Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 Page 4 of 13 Figure 1 Inhibition of rhabdoid tumor growth by flavopiridol and 4OH-Tam. A-C. Survival curves of MON (A), G401 (B), and A204 (C) cells treated with flavopiridol in the presence and absence of 4OH-Tam for two days. D. Table of IC50 values as approximated from A-C. Flavo = flavopiridol; 4OHT = 4OH-Tam. exposure. Notably, by day five of treatment, combina- tions of very low concentrations of flavopiridol (~ 0.2 nM) and 10 μM 4OH-Tam decreased cell survival by 80% in MON and 99% in G401 cells (Figure 2B and 2F). These results indicated that treatment with low concen- trations of flavopiridol in combination with 4OH-Tam for prolonged periods potently inhibits RT cell growth. Induction of apoptosis by flavopiridol and 4OH-Tam To determine the mechanism of growth inhibition by these drugs, we studied MON cells which showed inter- mediate responses to the combination of drugs with an intermediate level of 4OH-Tam (5 μM) and duration of treatment. MON cells were exposed to combinations of flavopiridol and 5 μM 4OH-Tam for two days and sub- jected to cell cycle analysis. Treatment of cells with 5 μM 4OH-Tam alone resulted in G1 arrest (p = 0.0117, Figure 3A). Our previous results indicated that flavopiri- dol at 200-400 nM induces G1 arrest [13]. However, treatment with 100 nM flavopiridol induced G2 arrest in the presence or absence of 4OH-Tam (p = 0.0039 and p = 0.0003, respectively, Figure 3A) indicating that the stage of cell cycle arrest induced is dose-dependent. These results also indicate that G2 arrest induced by fla- vopiridol is dominant over 4OH-Tam-induced G1 arrest. Analysis of the sub-G1 fraction of cells after single or combination treatment indicated that the percentage of sub-G1 cells increased in a dose-dependent manner upon treatment with flavopiridol, which was further enhanced by combination with 4OH-Tam (Figure 3B). Very high levels of cell death are induced by these treat- ments and this likely plays the major role, compared to induction of cell cycle arrest, in inducing cytotoxicity in response to treatment with these drugs. To determine if cell death was due to apoptosis, the cells were stained for annexin (indicative of apoptosis) and propidium iodide (indicative of necrosis) and the percentage of cells positive for either or both stains was determined by FACS (Figure 3C). While the percentage of necrotic or dead cells remained largely unchanged, there was a dose-dependent increase in annexin-positive cells demonstrating that combination treatment with flavopir- idol and 4OH-Tam induces significant levels of apoptosis. Induction of apoptosis by flavopiridol and 4OH-Tam is caspase-dependent Since it is known that flavopiridol can induce apoptosis by caspase-dependent or caspase-independent mechan- isms, we investigated the effect of a caspase inhibitor on drug-induced cytotoxicity. MON cells were exposed for two days to flavopiridol and 4OH-Tam in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK, and FACS analysis was carried out to assess the effects on cell cycle and apoptosis. The results indicated that drug-induced cell cycle arrest was unaffected (p = 0.0458 and p = 0.0025 for G1-arrest induced by 100 nM flavopiridol with and without 4OH-Tam respectively)