Blood samples from patients were obtained before treatment and on days 1, 3, and 8 of cycle 1. Whole blood was collected in sodium heparin Vacutainer tubes and shipped at ambient temperature to a central laboratory at MethylGene within 24 hours. Peripheral white cells were isolated using standard procedures. (A) Average histone H3 acetylation. Induction of histone acetylation was analyzed using an enzyme-linked immunosorbent assay (ELISA). Nonbiased data with standard error are shown. (B) Average percentage inhibition of HDAC enzyme activity. Whole-cell HDAC enzyme assays were performed using 8 × 105 isolated peripheral white cells per well, which were incubated with 0.3 mM Boc-Lys(ϵ-Ac)-AMC, a membrane-permeable HDAC substrate. After 1 hour at 37°C with 5% CO2, the reaction was quenched with 1 μM TSA, cells were lysed with 1% NP-40, and the deacetylated product was cleaved with Fluor-de-Lys-Developer. The reaction was allowed to develop for at least 15 minutes at 37°C with 5% CO2; then the fluorescent signal was read at Ex 360, Em 470, with cutoff of 435 nm on a fluorometer. Nonbiased data with standard error are shown.
