Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 Page 6 of 13 Figure 3 Induction of cell cycle arrest and apoptosis by flavopiridol and 4OH-Tam: A. Diploid cell cycle profile determined by FACS analysis of MON cells treated with flavopiridol and 4OH- Tam for two days; B. Percentage of MON cells at sub-G1 when exposed for two days to flavopiridol and 4OH-Tam C. Percentage of apoptotic (annexin positive), necrotic (PI positive) and dead (both annexin and PI positive) MON cells when exposed for two days to flavopiridol and 4OH-Tam. (Figure 4A), but induction of apoptosis by flavopiridol with or without 4OH-Tam was significantly inhibited by Z-VAD-FMK (p < 0.0001, Figure 4B). This established that cell death induced by flavopiridol, 4OH-Tam, or their combination is mediated by caspase-dependent apoptosis in RT cells. We further investigated the kinetics of induction of caspase 3 and 7 activities upon drug treatment. The results indicated that, while Figure 4 Effect of caspases on flavopiridol- and 4OH-Tam- induced cell cycle arrest and apoptosis: A. Diploid cell cycle profile determined by FACS analysis of MON cells treated with Flavopiridol and 4OH-Tam for two days in the presence of pan- caspase inhibitor Z-VAD-FMC (Compare to Figure 2A); B. Percentage of MON cells at sub-G1 when exposed for two days to flavopiridol and 4OH-Tam, in the absence and presence of pan-caspase inhibitor Z-VAD-FMC (CI). C. Kinetics of induction of caspase 3/7 activity in MON cells by flavopiridol and 4OH-Tam. The diagram illustrates the fold increase in caspase 3/7 activity at various time points after treatment. F100 = 100 nM flavopiridol; T5 = 5 μM 4OH-Tam. flavopiridol induced peak caspase 3 and 7 activities at six hours, 4OH-Tam exhibited delayed kinetics with peak induction at 12 hours. Interestingly, combination of 4OH-Tam and flavopiridol elevated caspase 3 and 7