ABSTRACT
This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Carcinoma, Hepatocellular/drug therapy , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Histones/metabolism , Liver Neoplasms/drug therapy , Tumor Suppressor Protein p53/metabolism , Acetylation , Benzothiazoles/pharmacology , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , DNA Damage , Humans , Hydroxamic Acids/pharmacology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Small Interfering/pharmacology , Toluene/analogs & derivatives , Toluene/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , VorinostatABSTRACT
This study describes the molecular mechanism by which treatment with 3-AB, a potent inhibitor of PARP, allows human osteosarcoma MG-63 cells to restrict growth and enter differentiation. Our findings show that in MG-63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3-AB-treatment, the poly(ADP-ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, beta-catenin, c-Jun, c-Myc and Id2) and upregulation of those implicated in the osteoblastic differentiation (p21/Waf1, osteopontin, osteocalcin, type I collagen, N-cadherins and alkaline phosphatase). Our study suggests that use of PARP inhibitors may induce a remodeling of chromatin with the reprogramming of gene expression and the activation of differentiation.