Hyperproliferation, uncontrolled inflammation and a distorted balance between cell survival and apoptosis could underlie a multitude of pathologies. Consequently, HDIs are perceived as promising drug candidates in the treatment of a wide range of diseases (reviewed by ), including fibroproliferative disorders, such as liver fibrosis, systemic sclerosis, pulmonary fibrosis and renal fibrosis. HDI-based treatment could also involve diseases of dysregulated immunity, i.e. autoimmune disorders (for review see [38, 72–74]). This is, however, not the end of the list. As demonstrated by recent research, HDIs may also be valuable tools in the prevention of neuronal degeneration as occurring in, e.g. Huntington’s disease [75–77]. It has also been suggested that HDIs could be helpful in combating infections with protozoan parasites  or with viruses, including HIV [79–81].
Physiological condition: effects of histone deacetylase inhibitors on primary hepatocytes
The majority of somatic cells in the mature organism stops dividing and enters a state of quiescence, i.e. G0-phase of the cell cycle. Some of these cells will irreversibly lose their ability to proliferate and finally progress into a state of senescence, whereas others retain their capacity to divide upon stimulation . Hepatocytes which represent the predominant cell type in the liver (∼80% of the organ mass) belong to the latter group of cells that are semi-permanently withdrawn from the cell cycle . These parenchymal cells perform multiple metabolic and secretory functions including protein synthesis, storage and transformation of carbohydrates, synthesis of cholesterol, bile salts and phospholipids, metabolism of endogenous substances and, most importantly, biotransformation of xenobiotics . In fact, the majority of drugs undergoes biotransformation in the liver. In particular oral drugs are absorbed by the gut and transported via portal circulation directly to the liver.
Due to the high proliferative capacity of the hepatocytes, the liver is among the few internal organs that are capable of natural regeneration of lost tissue. This capability is imperative for its survival. In the case of acute liver injury, hepatocyte proliferation is aimed at replacing necrotic and apoptotic cells . Basically, all types of cells in the liver, namely hepatic stellate cells (HSCs), Kupffer cells, Pit cells, bile duct epithelial cells and fenestrated endothelial cells, contribute to the regeneration of liver parenchyma, not only by their own proliferation, but also by stimulation of hepatocyte proliferation .
The in vivo regenerative response in the liver is manifested by a semi-synchronous cell-cycle re-entry of surviving hepatocytes, accompanied by an induction of a number of differentiation-promoting pathways [87, 88]. These allow preservation of metabolic homeostasis during the regeneration of the organ. In vitro, the proliferation and differentiation programmes are inversely related. Thus, in sharp contrast to the in vivo situation, cultured primary hepatocytes are unable to redifferentiate upon cell division and lose their liver-specific functions . Therefore, currently available in vitro models based on primary hepatocytes fail to effectively reproduce the in vivo status of the cells for longer periods, restricting their applicability in, for instance, long-term pharmaco-toxicological tests of new chemical entities and implying the use of animal-based tests.
Our previous research clearly shows that HDI therapy may be beneficial for cultured primary rat hepatocytes. Exposure of hepatocytes to HDIs allows overcoming the isolation procedure-induced cell cycle re-entry of the cells . As a consequence, mitogen-stimulated primary hepatocytes cease to proliferate in the presence of TSA. The specific timing of cell cycle arrest is dictated by the onset of exposure to the HDI involved . We found that hepatocytes exposed to TSA from cell seeding onwards respond with an early S-phase cell cycle arrest, as demonstrated by block of DNA replication and lack of the S/G2/M-phase marker cdk1 (Fig. 3). When HDI exposure is initiated already during the isolation process of the cells, namely when hepatocytes are still in the state of considerable quiescence, neither the proto-oncogene c-jun, nor cyclin D1 could be detected, pointing towards an early G1-phase cell cycle arrest . Interestingly, ω-carboxypenthyl p-dimethylaminobenzamide hydroxamate (4-Me2N-BAVAH), a metabolically more stable structural TSA analogue, is a more potent inhibitor of hepatocyte proliferation. Indeed, we found that 4-Me2N-BAVAH could already promote G1-cell cycle arrest when exposed from the time of plating onwards. Unexpectedly, p21Cip1, controlling the progression through the G1-phase and as such a potential mediator of the observed cell cycle arrest, was not induced. Instead, diminished activation of transcription factor NF-κβ seemed to underlie the transcriptional repression of the cyclin D1, although, the exact mechanism remains to be elucidated . HDACs regulate NF-κβ signalling pathways at multiple levels. For instance, both HDAC-1 and HDAC-3 have been implicated in the transcriptional silencing of NF-κβ-controlled genes [92–94]. In addition, HDAC-3 directly deacetylates the transcription factor promoting its export from the nucleus and sequestration by its inhibitor, Iκβ. Inhibition of deacetylase activity would augment the NF-κβ function by relieving HDAC-mediated repression. Nevertheless, a study in mouse epithelial JB6 cells revealed that HDIs down regulated cyclin D1 expression by diminishing NF-κβ-DNA binding . Another study could demonstrate that TSA prevented the phosphorylation of the inhibitor Iκβ and its subsequent degradation, resulting in a decreased NF-κβ activation . Whether similar mechanisms are responsible for the inhibition of NF-κβ activation in primary hepatocytes upon exposure to HDIs is still to be determined.
Our finding, namely a p21Cip1-independent cell cycle arrest in response to exposure of primary hepatocytes to HDIs, was confirmed by another group . Indeed, normal human skin fibroblasts and primary neuronal cells did not demonstrate p21Cip1 up-regulation upon HDI exposure. The latter findings may suggest that p21Cip1-independent cell cycle arrest is a common feature of HDAC inhibition in healthy, (primary) cells [23, 99]. Nevertheless, there is a subtle indication that HDACs control p21Cip1 expression in hepatic tissue. Indeed, forced expression of human HDAC-1 diminishes the expression of p21Cip1 mRNA in mouse liver [64, 65]. Unfortunately, the authors did not clarify whether this phenomenon was of a primary or secondary nature. Accordingly, the following question was not answered; was an increased HDAC-1 activity at the p21Cip1 gene promoter responsible for observed decrease of p21Cip1 mRNA or rather the diminished production of its upstream regulator, i.e. p53? Furthermore, the expression of p21Cip1 protein in response to HDAC-1 overexpression was not investigated [64, 65].
Recently, new light was shed on the role of HDAC-1 in the control of liver proliferation by Wang et al.. They showed that in the quiescent liver of aged mice HDAC-1 protein was elevated and interacted with the negative regulator of liver proliferation, namely CCAAT-enhancer-binding protein (C/EBP)-α. The latter guided the HDAC-1 enzyme to E2F-responsive gene promoters enabling deacetylation of local histones and subsequent transcriptional silencing of the corresponding genes. The expression of proliferation-specific transcription factors, i.e. Forkhead box M1 B (FoxM1B) and c-myc, was inhibited in the former manner reflecting a diminished ability of the liver to regenerate during the aging process. Furthermore, the observed increase of the HDAC-1 protein was cyclin D3-dependent and resulted from an intensified translation of HDAC-1 mRNA . Surprisingly, HDAC-1 was also elevated in the liver of young mice undergoing partial hepatectomy (PH) [64, 65, 101]. Nonetheless, as opposed to the previous study, a related transcription factor, namely C/EBP-β acted as a primary HDAC-1 interaction partner instead of C/EBP-α. Following PH, the HDAC-1-C/EBP-β complex repressed the transcription of the former C/EBP isoform, and by doing so abolished the C/EBP-α-mediated block of liver proliferation . A substantial role of HDAC-1 in this process was underlined by the fact that depletion of the enzyme by siRNA technology prevented cyclin D1 and PCNA induction upon PH, whereas C/EBP-α protein was expressed at high levels .
Our research group has shown that apart from cell proliferation, HDACs also modulate other aspects of hepatocyte physiology. As such, exposure of cultured adult rat hepatocytes to TSA improved both their xenobiotic biotransformation capacities and albumin secretion, and their gap junctional intercellular communication [102–104]. Additionally, an increased survival rate of hepatocytes was seen in conventional monolayer cultures by delaying activation of programmed cell death pathways [104, 105].