Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 and 3 activity, with maximum induction observed at 12 hours (Figure 7C and Additional File 1F and 1I). Abro- gation of p53 led to a further increase in caspase 2 and 3 activity after combination treatment, similar to that caused by flavopiridol alone (Figure 7C and Additional File 1F and 1I). 4OH-Tam alone caused only slight increases in caspase 2 and 3 activities in the presence or absence of p53 (Figure 7C and Additional File 1E and 1H). To demonstrate that induction of caspase 2 and 3 activities were specific, we treated MON cells in the presence and absence of p53 and also in the presence and absence of specific caspase 2 or 3 inhibitors. Cas- pase 2 and 3 activities were determined at the 24 hr. time point in all of the treatment conditions. We found that treatment of cells with caspase 2 or cas- pase 3 inhibitors significantly reduced the induction of caspase 2 or 3 activities upon drug treatment respectively. These results indicated that flavopiridol and 4OH-Tam drug treatment selectively induces cas- pases 2 and 3 in MON cells, as inhibitors to these caspases eliminated these increases (Additional File 1J and 1K). Conclusions Our report demonstrates, for the first time, that RT cell growth and survival is potently inhibited by combi- nation treatment with clinically achievable concentra- tions of 4OH-Tam (2.5 or 5 μM) and flavopiridol (< 200 nM). Since high concentrations of flavopiridol may cause significant toxicities, and since only low concentrations may be achievable in areas such as the brain due to the blood brain barrier, our results pro- vide a method to increase the efficacy of low concen- trations of flavopiridol. Flavopiridol and 4OH-Tam together induce a signifi- cant increase in RT cell death. Induction of cell death by flavopiridol and the combination of flavopiridol with 4OH-Tam is due to caspase-dependent apoptosis and caspase-profiling assays indicate that these treatments can potently induce caspases 2 and 3. In addition to inducing cell death, these treatments also induce cell cycle arrest. While our previous report indicated that 400 nM flavopiridol induces G1 arrest [13], this current report indicates that 100 nM flavopiridol is sufficient to cause G2 arrest indicating that flavopiridol potently induces cell cycle arrest in a dose-dependent manner. Flavopiridol-induced G2 arrest was correlated to down- regulation of cyclin B1 and up-regulation of p53 and p21Waf-1. On the contrary, 4OH-Tam inhibited p53 expression; however, this effect was nullified by addition of 100 nM flavopiridol, explaining the dominant effect of flavopiridol in mediating G2 arrest. These results sug- gest that the effect of flavopiridol in inducing p53 is Page 10 of 13 upstream of the mechanism by which 4OH-Tam inhibits p53. Interestingly, we found that p53 differentially regu- lated flavopiridol-mediated cell cycle arrest and apopto- sis. RNA interference analysis of p53 indicated that while flavopiridol-mediated G2 arrest was dependent on p53, flavopiridol-mediated apoptosis (but not that mediated by 4OH-Tam) was countered by p53. This is an intriguing observation since there was a clear enhancement of apoptosis induced by flavopiridol when p53 was abrogated by RNA interference (Figure 6D). Previously it has been reported that lack of p53 enhances radio-sensitivity via activation of E2F-1 and induction of caspase 8 activity in glioma cells [40]. Furthermore, radio-sensitivity of keratinocytes was enhanced by abrogation of p53 and was mediated by down-regulation of anti-apoptotic proteins Mcl-1 and Bcl-XL [41]. To our knowledge, this is the first report which indicates that down-modulation of p53 also enhances drug-induced apoptosis. Our results indicate that the potency of flavopiridol can be enhanced if p53 can be inhibited by some means. Most RTs express p53, though a percentage of RTs do show mutations within the p53 gene [44-46]. Some RT cell lines express p53 at high levels or with increased nuclear distribution, however, the p53 pathway has been tested and considered to be functionally intact [42,44]. The mutation status of p53 in MON RT cells has not been determined but the proper expression of p53 and responsiveness of p21Waf1 to p53 levels leads us to believe that the p53 pathway is intact in these cells. However, other studies indicated that the pro-apoptotic pathway downstream of p53 may be dysfunctional in MON cells but the exact nature of this defect is unknown [43]. Although this defect in the pro-apoptotic pathway downstream of p53 could account for the observed effects reported here, our observation indicates that flavopiridol-induced apoptosis is inhibited by p53. More experiments are needed to delineate the exact role of the p53 pathway in flavopiridol-induced cytotoxicity in MON and other RT cells. The relationship of flavopiridol and p53 in inducing apoptosis seems to be paradoxical in different cell lines and in different treatment approaches. Perhaps this is related to the paradoxical anti-apoptotic activities of p53 itself in various cancer cells [47]. The activities of p53 are cell type dependent and can be either pro-apoptotic and/or pro-survival. For example, in RT cells p53 is deleterious to flavopiridol-mediated apoptosis, but in other cancer cell lines flavopiridol-induced apoptosis is actually dependent on p53. An example is provided by Ambrosini et al who demonstrated that enhanced apop- tosis induced by a combination therapy of flavopiridol with a G1-arrest-inducing agent (namely SN-38) was