Figure 2.

Induction of apoptosis by the vorinostat + flavopiridol drug combination. A. Induction of apoptosis in the CHLA-90 cell line measured by TUNEL assay. Cells were pre-treated with vorinostat (2 μM) for 24 hrs and then flavopiridol (0.2 μM) was added for an additional 24 hrs. Apoptosis induced by vorinostat alone was assayed after a 48-hr exposure. For apoptosis inhibition, the pan-caspase BOC-d-fmk (40 μM) was added 2 hrs prior to vorinostat and an additional 40 μM was added 24 hrs later according to the manufacturer’s instructions. B. Immunoblot analysis of caspase-3 (35 kDa) and caspase-9 (47 kDa) is shown in representative cell lines with wt TP53 (CHLA-136) or mt TP53 (CHLA-90). Drug effects were determined according to activation of caspase-3 to a cleaved 19 kDa form, and caspase-9 to a cleaved 35 kDa form. Equal loading of protein was confirmed by β-actin expression. Cells were pre-treated with 2 μM vorinostat (V) for 24 hrs, and then 0.2 μM flavopiridol (Fl) was added for additional an 6, 9, 12, 16, 18, or 24 hrs. C – Vehicle treated controls.