Acetylation of PML Is Involved in Histone Deacetylase Inhibitor-mediated Apoptosis

Fumihiko Hayakawa,Issay Kitabayashi,Akihiro Abe,Tomoki Naoe,Pier Paolo Pandolfi

doi:10.1074/jbc.m802217200

Abstract

PML is a potent tumor suppressor and proapoptotic factor and is functionally regulated by post-translational modifications such as phosphorylation, sumoylation, and ubiquitination. Histone deacetylase (HDAC) inhibitors are a promising class of targeted anticancer agents and induce apoptosis in cancer cells by largely unknown mechanisms. We report here a novel post-transcriptional modification, acetylation, of PML. PML exists as an acetylated protein in HeLa cells, and its acetylation is enhanced by coexpression of p300 or treatment with a HDAC inhibitor, trichostatin A. Increased PML acetylation is associated with increased sumoylation of PML in vitro and in vivo. PML is involved in trichostatin A-induced apoptosis and PML with an acetylation-defective mutation shows an inability to mediate apoptosis, suggesting the importance of PML acetylation. Our work provides new insights into PML regulation by post-translational modification and new information about the therapeutic mechanism of HDAC inhibitors.

Highlights

PML Exists as an Acetylated Protein in HeLa Cells Treated with Trichostatin A—Histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) induce differentiation, growth arrest, and apoptosis of cancer cells
We focused on one of these targets, PML, a multifunctional protein that is involved in apoptosis, tumor suppression, and cell cycle regulation [2]
The data presented here demonstrate that acetylation of PML may enhance its sumoylation and play an important role in the control of PML-dependent apoptosis in response to TSA exposure

Summary

EXPERIMENTAL PROCEDURES

Antibodies, Plasmids, and Cell Culture—The sources of antibodies and plasmids and the cell culture conditions are detailed in the supplemental data. Transient Transfection, Immunoprecipitation, Immunoblotting, Immunofluorescence, and in Vitro and in Vivo Acetylation Assays—These were performed as described previously [21, 28] except transient transfections into PMLϪ/Ϫ MEFs were performed by nucleofection using the Nucleofector system (Amaxa Biosystems) according to the manufacturer’s instructions. Antibody Array Assay and Detection and Quantification of Apoptosis—These are detailed in the supplemental data. In Vitro Sumoylation Assay—The in vitro sumoylation assay was performed essentially as described previously [21], except recombinant SUMO E1 ligase purchased from BIOMOL International was used instead of HeLa cell lysate. Cell Sorting—Cell sorting was performed using BD FACSAria Cell Sorter (BD Biosciences) according to the manufacturer’s instructions

RESULTS

PML demonstrated that acetylated

DISCUSSION