VEGF induces Hsp90 expression on HL-60 cells and primary leukemias.

(A) VEGF induces Hsp90 expression on HL-60 cells. Results of Western blot analysis of Hsp90 expression level on HL-60 cells are shown. Protein extracts were obtained after 3, 6, 12, and 24 hours incubation of serum-starved HL-60 cells in the absence or presence of VEGF. Western blotting results were quantified by densitometry and are shown as the ratio between densitometry values in treated versus untreated (control) cells. Each experiment was repeated 3 times, and the SD between the different ratios was calculated and is shown. (B) VEGF induces Hsp90 expression also on primary leukemias and 2 other leukemic cell lines. Total protein extracts were obtained after 12 hours of VEGF stimulation, and Hsp90 was detected by Western blotting, as above. (C) VEGF increases Hsp90 expression by interacting with KDR and activating the MAP kinase pathway. Protein extracts were obtained from HL-60 cells, which were cultured in serum-free RPMI for 3 hours (control), in presence of 50 ng/mL VEGF, VEGF plus 1 μg/mL KDR antibody, or 100 ng/mL (PLGF). To determine the molecular pathways by which VEGF induced Hsp90 expression, protein extracts were also incubated with an MAP kinase inhibitor (PD98059, used at 30 μM), wortmanin (PI3 kinase inhibitor, used at 30 nM), or LY294002 (PI3 kinase inhibitor, used at 3 μM). Western blotting results were quantified by densitometry and are shown as the ratio between densitometry values in treated versus untreated (control) cells. Each experiment was repeated 3 times, and the SD between the different ratios was calculated and is shown.