Joanna Fraczek

Biology and pathobiology of gap junctional channels in hepatocytes

The present review provides the state of the art of the current knowledge concerning gap junctional channels and their roles in liver functioning. In the first part, we summarize some relevant biochemical properties of hepatic gap junctional channels, including their structure and regulation. In the second part, we discuss the involvement of gap junctional channels in the occurrence of liver cell growth, liver cell differentiation, and liver cell death. We further exemplify their relevance in hepatic pathophysiology. Finally, a number of directions for future liver gap junctional channel research are proposed, and the up‐regulation of gap junctional channel activity as a novel strategy in (liver) cancer therapy is illustrated. (HEPATOLOGY 2007.)

Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation

Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cells differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro.

Toxicological and metabolic considerations for histone deacetylase inhibitors

Introduction: Vorinostat and romidepsin were the first histone deacetylase (HDAC) inhibitors (HDi) that fulfilled the preclinical promise of anticancer potential in clinical trials. Nevertheless, they merely opened a new chapter in the history of cancer therapy. Demonstration of their antitumor activity was a straightforward task in in vitro setting. Proving their efficacy in vivo was much more difficult, since the effects of an administrated drug strongly depend on its absorption, distribution, metabolism and excretion. Areas covered: This article summarizes clinical data on the pharmacokinetic properties of HDi that are currently at more advanced stages of clinical development. Specific attention is paid to the metabolic pathways. Moreover, a comprehensive overview of HDi-related adverse effects is given. Expert opinion: At this moment, HDi form one of the most interesting classes of therapeutics, yet their efficacy and safety profiles could still be improved by i) designing better formulations, ii) more extensive characterization of their disposition at the preclinical stage, iii) targeting of individual disease-related deacetylase isoforms and/or their complexes, iv) selecting a target patient population with the highest probability of response based on molecular signatures.

Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes

SummaryBoth, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

SummaryThe vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Histone deacetylase inhibitors in multiple myeloma

Novel drugs such as bortezomib and high-dose chemotherapy combined with stem cell transplantation improved the outcome of multiple myeloma patients in the past decade. However, multiple myeloma often remains incurable due to the development of drug resistance governed by the bone marrow microenvironment. Therefore targeting new pathways to overcome this resistance is needed. Histone deacetylase (HDAC) inhibitors represent a new class of anti-myeloma agents. Inhibiting HDACs results in histone hyperacetylation and alterations in chromatine structure, which, in turn, cause growth arrest differentiation and/or apoptosis in several tumor cells. Here we summarize the molecular actions of HDACi as a single agent or in combination with other drugs in different in vitro and in vivo myeloma models and in (pre-)clinical trials.

Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4-Me2N-BAVAH, a hydroxamate-based HDAC-inhibitor.

Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity. In the present study, the differentiation-stabilizing effect of a new histone deacetylase inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), a structural Trichostatin A (TSA)-analogue with a more favourable pharmaco-toxicological profile, was studied at a genome-wide scale by means of microarray analysis. Several genes coding for xenobiotic biotransformation enzymes were found to be positively regulated upon exposure to 4-Me(2)N-BAVAH. For CYP1A1/2B1/3A2, these observations were confirmed by qRT-PCR and immunoblot analysis. In addition, significantly higher 7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-dealkylase activity levels were measured. These effects were accompanied by an increased expression of CCAAT/enhancer binding protein alpha and hepatic nuclear factor (HNF)4α, but not of HNF1α. Finally, 4-Me(2)N-BAVAH was found to induce histone H3 acetylation at the proximal promoter of the albumin, CYP1A1 and CYP2B1 genes, suggesting that chromatin remodelling is directly involved in the transcriptional regulation of these genes. In conclusion, histone deacetylase inhibitors prove to be efficient agents for better maintaining a differentiated hepatic phenotype in rat hepatocyte cultures.

Epigenetic Regulation of Myeloma Within Its Bone Marrow Microenvironment

Epigenetic mechanisms play a crucial role in the normal development of the mammalian organism and are essential for maintaining the cell identity and normal functionality. Global changes in the epigenetic landscape associated with aberrant gene expression are a hallmark of cancer. Current knowledge indicates that both epigenetic alterations and genetic aberrations play an important role in the onset and progression of cancer. Recent findings have demonstrated that in cancer extensive reprogramming of all components of the epigenetic machinery (including DNA methylation, histone modifications and miRNA expression) takes place and have furthermore revealed the existence of a dynamic interplay between the different components. However, the exact sequence of events and underlying molecular mechanism contributing to carcinogenesis are only just beginning to be uncovered. Interestingly, the reversal of aberrant epigenetic modifications has emerged as a potential treatment strategy of cancer. Here, we describe the role of the epigenetic alterations in the pathogenesis of cancer focusing on the hematological malignancy multiple myeloma. In addition, recent advances regarding the relationship between histone modifications, chromatin-modifying enzymes, DNA methylation and miRNA expression are discussed.

Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture.

A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described.