Effects of pharmacologic inhibitors on BCR/ABL-dependent expression of HIF-1α mRNA and HIF-1α transcriptional activity.

(A) Ton.B210-X cells were starved or grown in the presence of doxycycline and were incubated with the MEK-inhibitor PD98059 (25 μM), the PI3-kinase inhibitor LY294002 (20 μM), DMSO, or control medium for 16 hours. A densitometric evaluation of HIF-1α mRNA is shown below. (B) The effect of rapamycin (20 nM) on BCR/ABL-dependent expression of HIF-1α mRNA in Ton.B210-X cells is shown. Northern blotting was performed using a murine HIF-1α probe. After reprobing for β-actin, autoradiograms were subjected to densitometry, the results of which are shown below the blot analyses. (C) Effect of inhibitors on BCR/ABL-induced HRE-promoter activity. Ton.B210-X cells were starved or induced to express BCR/ABL and were incubated with inhibitors as indicated (50 μM PD98059; 20 μM LY294002; 20 nM rapamycin). After 48 hours, cells were assayed for luciferase and βGal activities. HRE transcriptional activity was reported as the ratio HRE-Luc/pCMV-βGal and was expressed as percentage of control (starved cells). Results represent the means ± SD of 3 independent experiments.