Regulation of the expression of claudin 23 by the enhancer of zeste 2 polycomb group protein in colorectal cancer.

Altered epigenetic mechanisms, similar to gene mutations, contribute to the pathogenesis and molecular heterogeneity of neoplasms, including colorectal cancer (CRC). Enhancer of zeste 2 (EZH2) is a histone methyltransferase, which is involved in epigenetic gene silencing and is aberrantly expressed in CRC. Therefore, the identification of the genes regulated by EZH2 in CRC is important to improve current understanding of its role in cancer epigenetics. The present study used chromatin immunoprecipitation (ChIP) followed by deep sequencing to assess genome-wide EZH2­DNA interactions in healthy or CRC mucosa samples. In total, 86.9/61.6 and 92.5/62.6 million tags were sequenced/mapped in healthy and CRC mucosa samples, respectively. The EZH2-binding densities were correlated with transcriptomic datasets and this demonstrated that the claudin-23 (CLDN23) gene, which encodes a component of cell-cell adhesion structures, was occupied by EZH2 and significantly silenced in CRC tissue. The measurement of DNA methylation at the CLDN23 promoter using pyrosequencing excluded the possibility that silencing of this gene in CRC patient samples was a result of DNA hypermethylation. Following treatment of the Colo205 and HT-29 CRC cell lines, with the EZH2 inhibitor, GSK126, the level of histone H3 lysine 27 trimethylation (H3K27me3) was reduced and the mRNA and protein expression levels of CLDN23 were increased. ChIP analysis confirmed that the level of H3K27m3 along the CLDN23 gene was decreased in the GSK126-treated cell lines. Furthermore, ChIP analysis of these samples detected histone H3 lysine 4 trimethylation (H3K4me3) at the CLDN23 promoter, demonstrating that the balance between H3K27me3 and H3K4me3 may underlie the regulation of the expression of CLDN23. The present study demonstrated an epigenetic link between the activity of the EZH2 methyltransferase at the CLDN23 locus and the expression of CLDN23 in CRC tissue.

The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice.

The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.

Quantitative trait loci analysis for peripheral blood parameters in a (BALB/cW × C57BL/6J-Mpl (hlb219)/J) F(2) mice.

The genetic basis of the peripheral blood cell parameters is not fully elucidated. Thus, it is essential to research the correlation between blood cell counts levels and the genome in laboratory animals and subsequently in humans. In the present study, we examined 288 F(2) mice from a cross between BALB/cW and C57BL/6J-Mpl(hlb219)/J. The C57BL/6J-Mpl (hlb219)/J strain is a mouse model of thrombocytopenia. We found very strong correlations for PLT counts and revealed some highly significant correlations for RBC counts. On the basis of the obtained results, we presume that genetic control of erythrocyte parameters is divided into two pathways: first, the morphological determinants responsible for the red blood cell count (RBC), hematocrit (HCT), and mean corpuscular volume (MCV), and second, the functional pathway determining the hemoglobin content (HGB). The locus on Chromosome 4 is the only detected quantitative trait locus (QTL) influencing the analyzed platelets parameters. We also detected highly significant correlations for erythrocyte parameters on Chromosome 1 (RBC, MCV, MCH), Chr 7 (HGB), Chr 9 (MCHC), Chr 11 (RBC), and Chr 17 (MCH). Finally, with regards to the given correlations, using the Mouse Genome Database resource, we proposed candidate genes with possible meaning for the level of these parameters: cytokine receptor genes (e.g., Mpl), transcription factor genes (e.g., Xbp1, Ikzf1), hemoglobin chain genes (e.g., Hbb-b1, Hbb-ar), and many others localized in the confidence intervals of found QTLs.