC exposed to 0 to 50 ng/mL VEGF for 24 hours or to 10 ng/mL TNF-α for 30 minutes. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.Įlectrophoretic mobility shift assay. Nuclear factor-κB (NF-κB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-κB down-regulated CXCL8 and CXCL1 expression levels. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-α (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. These results cannot be attributed to the role of Bcl-2 in cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2–transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. We initially attributed these results to Bcl-2–mediated endothelial cell survival. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |