Induction and superinduction of growth arrest and DNA damage gene 45 (GADD45) α and β messenger RNAs by histone deacetylase inhibitors trichostatin A (TSA) and butyrate in SW620 human colon carcinoma cells
Introduction
Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are two classes of protein-modifying enzymes that catalyze the addition and removal of acetyl groups onto and from ϵ-NH2 of lysine residues of proteins including histone, transcription factors and co-factors [1], [2], [3], [4], [5], [6]. Recent evidences suggested important roles for these two families of enzymes in diverse cellular functions such as chromatin structure remodeling, gene silencing, cell proliferation, apoptosis and cellular signal transduction [6], [7], [8], [9].
Ten different HDACs have been identified [6], [8], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20] in human. They are classified into two groups: class I HDAC encompasses HDAC1–3 and 8, while class II HDAC contains HDAC4–7, 9 and 10. Class I HDACs are smaller proteins with approximately 300–500 amino acids, while class II HDACs are larger proteins that have close to 1000 amino acids. The non-catalytic N-terminus may play a regulatory role. Recent investigations demonstrate that HDAC4 and 5 are phosphorylated by CaM-kinase and phosphorylation modulates translocation of HDACs from cytoplasm into nucleus [21], [22], [23]. In addition to these two classes of HDACs, a new type of HDAC was identified in yeast, which requires nicotinamide adenine dinucleotide (NAD) as a cofactor for deacetylase enzymatic activity [8], [24], [25].
Histone deacetylase inhibitors such as trichostatin A (TSA) and butyrate profoundly affect diverse aspects of cell physiology. TSA and butyrate have been shown to inhibit cancer cell growth in vitro [26], [27] and in vivo [28], [29], revert oncogene-transformed cell morphology [26], [30], [31], induce apoptosis [9], [32] and enhance cell differentiation [33], [34], [35]. The effects of TSA and butyrate on cells are presumed to be primarily mediated by inhibition of histone deacetylases. Several investigators have demonstrated that the Cdk inhibitor p21(cip/waf) is a target gene of TSA [36], [37], [38], [39]. TSA induced p21(cip/waf) messenger RNA (mRNA) and protein synthesis and this seemed to correlate with the growth-arresting effect of TSA. It was hypothesized that TSA induces cell growth arrest primarily via p21(cip/waf) induction [40], [41]. Indeed, in HCT116 human colon cells with the p21(cip/waf) gene deleted, the growth inhibitory effect of both TSA and butyrate was diminished [40]. However, in primary mouse embryonic fibroblast derived from p21(cip/waf) knockout mice, butyrate still induced G1 arrest [42]. In addition, Kim et al. [43] observed that in p21 (−/−) colon carcinoma, TSA was capable of inducing apoptosis. Thus the role of p21(waf/cip) in TSA-induced cell growth arrest and apoptosis remains to be fully understood and it may not be the sole target gene responsible for TSA- and/or butyrate-induced cell growth arrest.
Here we describe the identification of growth arrest and DNA damage gene 45α (GADD45α) and β as TSA and butyrate-inducible genes. Our results indicated TSA was a potent inducer of apoptosis for human colon carcinoma cell line SW620 and in this cell line, GADD45α and β were rapidly induced by TSA and butyrate. This induction was independent of protein synthesis. In addition, cyclohexamide (CHX) super-induced GADD45 in the presence of TSA and butyrate. Two protein kinase inhibitors, H7 and H8, were found to block both the induction of GADD45 mRNA and apoptosis induced by TSA. These data suggest that GADD45 may play an important role in TSA-induced cell growth arrest and apoptosis.
Section snippets
Materials
Nylon membrane (Biotran) was purchased from ICN Biotechnologies (Costa Mesa, CA). α-32P-dATP (3000 Ci/mmol) was purchased from NEN-Dupont (Boston, MA). Oligo-dT agarose was obtained from Pharmacia Biotech (Piscataway, NJ). Routine molecular cloning and sequence analyzes were performed as previously described [44]. Random priming labeling kits were obtained from Promega Inc. (Madison, WI). TSA and butyrate were purchased from Sigma Inc. (St. Louis, MO). H7, H8 and curcumin were obtained from
Expression of HDACs in SW620 cells
To gain insight into the effects of HDAC inhibitors on colon cancer cells, we examine the expression profile of different HDACs in SW620 cell line. Northern blot analysis was performed to compare the level of expression of different HDACs. Fig. 1 showed that several HDACs were prominently expressed in SW620 cells, including HDAC1–3, whereas the expression levels of HDAC4–6 and 8 were much lower. A modest increase of HDAC3 mRNA was observed when SW620 cells were treated with TSA. This result was
Discussion
HDAC inhibitors have been proposed to be potential anti-cancer therapeutics and several inhibitors are being evaluated in human clinical trials [54], [55]. However, it is still not completely understood how a HDAC inhibitor induces cell growth arrest and apoptosis. The prevailing hypothesis is that HDAC inhibitors influence transcription of important regulatory genes that in some way lead to cell arrest and apoptosis. In support of this view, de novo protein synthesis was found to be required
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