Effects of rapamycin on BCR/ABL-induced VEGF gene expression.

(A) Ton.B210-X cells induced to express BCR/ABL were treated with rapamycin (20 nM) (BCR/ABL + rapamycin) or control medium (BCR/ABL) for 24 hours and then subjected to Northern blot analysis using a murine VEGF probe. (B) Primary peripheral blood mononuclear cells obtained from a patient with BCR/ABL⁺ CML were cultured in the presence (rapamycin) or absence (Control) of rapamycin (10 nM) for 12 hours. Cells were subjected to Northern blot analysis using a human VEGF cDNA probe. The β-actin loading control and densitometric quantifications of VEGF mRNA expression are shown below the blot analyses. (C) VEGF reporter gene activity was measured in Ton.B210-X cells. Cells induced to express BCR/ABL were incubated with rapamycin (20 nM) or control medium for 24 hours. Induced and starved Ton.B210-X cells (not expressing BCR/ABL) were examined for VEGF reporter gene activity. Results represent the means ± SD of 3 independent experiments. (D) Ton.B210-X cells induced to express BCR/ABL were cultured in the presence (BCR/ABL + rapamycin) or absence (BCR/ABL) of rapamycin (20 nM) for 36 hours. Starved Ton.B210-X cells were also examined. After incubation, cell-free supernatants were collected and examined for secreted VEGF by ELISA. Results represent the means ± SD of 3 independent experiments.