The bromodomain and extra terminal (BET) family protein bromodomain containing protein 4 (BRD4) is an epigenetic regulator recently identified as a therapeutic target for several hematological cancers, notably mixed lineage leukemia-fusion acute myeloid leukemia (MLL-AML). resulting in cell cycle arrest and apoptosis in a c-MYC-independent manner. Our data suggest that BET bromodomain inhibition might enhance current chemotherapy strategies in AML, notably in poor-risk DNMT3A/NPM1-mutated disease. for 5 min at 4C, and the resultant supernatants were frozen in liquid nitrogen until use. For immunoprecipitation experiments cells were pretreated with 500 nmol/L daunorubicin and 400 nmol/L trichostatin A (TSA) for 24 h to induce p53 expression and hyper-acetylation prior to preparation of cell extracts. Immunoprecipitation Equal amounts of total protein extracts from OCI-AML3 or Hela cells were precleared with protein A/G-Sepharose beads (GE Healthcare) at 4C for 1 h. Supernatants were immunoprecipitated for 2 h at 4C with a BRD4-specific antibody or with rabbit immunoglobulin as a negative control. The proteinCantibody complexes were pulled down by adding protein A/G-Sepharose beads. Sample pellets were then subjected to several washes in PBS. The immunocomplexes were recovered from the protein A/G-Sepharose beads by boiling the samples in electrophoresis loading buffer. The immunocomplexes were analyzed by Western blot using anti-p53 antibodies. Western blotting OCI-AML3 cells were treated with 0.5 mol/L JQ1 for 24 h and then harvested. Samples were then diluted in loading buffer (with -mercaptoethanol), boiled for 5 min, and subjected to gel electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). A quantity of 36 g of cell extract was loaded per lane. Proteins were electrophoretically transferred onto PVDF for 1 h at 150 mA constant current. Immunolabeling was achieved by blocking the gel with 5% milk for 1 h at room temperature and then rotated overnight at 4C with primary antibodies diluted 1:2000 in Tris-buffered saline 0.1% Tween20. Secondary antibodies were used at a dilution of 1:20,000. Blots were developed using a Vectastain ABC kit or a chemiluminescent detection kit (Vector labs, Peterborough, U.K.). Statistical analysis All values are shown as mean SEM from at least three independent experiments (or a representative experiment of three is shown) and considered significant if < 0.05. Significance between groups was calculated using Student's t-tests. Results JQ1 inhibits proliferation of the AML cell line OCI-AML3 in a dose-dependent manner The bromodomain inhibitor JQ1 has MSDC-0160 IC50 been reported to inhibit the proliferation of many leukemia cells lines, particularly those containing MLL mutations . We performed experiments on OCI-AML3, a p53-wildtype AML cell line that carries mutations in DNMT3A (R882C) and NPM1c (exon-12) genes . In preliminary experiments, a timeCresponse curve to analyze the effect of Rabbit Polyclonal to TACC1 1 mol/L JQ1 on OCI-AML3 cell viability was carried out using WST-1 assays. In subsequent experiments, cells were analyzed at 72 h as this treatment time caused a significant decrease in viability (Fig. ?(Fig.1A).1A). We found that JQ1 caused a dose-dependent decrease in cell viability with an IC50 of 500 nmol/L (Fig. ?(Fig.1B).1B). Control experiments using the inactive enantiomer (C)-JQ1 or vehicle had no effect. For comparative purposes, K562 and Raji cells were used in additional experiments. The p53-mutated K562 cell line responded poorly to the compound as previously reported  with an IC50 of >2 mol/L whereas the Raji cell line was moderately sensitive with an IC50 of 1C2 mol/L (Fig. ?(Fig.11B). Figure 1 JQ1 mediates cell death in OCI-AML3 cells. (A) OCI-AML3 cells were treated with 1 mol/L JQ1 for 96 h and then cell viability was measured using the WST-1 assay. (B) A timeCresponse curve for 1 mol/L JQ1 is shown. (C) Growth curves … Growth curves confirmed that the decrease in cell viability observed in JQ1-treated OCI-AML3 cells was due to cell death (Fig. MSDC-0160 IC50 ?(Fig.1C).1C). Flow cytometry analysis using propidium iodide and annexin V staining confirmed that death was by apoptosis (Fig. ?(Fig.11D). JQ1 induces double-stranded DNA breaks and pan-nuclear H2AX staining In order to determine the mechanism of action of JQ1, we carried out experiments to look for DNA damage responses. OCI-AML3 cells treated with 0.25 mol/L JQ1 were immunolabeled for H2AX phosphorylation and 53BP1. JQ1 induced a robust increase in pan-nuclear H2AX staining indicative of an early apoptotic response (Fig. ?(Fig.2A2A and B). This was accompanied by a significant increase in 53BP1 foci (Fig. ?(Fig.2C)2C) but not H2AX foci. Only 10% of cells with pan-nuclear MSDC-0160 IC50 H2AX staining had 53BP1 foci. No increase in pan-nuclear H2AX staining was observed at 48 h although.