Natalia Calonghi

9HSA is an endogenous product of lipid peroxidation identified in several human and murine cell lines. 9HSA exogenous administration to a human colon adenocarcinoma cell line (HT29) arrests the cells in G0/G1, the block of the cell cycle progression being characterized by an increase in the expression of p21WAF1 in an immediate-early, p53-independent fashion. The agents that promote p21 transcription by p53-independent mechanisms induce binding of different transcription factors to specific cis-acting elements located within the p21 promoter. The Sp1-3 site has been shown to be required for p21 induction by TGFbeta, calcium, lovastatin, and the histone deacetylase HDAC1 inhibitors. Acetylation of core nucleosomal histones is regulated by the opposing activities of histone acetyltransferases (HATs) and deacetylases (HDACs). HDACs catalyze removal of an acetyl group from the epsilon-amino group of lysine side chains of histones H2A, H2B, H3, and H4, thereby reconstituting the positive charge on lysine. Transcriptionally silent chromatin is composed of nucleosomes in which the histones have low levels of acetylation on the lysine residues of their amino-terminal tails. Acetylation of histone proteins neutralizes the positive charge on lysine residues and disrupts nucleosome structure, allowing unfolding of the associated DNA, access to transcription factors, and changes in gene expression. Several studies show that HDAC1 inhibitors induce G1 and/or G2 arrest, apoptosis and/or differentiation in many tumour cells. Various compounds have been identified as HADC1 inhibitors and charachterized for their anticancer potential: short-chain fatty acids (butyrate, phenylbutyrate, valproic acid), trapoxin (TPX), hydroxamic acids (suberoylanilide hydroxamic acid (SAHA),  piroxammide, trichostatin A (TSA), synthetic hybrids of hydroxamic acids and cyclic tetrapeptides).

In HT29 cells, such agents induce effects overimposable to those induced by 9HSA, whose activity as an HDAC1 inhibitor has been recently reported. The riconstruction of the 3D model of the human enzyme, not yet crystallized, and docking studies have evidenced that the hydroxyacid can interact with the catalytic site and inhibit its activity. HDAC1 inhibition causes growth arrest in G0/G1 in HT29 cells, increase of p21WAF1 but not of p27 KIP1 transcription, and triggering of cell differentiation toward a more benigne phenotype. Such effects have been found to be associated to a rapid acetylation of histone H4 and of several isoforms of H3 histones, not yet characterized. Preliminary data show that 9HSA treatment alters the binding of cyclin D1 to HDAC1. Cyclin D1 overexpression is found in various human cancers, is required for tumorigenesis, and is correlated with tumor invasion. In cancers from different tissues, and in particular in human colon cancer, cyclin D1 overexpression results rather from induction of oncogenic signals than from a clonal somathic mutation or rearrangements in the cyclin D1 gene.  Oncogenic factors, such as Ras, Src, ErbB2, beta-catenin, oncogenic Stats, and SV40 small t antigen, induce cyclin D1 expression through the binding to a distinct DNA sequence in the cyclin D1 promoter. Induction of cyclin D1 is growth factor-dependent and tightly regulated either at the activation level of transcription, or of protein expression, or of cellular localization. The protein appears in early G1, is rapidly induced following mitogen stimulation, and rapidly declines when these factors are withdrawn. In particular the abundance of cyclin D1 is caused by growth factors such as EGF, IGF1, IGF2, amino acids, lysophosphatidic acid (LPA), estrogens, androgens, retinoic acid, secreted factors from adipocytes, and gastrointestinal hormones such as gastrin. The main role of cyclin D1 is played via its association with Cdk4 that initiates Rb phosphorylation, relieves HDAC-binding and allows transcriptional activation of the S-phase genes. Following its association with Cdk4, cyclin D1 is phosphorylated on Thr 286 by glicogen synthetase 3beta (GSK-3beta); this represents a mechanism by which the protein is translocated out of the nucleus to induce its own proteasomal degradation. Interestingly, free cyclin D1 is ubiquitinated by a different mechanism that does not require GSK-3beta or the phosphorylation on Thr286; this seems to suggest the existence of two different pools of cyclin D1, free and bound to Cdk4, regulated by different mechanisms and with possible different functions. In particular, at the nuclear level cyclin D1 regulates cell growth, metabolism, and differentiation through its interaction with more than 30 transcription factors, coactivators, and corepressors that govern histone acetylation and chromatin remodeling proteins.

To date, the better understood mechanism that involves cyclin D1 in transcription is that by which the protein inhibits adipocytes differentiation through PPARgamma (peroxisome proliferator-activated receptor gamma) repression, and HDAC1 and histone-metiltransferases recruitment to PPARgamma response element (PPARE) of lipoprotein lipase (LPL) promoter. Recent studies have shown that cyclin D1 coprecipitates with HDAC1 and that their interaction is functionally relevant, because it regulates the acetylation of  Lysine 9 of histone H3 in the chromatin proximal to the PPARE of the LPL promoter. PPARgamma is a nuclear receptor that forms an eterodimer with the retinoic acid receptor, and the complex binds to the PPARE promoters of the target genes. In the colon, PPARgamma expression varies along the crypt axis, its level being higher in postmitotic cells that form the intestinal lumen. The ligand dependent activation of such receptor lowers proliferation in several cell lines, and in some cases growth inhibition associates to differentiation. 9HSA inhibition of HDAC1 can on one hand determine chromatin modification by hyperacetylation of specific histones, and on the other favour cyclin D1 access to promoters that it regulates, thus triggering precise programmes of differentiation and growth arrest.