The pharmaceutical potential of histone deacetylase inhibitors.

Protein acetylation, catalyzed by the opposing activities of histone deacetylases (HDAC) and histone acetyltransferases, is now recognized to be an important epigenetic modulator of gene transcriptional activity and cell function. As a result of the intense search for HDAC inhibitors (HDACi) during the past fifteen years, a large number of structurally divergent classes with variable potencies and isoenzyme selectivities have been identified. They occupy an important and promising position in a number of therapeutic areas.

Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes.

Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated.

Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.

Primary hepatocytes and their cultures are a simple but versatile, well-controlled, and relatively easy to handle in vitro system that is well-accepted for investigating xenobiotic biotransformation, enzyme induction and inhibition, and (biotransformation-mediated) hepatotoxicity. In addition, hepatocyte cultures have proven to be valuable tools in the study of liver physiology, viral hepatitis, and liver regeneration and are proposed as an alternative to orthotopic liver transplantation. It has been observed, however, that a number of liver-specific functions are progressively lost with time when hepatocytes are isolated and cultivated.

Hepatocytes in suspension.

Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the "gold standard" for investigating xenobiotic biotransformation and metabolic bioactivation. When used in suspension, they provide an easy-to-handle and relatively cheap in vitro system that can be used for up to 4 h.

Rat hepatocyte cultures: collagen gel sandwich and immobilization cultures.

Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g.

Rat hepatocyte cultures: conventional monolayer and cocultures with rat liver epithelial cells.

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended lifespan and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions.

Isolation of rat hepatocytes.

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined.

Rat Hepatocyte Cultures : Collagen Gel Sandwich and Immobilization Cultures.

Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g.

Rat Hepatocyte Cultures : Conventional Monolayer and Cocultures With Rat Liver Epithelial Cells.

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended life-span and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions.

Differential effects of histone deacetylase inhibitors in tumor and normal cells-what is the toxicological relevance?

Histone deacetylase (HDAC) inhibitors target key steps of tumor development: They inhibit proliferation, induce differentiation and/or apoptosis, and exhibit potent antimetastatic and antiangiogenic properties in transformed cells in vitro and in vivo. Preliminary studies in animal models have revealed a relatively high tumor selectivity of HDAC inhibitors, strenghtening their promising potential in cancer chemotherapy. Until now, preclinical in vitro research has almost exclusively been performed in cancer cell lines and oncogene-transformed cells.

Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium.

Isolated hepatocytes in suspension express most of the functional activities of the intact liver and offer an easy-to-handle in vitro system for investigating both the biotransformation and damaging effects induced after a single exposure to xenobiotics upto 3-4h. There is, however, a general lack of consensus with respect to the choice of a suitable suspension medium. This motivated us to perform a comparative study of the effects of five frequently used bicarbonate-based media (Ca(2+)-containing Krebs-Henseleit buffer (KHB) with or without 25mM HEPES, 10mM glucose and 2% (g/v) BSA supplements, and Williams' E culture medium) on the viability (LDH leakage, caspase-3 processing and activity, Bid/Bax expression) and functionality (energy status, glutathione content, phases I and II biotransformation) of freshly isolated rat hepatocytes in suspension upto 3h.

Trichostatin A-like hydroxamate histone deacetylase inhibitors as therapeutic agents: toxicological point of view.

Modulation of chromatin structure through histone acetylation/deacetylation is known to be one of the major mechanisms involved in the regulation of gene expression. Two opposing enzyme activities determine the acetylation state of histones: histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively acetylating or deacetylating the epsilon-amino groups of lysine residues located in the amino-terminal tails of the histones. In general, transcriptionally active chromatin is associated with hyperacetylated histones, whilst silenced chromatin is linked to hypoacetylated histones.

Rat hepatocyte suspensions as a suitable in vitro model for studying the biotransformation of histone deacetylase inhibitors.

This paper focuses on the use of liver-derived in vitro systems for biotransformation studies during early drug development, as exemplified by the two molecules recently studied in our laboratory: Trichostatin A (TSA) and its structural analogue 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me2N-BAVAH). Phase I biotransformation of TSA, a histone deacetylase inhibitor with promising antifibrotic and antitumoural properties, was investigated in liver microsomal (rat and human) and in hepatocyte (rat) suspensions. Within 40 minutes, 50 microM of TSA was completely metabolised by 2 x 10(6) hepatocytes/ml.

Proliferation of epidermal growth factor-stimulated hepatocytes in a hormonally defined serum-free medium.

The present study shows that adult rat hepatocytes in primary culture, which normally exhibit a restricted capacity to proliferate, can proceed through the cell cycle when cultured in a mixture of minimal essential medium (MEM) and Medium 199 (MEM-M199; 3:1, v/v), containing epidermal growth factor (EGF; 50 ng/ml), low glucose (0.75 g/l) and low levels of inorganic salts, amino acids and vitamins. Under these conditions, hepatocytes flatten and cell extensions appear.

Trichostatin A induces differential cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.

Background/aims: The effects of Trichostatin A (TSA), a drug candidate for cancer therapy, on proliferation and survival of primary hepatocytes, the major site of xenobiotic biotransformation and primary target of drug-induced toxicity, were investigated.

Methods: DNA replication was measured using [methyl-3H]-thymidine incorporation. Cell cycle markers were analyzed by Western and Northern blottings.