Figure 4.

Sensitisation of neuroblastoma cells to ABT-737 in hypoxia is variably dependent on HIF-1alpha. A. SH-EP1 cells transiently transfected with siRNA to HIF-1α show no protein expression of HIF-1α over a 64 hr timecourse in hypoxia, and no hypoxia-induced up-regulation of the known HIF-1 transcriptional targets CA IX and GLUT-1. Actin is shown as a loading control. Blots are representative of 3 independent experiments. B. Sensitisation to ABT-737 by 48 hr exposure to hypoxia is lost in SH-EP 1 cells transiently transfected with siRNA to HIF-1α. The mean IC50 to ABT-737 calculated from 3 separate SRB assays is shown in normoxia and hypoxia for wild type, non-target siRNA transfected, and HIF-1α siRNA transfected SH-EP1 cells. The difference between the IC50 in SRB in normoxia and hypoxia was significant by Student t-test (p<0.006) for wild type and non-target controls, but no difference was seen between normoxia and hypoxia in HIF-1α siRNA transfected SH-EP1 cells. C. Sensitisation of SH-EP1 cells to ABT-737-induced apoptosis is lost in SH-EP 1 cells transiently transfected with siRNA to HIF-1α. % of annexin V/7-AAD positive cells (mean of 3 independent experiments) in mock treated, non-target siRNA transfected, and HIF-1α siRNA transfected SH-EP1 cells is shown after 24 hr exposure to ABT-737 at the normoxic IC50 concentration in normoxia and hypoxia. The increase in apoptosis in hypoxia in mock and non-target transfected SH-EP1 cells is significant by Student t-test (p<0.02), and the reduction in ABT-737-induce apoptosis in hypoxia in HIF-1α siRNA transfected SH-EP1 cells in comparison with non-target transfected cells is also significant by Student t-test (p<0.008).