Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 recruiting the HDAC1 complex [7-10]. We have found that cyclin D1 is de-repressed in human and mouse RTs and is required for rhabdoid tumorigenesis in mouse models [9,11,12]. Such studies indicated that therapeutic targeting of cyclin D1 and its pathway could be an effec- tive and novel therapeutic strategy for RTs. We previously reported that down-modulating cyclin D1 and inhibiting cyclin dependent kinases (cdks) using either flavopiridol or a combination of N-(4-hydroxy- phenyl)retinamide (4-HPR) with 4OH-Tam is effective in inhibiting RTs in vitro and in xenograft tumor mod- els in vivo [11,13]. The effectiveness of 4-HPR and flavo- piridol was correlated with down-modulation of cyclin D1 in xenograft tumors [13]. Flavopiridol is one of the first cdk inhibitors to enter clinical trials. Although early clinical trials were unsuc- cessful, design of a novel schedule of administration based on the in vitro and in vivo pharmacokinetic mod- eling of flavopiridol’s effect has shown promising effi- cacy in refractory chronic lymphocytic leukemia [14]. Phase I trials of flavopiridol in children have revealed that its toxicity profile, pharmacokinetics, and maximum tolerable dose were similar to that in adults, indicating that using flavopiridol in RT patients, a largely pediatric population, is feasible [15]. The effects of flavopiridol on cancer cells are varied and cell type dependent. In many cell lines flavopiridol leads to G1 arrest due to down-modulation of cyclin D1 and inhibition of its pathway by various mechanisms [16-24]. In other cells, flavopiridol induces G2 arrest, in part due to its potent ability to inhibit cdk 7, 8 and 9 activities [25]. Flavopiridol also inhibits transcription of Mdm-2 resulting in the accumulation of its proteolytic target, p53, which triggers p21Waf1 up-regulation, cyclin B1 down-regulation, and ultimately G2 arrest [26]. Fla- vopiridol can induce apoptosis at nanomolar concentra- tions and its pro-apoptotic action is either caspase- dependent or -independent [25]. Flavopiridol can trigger apoptosis by activation of caspases 2, 3 and 8 [27] or by activation of apoptosis inducing factor (AIF) via its release from the mitochondria [28]. At this point, the mechanism of action of flavopiridol in RT cells is not completely understood. Since flavopiridol can be toxic at high doses, recent studies have focused on combining low concentrations of flavopiridol with other anti-neoplastic agents [29,30]. In this report, we tested the combination of flavopiridol with 4OH-Tam to determine its ability to increase ther- apeutic efficacy against RT cells. 4OH-Tam inhibits tumor cell growth in part by deregulating cyclins and cdks. Breast cancer cells over-expressing cyclin D1 are resistant to 4OH-Tam and the level of cyclin D1 is negatively correlated to responsiveness to 4OH-Tam [31-34]. 4OH-Tam suppresses the growth of estrogen- Page 2 of 13 receptor positive tumors by down-modulating cyclin D1 [35]. Furthermore, treatment of tumor cell lines with 10μM 4OH-Tam induces the expression of p21Waf1 and p27Kip1, which are known to block the effects of cyclin D1 [36]. Previous studies have demonstrated that 4OH- Tam is effective in inducing cytotoxic effects in RT cells [37]. In this study it was demonstrated that the expres- sion of ERa receptor in RT cells is variable and that the cytotoxic effects of 4OH-Tam are independent of ERa expression [37]. Since the efficacy of flavopiridol in xenograft RTs was correlated with down-modulation of cyclin D1 and up-regulation of p21Waf1[13], we consid- ered combining 4OH-Tam with flavopiridol to enhance its therapeutic efficacy in RT cells. We report here that the combination of flavopiridol with 4OH-Tam potently inhibited the survival of RT cells. A low concentration of flavopiridol (100 nM) induced G2 arrest in RT cells in a p53-dependent man- ner and resulted in a moderate amount of apoptosis. Addition of 4OH-Tam significantly increased flavopiri- dol-mediated apoptosis. Down-modulation of p53 did not affect 4OH-Tam-induced cytotoxicity, but signifi- cantly enhanced flavopiridol-mediated apoptosis. Furthermore, we found that the increased cytotoxic effects of flavopiridol and 4OH-Tam correlated with augmentation of caspase 2 and 3 activities and that these effects were independent of p53. These studies indicate that the combination of flavopiridol and 4OH- Tam can be effective in potently inhibiting RT cell growth and is potentially a novel therapeutic strategy against RTs. Methods Cell culture and drug treatment MON [6], G401 (American Type Culture Collection), and A204 cells (American Type Culture Collection) were maintained in RPMI supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomy- cin, and 2 mM L-glutamine. For drug studies, the cells were transferred one day before drug treatment to RPMI containing 10% charcoal and dextran treated fetal bovine serum (HyClone, Cat. SH30068.03). Flavopiridol was obtained from the CTEP program at NCI (courtesy of Dr. Colevas). 4OH-Tam and pan-caspase inhibitor z-VAD-FMK were purchased from SIGMA (Catalogue #H7904) and Promega (Catalogue #G7232) respectively. Cell Survival analysis 8,000 cells/well (MON and A204) or 6,500 cells/well (G401) were plated in 96-well plates and treated with different concentrations/combinations of drugs, using the epMotion 5070 automated liquid handler system (Eppendorf). The CellTiter 96 AQueous One Solution Cell Proliferation Assay was used to determine cell