These combinations showed strong synergistic effects on tumor cell growth and represent a promising potential tool for the treatment of rhabdoid tumors. Methods Cell lines Rhabdoid tumor cell always find useful information lines BT12 and BT16, G401 and A204 were cultured in DMEM high glucose formulation, supplemented with 10% fetal bovine serum, 2% glutamine and no additional antibiotics. The cells were cultured at 37 C in a humidified atmosphere with 5% CO2. A204 and G401 were obtained from ATCC. BT12 and BT16 were a gift from Dr. P. Houghton. Mouse embryonic stem cell line OG2 was cultured to the distributors recommendation in DMEM with Glutamax, non essential aminoacids, mercaptoethanol, PenStrep and LIF. For differentiation of ESCs OG2 cells were cultured at least five days without LIF. OG2 cell line was a gift from Hans Sch?ler.

The identity of all cell lines was verified using ST PCR. All experiments using cell lines in this publication were at least performed using three independent replicates. Histone deacetylase inhibitors, Cyclin D inhibitors and chemotherapy Suberoylanilindehydroxamic acid, Trichostatin A, N retinamide and 4 Hydroxy Tamoxifen were reconstituted in 100% ethanol, as a 10 mM solutions. M344 was synthesized by one of us. Doxorubicin was purchased from Merck. Cytotoxicity assay Cell suspensions were seeded into four 96 well plates. Cells were allowed to reach exponential growth before 100 ul of cell culture medium containing the drugs at different concentrations were added. Each drug concentration was tested in 3 biological replicates.

For experiments with combined treatment we used compound 1 in increasing concentrations as in single compound experiments. Compound 2 was used at 1 10 of the concentration of compound 1. After 0, 24, 48 and 72 hr cells were incubated 3 hr with 10 ul MTT reagent. Metabolically active cells cleaved the yellow tetrazolium salt to a purple formazan dye. A decrease in the number of living cells correlated with the number of purple formazan crystals. Crystals were dissolved in 100ullysis buffer. The specimen was evaluated spectrophotometrically at 570 nm and a reference of 650 nm using a Multiskan Ascent multiplate reader. Analysis of combined drug effects on cytotoxicity To evaluate drug combination effects we analyzed cytotox icity assay data using the median effect method by Chou and Talalay.

We employed three biological replicates of the cytotoxicity assay for each experiment. The fraction of unaffected cells was defined as the proportion of living cells compared to the control. The combination index indicates synergism if CI 1, antagonism for CI 1 and an additive effect for CI 1. Values of the CI were determined at the IC50 concentration. The method was implemented AV-951 in the statistical software R. Western blots For differentiation of mouse embryonic stem cell line OG2 cells were grown without LIF.