Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 eliminated the flavopiridiol-induced phosphorylation of pRb at the 25 nM concentration (Figure 5A lane 7). However, 4OH-Tam had no effect on 100 nM flavopiri- dol-induced inhibition of pRB phosphorylation, consis- tent with the observed dominant effect of 100 nM flavopiridol in inducing G2 arrest. Flavopiridol induced p21Waf-1 expression in RT cells Our previous analysis indicated that flavopiridol efficacy in mouse xenografts correlated to both down-modula- tion of cyclin D1 and up-regulation of p21Waf-1 [13]. Therefore, we examined p21Waf-1 levels upon flavopiri- dol treatment with and without 4OH-Tam. We found that p21Waf-1 was up-regulated in a dose-dependent manner upon treatment with flavopiridol (Figure 5A, lanes 1-4), but 4OH-Tam alone only modestly up-regu- lated p21Waf-1(Figure 5A, compare lanes 1 and 5). We also investigated the accumulation of nuclear p21Waf-1 upon drug treatment by immunocytochemical analysis. The results indicated that flavopiridol treatment led to increased nuclear p21Waf-1, which was further increased upon addition of 4OH-Tam (Figure 5B and 5C). These results were confirmed by quantifying the percentage of cells with nuclear p21Waf-1 (Figure 5C) and, taken together, indicate that combination treatment results in both increased protein levels and increased nuclear loca- lization of p21Waf-1. Abrogation of p53 inhibited cell cycle arrest but enhanced apoptosis induced by flavopiridol The above studies pointed to the involvement of p21Waf-1 in flavopiridol or flavopiridol plus 4OH-Tam-mediated cytotoxicity. It has been established that p21Waf-1 is a downstream effector of p53 [38]. Furthermore, tumori- genesis in Ini1 heterozygous mice is enhanced upon abrogation of p53 [39]. Since p53 is up regulated upon flavopiridol treatment, we further investigated its role. To determine whether p53 is necessary for flavopiridol- induced cell cycle arrest and/or apoptosis, RNA interfer- ence was used to down-modulate p53 expression. Trans- fection of RT cells with p53 siRNA slightly enhanced cell proliferation (data not shown). As expected, flavopiridol markedly increased p53 expression in cells transfected with control siRNA (Cy3), but not in cells transfected with p53 siRNA (Figure 6A, compare lanes 2 and 6). FACS analysis of cells transfected with control or p53 siRNA and treated with single or combination drugs for two days indicated that, while control siRNA did not affect drug-induced cell cycle arrest (p = 0.0221 and p = 0.0035 for G1-arrest induced by 100 nM flavopiridol with and without 4-OH-Tam respectively, Figure 6B), knock- down of p53 resulted in abrogation of flavopiridol- mediated G2 arrest both in the presence and absence of 4OH-Tam (p = 1.000 (not significant), Figure 6C). These Page 8 of 13 results indicated that p53 is necessary for flavopiridol- mediated G2 arrest in RT cells. Interestingly, knock-down of p53 demonstrated differ- ential effects on flavopiridol and 4OH-Tam-mediated apoptosis in RT cells. Knock-down of p53 had no effect on 4OH-Tam-induced apoptosis indicating that 4OH- Tam-induced apoptosis is independent of p53 (Figure 6D). However, p53-knockdown enhanced flavopiridol- mediated apoptosis in the presence or absence of 4OH- Tam (Figure 6D). These results indicated that p53 is detrimental to flavopiridol-induced apoptosis in MON cells. Additionally, these results indicated that flavopiri- dol and 4OH-Tam induce apoptosis in RT cells by two different mechanisms; flavopiridol-mediated apoptosis being inhibited by p53 and 4OH-Tam-induced apoptosis being independent of p53. Role of caspases in flavopiridol and 4OH-Tam induced apoptosis Expression of p53 has been associated with resistance to radiation-induced apoptosis in some cancers including gliomas and keratinomas [40,41]. Many RTs are resis- tant to chemotherapy and radiotherapy; however the mechanistic basis for this resistance is not clearly under- stood [42,43]. Based on our results we surmised that induction of p53 by flavopiridol is counter-productive to induction of apoptosis in RT cells. It has been reported that p53 is expressed in a majority of RTs and sequence analysis of mRNA does not show any abnormality in the p53 coding region [44,45]. Therefore, understanding the role of p53 in inhibiting drug-induced apoptosis might shed light on the mechanism of drug resistance exhib- ited by these tumors. Since flavopiridol increased p53 levels in RT cells, which was inversely correlated to induction of apoptosis, we explored the possibility that flavopiridol induced apoptosis through specific caspases. We profiled the kinetics of induction of caspase 2, 3, 8 and 9 activities in RT cells in the presence or absence of p53 and upon treatment with flavopiridol, 4OH-Tam, or their combination. Analysis of the activation kinetics of caspases 2, 3, 8 and 9 indicated that neither flavopiridol nor 4OH-Tam induced caspase 8 and 9 activities in RT cells (Figure 7A and 7B and Additional File 1A-C). Induction of caspases 2 and 3 was affected by treatment with flavopiridol and flavopiridol with 4OH-Tam (p = 0.0022 for caspase 2 and p.0001 for caspase 3) and was correlated to p53 levels (p = 0.0074 for caspse 2 and p < 0.0001 for cas- pase 3) (Figure 7C and 7D). Flavopiridol increased cas- pase 2 and 3 activity two-fold within 6 hours and abrogation of p53 allowed flavopiridol to increase cas- pase 2 and 3 activity four-fold in the same time (Addi- tional File 1D and 1G). Exposure of cells to flavopiridol with 4OH-Tam caused an overall increase in caspase 2