Description

Myelodysplastic syndromes, Mesothelioma,Cancer

Immunoprecipitation-HDAC assays

The lysate of Jurkat cells is incubated for 1 hour on ice and cleared by centrifugation at 12,000 g for 10 minutes at 4 °C. Supernatants are precleared with 30 μL of 50% protein G-Sepharose slurry for 1 hour at 4 °C. Beads are pelleted by centrifugation and supernatants are incubated for 1 hour at 4 °C with 10 μg of IgG fraction from anti-HDAC1 or HDAC3 polyclonal antisera (preincubated 2 hours at room temperature with either the homologous or heterologous immunizing peptide). Both antisera are raised in rabbits against the carboxylterminal peptide of HDAC1 and HDAC3 by using synthetic peptides coupled to keyhole limpet hemocyanin. 30 μL of 50% protein G-Sepharose slurry is added for 1 hour at 4 °C. Immune complexes are pelleted by centrifugation and washed three times with 1 mL of lysis buffer. Beads are resuspended in 200 μL of HDAC buffer (20 mM Tris-HCl, pH 8.0/150 mM NaCl/10% glycerol), and the HDAC assay is performed with an ³H-acetylated peptide corresponding to amino acids 1-24 of histone H4. Released [³H]acetic acid is quantified by scintillation counting. For inhibitions studies, the immunoprecipitated complexes are preincubated with the different concentrations of Vorinostat for 30 minutes at 4 °C.

Cell Lines

LNCaP, PC-3, and TSU-Pr1

Concentrations

Dissolved in DMSO, final concentrations ~7.5 μM

Incubation Time

1, 2, 3 and 4 days

Methods

Animal Models

Male BALB/c nude (nu/nu) mice implanted with CWR22 tumor cells

Formulation

Dissolved in DMSO

Doses

~100 mg/kg/day

Administration

Injection i.p.