Proteins implicated in the increase of adhesivity induced by suberoylanilide hydroxamic acid in leukemic cells D. Grebeňová a , P. Röselová a , M. Pluskalová a , P. Halada b , D. Rösel c , J. Suttnar a , B. Brodská a , P. Otevřelová a , K. Kuželová a, ⁎ a Institute of Hematology and Blood Transfusion, Prague, Czech Republic b Institute of Microbiology, Academy of Science of the Czech Republic, Prague, Czech Republic c Charles University, Faculty of Science, Prague, Czech Republic ARTICLE INFO ABSTRACT Article history: Received 24 January 2012 Accepted 12 September 2012 Available online 25 September 2012 We have previously shown that suberoylanilide hydroxamic acid (SAHA) treatment increases the adhesivity of leukemic cells to fibronectin at clinically relevant concentrations. Now, we present the results of the proteomic analysis of SAHA effects on leukemic cell lines using 2-DE and ProteomLab PF2D system. Histone acetylation at all studied acetylation sites reached the maximal level after 5 to 10 h of SAHA treatment. No difference in histone acetylation between subtoxic and toxic SAHA doses was observed. SAHA treatment induced cofilin phosphorylation at Ser3, an increase in vimentin and paxillin expression and a decrease in stathmin expression as confirmed by western-blotting and immunofluorescence microscopy. The interaction of cofilin with 14-3-3 epsilon was documented using both Duolink system and coimmunoprecipitation. However, this interaction was independent of cofilin Ser3 phosphorylation and the amount of 14-3-3-ε-bound cofilin did not rise following SAHA treatment. SAHA-induced increase in the cell adhesivity was associated with an increase in PAK phosphorylation in CML-T1 cells and was abrogated by simultaneous treatment with IPA-3, a PAK inhibitor. The effects of SAHA on JURL-MK1 cells were similar to those of other histone deacetylase inhibitors, tubastatin A and sodium butyrate. The proteome analysis also revealed several potential non-histone targets of histone deacetylases. © 2012 Elsevier B.V. All rights reserved. Keywords: SAHA Adhesion Cofilin Vimentin PAK HDAC inhibitor 1. Introduction Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysines of various proteins. The most prominent HDAC targets are histones, which are tightly bound to nuclear DNA and are important for its organization into nucleosomes. Different posttranslational modifications, including acetylation of core histones, regulate the rate of gene transcription, thus HDAC inhibition results in changes in the expression level of a large number of genes [1]. HDACs target not only the histones, but also many other protein substrates, e.g. the heat shock protein HSP90 or tubulin [2]. HDAC inhibitors (HDACi) are emerging as promising drugs in treatment of cancer and as anti-inflammatory agents [3,4]. HDACi induce different and pleiotropic effects in various transformed cells including growth arrest, activation of the extrinsic or intrinsic apoptotic pathways, autophagy, reactive oxygen species-induced cell death, mitotic cell death and senescence [5]. On the other hand, normal cells are considerably more resistant to HDACi. Suberoylanilide hydroxamic acid (SAHA) was the first HDACi approved by U.S. Food and Drug JOURNAL OF PROTEOMICS 77 (2012) 406 – 422 ⁎ Corresponding author at: Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague, Czech Republic. Tel.: +420 221 977 285; fax: +420 221 977 113. E-mail address: Katerina.Kuzelova@uhkt.cz (K. Kuželová). 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2012.09.014 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot