Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 dependent on p53 [48]. In this study the combination of flavopiridol and SN-38 was tested on isogenic pairs of cells differing only in p53. They found that the enhanced apoptosis by combination of flavopiridol and SN-38 was observed only in p53+/+ cells. Similar to SN- 38, 4OH-Tam also induces G1 arrest in our system, however; the effects of combining flavopiridol with 4OH-Tam obtained in RT cells are different in terms of inducing apoptosis. While flavopiridol-mediated G2 arrest was dependent on p53, flavopiridol-induced apop- tosis was abrogated by p53 (Figure 6C and 6D). On the contrary, p53 had no effect on 4OH-Tam-mediated apoptosis. Because of these results, we believe that p53 has compromising effects on flavopiridol-induced apop- tosis in RT cells, similar to the effect of p53 in protect- ing cells from radiation induced apoptosis as discussed above. Our studies involving p53 knock-down indicate that stimulation of p53 by flavopiridol limits its ability to induce apoptosis in RT cells. Thus, it is possible that 4OH-Tam increases the effects of flavopiridol because it down-modulates p53 in addition to inducing apoptosis by p53-independent mechanisms. This new understand- ing of p53’s role in drug-induced apoptosis in RT cells might shed light on the mechanism of resistance to therapies exhibited by these tumors. Also, evaluation of p53 levels induced by flavopiridol and other treatments may be necessary to implement effective treatment stra- tegies for RTs. Flavopiridol induces apoptosis by additional p53-inde- pendent mechanisms. It is able to block RNA polymer- ase II phosphorylation by inhibiting cdk 9, thereby blocking transcriptional elongation. This activity, as well as flavopiridol’s ability to reduce antiapoptotic protein MCL-1, has been implicated in the induction of apopto- sis in multiple myeloma cell lines [49]. Additionally, induction of the mitochondrial permeability transition by flavopiridol has been correlated with induction of apoptosis in chronic lymphocytic leukemia cells [50]. These functions of flavopiridol may also contribute to the apoptosis occurring in RT cells in a p53-indepen- dent manner. RTs are notoriously resistant to therapeutic interven- tions [3]. Potent chemotherapy in combination with sur- gery and radiotherapy have proven futile in increasing survival rates and only a handful of RT survivors have been reported [2]. Therefore, efforts to develop molecu- larly targeted therapies are needed. Based on the mole- cular understanding of RTs, it is known that INI1/ hSNF5 mediates tumor suppression in part by targeting cyclins and cdks [7-9]. Furthermore, cyclin D1 is up- regulated in, and necessary for, rhabdoid tumorigenesis [9,12]. Thus, it appears that therapeutically inhibiting the cyclin/cdk pathway is a novel, targeted treatment Page 11 of 13 strategy for RTs. Our report suggests that combination of flavopiridol and 4OH-Tam could be used as a novel combination therapy for RTs. Furthermore, this combi- nation could be effective in inducing apoptosis in other tumor models, especially those lacking p53. At this point, the concentrations of 4OH-Tam required to increase the effects of flavopiridol appear to be high. Nevertheless, in the pediatric population, where RTs most often occur, it has been reported that high doses of tamoxifen (100 mg/m2 twice a day) can be administered with minimal toxicity [51]. Furthermore, in cases where sustained high concentrations (≥ 10 uM) of 4OH-Tam may not be attained, alternative formulations, such as a liposomal formulation, of both tamoxifen and 4OH-Tam have been used that would result in the delivery of high concentrations directly to the tumor [52]. Therefore, further preclinical and clinical studies to test the efficacy of these drugs in children may lead to the development of definitive therapeutic strategies against RTs that may improve prognosis. Additional material Additional File 1: Flavopiridol- and 4OH-Tam-induced caspase profiles in MON cells: A-I. Panels represent kinetics of induction of caspase activities in MON cells transfected with control (+Cy3) or p53 (+p53) siRNAs and then treated with 100 nM Flavopiridol (F100), 5 μM 4OH-Tam (T5), or both (F100T5) for two days. J-K. Graphic representation of caspase 2 and 3 activities induced by the drugs (as in A-I) in the presence of control (Cy3) or p53 siRNA (p53), at 24 hr. time point, in the presence or absence of specific caspase 2 and 3 inhibitors, respectively. The values illustrate the average of two independent experiments. Acknowledgements The authors thank Dr. Prasad at AECOM for critically reading the manuscript and Dr. Kitsis at AECOM for useful discussions, Dr. Colevas at CTEP, NCI for providing flavopiridol. This work was supported by grants from ACS (#CCG- 10493) and Children’s Brain Tumor Foundation (CBTF, NY) to G.V.K, who is a Mark Trauner faculty scholar and a recipient of the Irma T. Hirschl Career Scientist Award. M.E.S. is supported by Institutional training grant, NIGMS (T32 GM 07491). Author details 1Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 USA. 2Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 USA. 3Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 USA. Authors’ contributions VC carried out FACS analysis, most of the immunoblot analysis, caspase profiling, p53 and p21 analysis, as well as participated in the design of the study, analyzed and interpreted the data, and participated in the initial preparation of the manuscript. MES participated in the design of the study, analyzed and interpreted part of the data, drafted the manuscript, carried out cell survival assays, and part of the immunoblot analysis. ZZ initiated the studies on the treatment of rhabdoid tumor cells with combination of flavopiridol and 4OH-Tamoxifen in the laboratory. DM participated in part of the survival assay, SM participated in the design of the study, provided guidance in the initial part of the study and preparation of the manuscript. GVK conceived of the study, designed the experiments, analyzed and