BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 673–677 (1997) ARTICLE NO. RC976515 Down-Regulation of Protein Kinase CKII Activity by Sodium Butyrate Gian Luigi Russo,* , † ,1 Valentina Della Pietra,* , † Ciro Mercurio,† ,2 Fulvio Della Ragione,† Daniel R. Marshak,‡ Adriana Oliva,† and Vincenzo Zappia* , † *I.S.A. Institute of Food Science and Technology, National Research Council, Avellino, Italy; †Institute of Biochemistry of Macromolecules, Second University of Naples, Naples, Italy; and ‡Osiris Therapeutics, Inc., Baltimore, Maryland 21231 Received March 6, 1997 greatly increased since the demonstration that the mol- Butyrate, a dietary fiber derivative, is a well-known ecule exerts a potent differentiating and antiprolifera- differentiating agent in cultured cell lines. In addition, tive effect both in vitro and in vivo (3-5). its antineoplastic activity toward colon-rectum can- Despite the quantity of data describing the anti-can- cers has been documented both in vivo and in vitro. cer effects of butyric acid, its mechanism(s) of action is Despite the large amount of information on the poten- still unknown. The molecule affects gene expression at tial clinical efficacy of butyrate, its mechanism of ac- different levels (3), but few data are available on its tion at the molecular level has only been partially intracellular targets. investigated. Here, we show that serine/threonine pro- A number of circumstantial observations led us to the tein kinase CKII is a target of butyrate activity. In the hypothesis that the serine/threonine protein kinase CKII human adenocarcinoma cell line, HT29, treated with (formerly known as casein kinase II) could be a potential 2 mM sodium butyrate, CKII activity decreases 50% at candidate for butyrate activity. Firstly, sodium butyrate 24 and 48 hours after drug addition. The enzyme down- (NaBt) induces cell cycle arrest in early G1 (6, 7); CKII regulation is not due to changes in protein amount is involved in cell cycle regulation in HeLa cell by phos- since the levels of the different CKII subunits remain phorylating Cdc2 kinase in G1 (8), and in the yeast Sac- constant during butyrate treatment. The data re- charomyces cerevisiae, CKII is essential for cell cycle pro- ported provide the first evidence that CKII down-regu- gression (9). Secondly, NaBt is a potent differentiating lation is involved in the signal transduction pathway agent, and CKII activity is reported to be regulated by started by butyrate.  1997 Academic Press several exogenous factors that induce post-translational modifications of its subunits (10, 11). Thirdly, the expres- sion of several proto-oncogenes is modified following Since 1960, epidemiological data have demonstrated treatment of cell lines with butyrate (3); the same tran- a protective effect of dietary fiber intake against colon scriptional factors such as Jun and Myc are also regu- cancer development (1,2). Among the possible mecha- lated by CKII phosphorylation (10, 12). Finally, CKII ac- nisms evoked to explain the role of dietary fiber in car- tivity is high in solid tumors (13), in some forms of human cinogenesis, the local production of butyric acid proba- leukemia (14), and more recently, its catalytic subunit, bly plays an important role. Butyric acid is a short a, causes neonatal leukemia when co-expressed with c- chain fatty acid produced by bacterial degradation of myc in transgenic mouse (15). poorly fermentable dietary fibers in the region of colon- In this report, we studied the effect of NaBt on CKII rectum (3). Interest in the action of butyrate was regulation in the HT29 cell line derived from a human colon adenocarcinoma (16). We demonstrated that NaBt, at the concentration of 2 mM, down-regulates 1 Corresponding author. Istituto Scienze Dell’Alimentazione- significantly CKII activity, leading to hypothesize that CNR, via Roma 52 A-C, 83100, Avellino, Italy. Fax: /39 825 781585. changes in CKII activity represent an important step E-mail: glrusso@alpha.szn.it. in mediating butyrate mechanism of action. 2 Present address: European Institute of Oncology, 20141, Milan, Italy. MATERIALS AND METHODS Abbreviations: CKII, casein kinase II; NaBt, sodium butyrate; PBS, phosphate buffer saline solution; PAGE, polyacrylamide gel Cell culture. HT29 cells (American Type Cell Collection, Rock- ville, MD) derived from a human colon adenocarcinoma were rou- electrophoresis. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 673