Regulation of Chk2 gene expression in lymphoid malignancies: involvement of epigenetic mechanisms in Hodgkin's lymphoma cell lines

The tumor suppressor Chk2 kinase plays crucial roles in regulating cell-cycle checkpoints and apoptosis following DNA damage. We investigated the expression levels of the genes encoding Chk2 and several cell-cycle regulators in nine cell lines from lymphoid malignancies, including three Hodgkin's lymphoma (HL) lines. We found that all HL cell lines exhibited a drastic reduction in Chk2 expression without any apparent mutation of the Chk2 gene. However, expression of Chk2 in HL cells was restored following treatment with the histone deacetylase inhibitors trichostatin A (TsA) and sodium butyrate (SB), or with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC). Chromatin-immunoprecipitation (Chip) assays revealed that treatment of HL cells with TsA, SB or 5Aza-dC resulted in increased levels of acetylated histones H3 and H4, and decreased levels of dimethylated H3 lysine 9 at the Chk2 promoter. These results indicate that expression of the Chk2 gene is downregulated in HL cells via epigenetic mechanisms.     

Nucleosome destabilization in the epigenetic regulation of gene expression

Assembly, mobilization and disassembly of nucleosomes can influence the regulation of gene expression and other processes that act on eukaryotic DNA. Distinct nucleosome-assembly pathways deposit dimeric subunits behind the replication fork or at sites of active processes that mobilize pre-existing nucleosomes. Replication-coupled nucleosome assembly appears to be the default process that maintains silent chromatin, counteracted by active processes that destabilize nucleosomes. Nucleosome stability is regulated by the combined effects of nucleosome-positioning sequences, histone chaperones, ATP-dependent nucleosome remodellers, post-translational modifications and histone variants. Recent studies suggest that histone turnover helps to maintain continuous access to sequence-specific DNA-binding proteins that regulate epigenetic inheritance, providing a dynamic alternative to histone-marking models for the propagation of active chromatin.     

Targeting proteasome inhibition in hematologic malignancies

Proteasome inhibitors represent potential novel anti-cancer therapy. These agents inhibit the degradation of multi-ubiquitinated target proteins mediating cell cycle progression, apoptosis, NF-kappa B activation, inflammation, cell cycle regulatory proteins such as cyclins and cyclin dependent kinase inhibitors, as well as immune surveillance; and regulate anti-apoptosis and cell cycle progression. Proteasome inhibitors also directly induce caspase-dependent apoptosis of tumor cells, despite the accumulation of p21 and p27 and irrespective of the p53 wild type or mutant status. Recent studies demonstrate that PS-341, peptide boronate, has remarkable anti-tumor activity in preclinical and clinical studies, not only in multiple myeloma but also in other malignancies.     

Stochastic and epigenetic changes of gene expression in Arabidopsis polyploids

Polyploidization is an abrupt speciation mechanism for eukaryotes and is especially common in plants. However, little is known about patterns and mechanisms of gene regulation during early stages of polyploid formation. Here we analyzed differential expression patterns of the progenitors' genes among successive selfing generations and independent lineages. The synthetic Arabidopsis allotetraploid lines were produced by a genetic cross between A. thaliana and A. arenosa autotetraploids. We found that some progenitors' genes are differentially expressed in early generations, whereas other genes are silenced in late generations or among different siblings within a selfing generation, suggesting that the silencing of progenitors' genes is rapidly and/or stochastically established. Moreover, a subset of genes is affected in autotetraploid and multiple independent allotetraploid lines and in A. suecica, a natural allotetraploid derived from A. thaliana and A. arenosa, indicating locus-specific susceptibility to ploidy-dependent gene regulation. The role of DNA methylation in silencing progenitors' genes is tested in DNA-hypomethylation transgenic lines of A. suecica using RNA interference (RNAi). Two silenced genes are reactivated in both ddm1- and met1-RNAi lines, consistent with the demethylation of centromeric repeats and gene-specific regions in the genome. A rapid and stochastic process of differential gene expression is reinforced by epigenetic regulation during polyploid formation and evolution.     

Targeting epigenetic mechanisms to overcome venetoclax resistance

The BH-3 mimetic venetoclax overcomes apoptosis and therapy resistance caused by high expression of BCL2 or loss of BH3-only protein function. Although a promising therapy for hematologic malignancies, increased expression of anti-apoptotic MCL-1 or BCL-XL, as well as other resistance mechanisms prevent a durable response to venetoclax. Recent studies demonstrate that agents targeting epigenetic mechanisms such as DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone methyltransferase EZH2 inhibitors, or bromodomain reader protein inhibitors may disable oncogenic gene expression signatures responsible for venetoclax resistance. Combination therapies including venetoclax and epigenetic therapies are effective in preclinical models and the subject of many current clinical trials. Here we review epigenetic strategies to overcome venetoclax resistance mechanisms in hematologic malignancies.     

Targeting of gene expression by siRNA in CML primary cells

Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells—mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22–37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.     

Review of Non-coding RNAs and the epigenetic regulation of gene expression

Advances in sequencing and detection technology over the past two decades, highlighted by the data explosion brought about by the human genome project, have transformed what was previously assumed to be a relatively simple genetic landscape into a new picture where the so-called “dark matter” of the genome has stolen the spotlight from the not so hip protein-coding genes. The simplified central dogma of molecular biology, in which a gene encodes for a protein via a messenger RNA (mRNA), is still at the core of genetics but is now caught in a much more complex web of regulation by the genomic region previously known as “junk” DNA. Books such as Non-coding RNAs and epigenetic regulation of gene expression, published by Caister Academic Press, become essential guidelines to help us understand the current status of the very fast paced field of RNA research, which has only just started to uncover the roles of non-coding RNAs (ncRNAs) in the regulation of gene expression.     

Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

Background  

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (β-actin, VEGF, oct4, TERT, H19 and Igf2) and a repetitive sequence (art2) in five organs (heart, liver, spleen, lung and kidney) from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3), the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3) died after the perinatal period. Normally reproduced cattle served as a control group (n = 3).