Histone Deacetylase Inhibitors Increase p27^Kip1 by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation

Figure 1

Effect of inhibitors of HDACs on cellular growth and phenotype. (a) K562 cells were plated at 300.000 cells/mL and incubated without (Con) and with 1 mM butyric acid (BuA), 2 mM phenylbutyric acid (PBuA), MS-275 (1 μM), 2 mM valproic acid (VPA), 100 μM splitomicin (Splito), and 10 mM nicotinamide (NAM). The effect on the growth is expressed as percentage of control after 2 days of incubation. The reported data represent the mean values ± S.D. of three independent experiments, with each experiment performed in duplicate. Con, untreated cells; compared to Con; ns, not significant compared to Con. (b) Various cell lines were incubated without (Con) and with BuA, Splito, and NAM at the concentrations reported in (a). The effect on the growth is expressed as percentage of control after 2 days of incubation. The reported data represent the mean values ± S.D. of three independent experiments, with each experiment performed in duplicate. Con, untreated cells; compared to Con; ns, not significant compared to Con. (c) K562 cells were plated and treated as in (a). Cell extracts were prepared as in Materials and Methods and analyzed by immunoblotting for the content of acetyl-histone H3 (AcetylH3). Actin was evaluated for confirming equal protein loading. (d) The panel reports the flow cytometric analyses of K562 cells treated with BuA, PBuA, VPA, and MS-275 (at concentrations reported in (a)) for different time intervals. At each time (24 and 48 hours) the percentage of cells in a specific phase is showed. Time 0 represents the percentage of specific phase of untreated growing cells. The reported data represent the mean values of three independent experiments. The percentage of subG1 cells is lower than 2% in all the FACS analyses. (e) K562 cells were incubated without (Con) and with BuA (1 mM) for the reported time intervals. Total cell extracts were then prepared as in Materials and Methods and analyzed by immunoblotting for PARP and PARP cleavage product (as marker of apoptosis). 1 μM staurosporine-treated cell extract was used as a positive control. (f) K562 cells were incubated without (Con) and with BuA (1 mM) for 16 and 24 hours. Total cell extracts were prepared as in Materials and Methods and analyzed by immunoblotting for (p27), (p21), and (p57). For the densitometric quantitation of analysis, three identical experiments were performed and the relative films were scanned and analyzed by ImageJ64 software. The data reported (as pixel) represent the mean values ± S.D. compared to untreated cells (Con).

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