The Effect of Chronic Mild Stress and Escitalopram on the Expression and Methylation Levels of Genes Involved in the Oxidative and Nitrosative Stresses as Well as Tryptophan Catabolites Pathway in the Blood and Brain Structures

Previous studies suggest that depression may be associated with reactive oxygen species overproduction and disorders of the tryptophan catabolites pathway. Moreover, one-third of patients do not respond to conventional pharmacotherapy. Therefore, the study investigates the molecular effect of escitalopram on the expression of Cat, Gpx1/4, Nos1/2, Tph1/2, Ido1, Kmo, and Kynu and promoter methylation in the hippocampus, amygdala, cerebral cortex, and blood of rats exposed to CMS (chronic mild stress). The animals were exposed to CMS for two or seven weeks followed by escitalopram treatment for five weeks. The mRNA and protein expression of the genes were analysed using the TaqMan Gene Expression Assay and Western blotting, while the methylation was determined using methylation-sensitive high-resolution melting. The CMS caused an increase of Gpx1 and Nos1 mRNA expression in the hippocampus, which was normalised by escitalopram administration. Moreover, Tph1 and Tph2 mRNA expression in the cerebral cortex was increased in stressed rats after escitalopram therapy. The methylation status of the Cat promoter was decreased in the hippocampus and cerebral cortex of the rats after escitalopram therapy. The Gpx4 protein levels were decreased following escitalopram compared to the stressed/saline group. It appears that CMS and escitalopram influence the expression and methylation of the studied genes.

ITGA4 gene methylation status in chronic lymphocytic leukemia

Background: We aimed to investigate ITGA4 gene expression pattern and to explore its methylation heterogeneity in chronic lymphocytic leukemia (CLL). Patients & methods: Eighty one CLL patients and 75 healthy subjects were enrolled and prognostic evaluation of patients was assessed. ITGA4 q-realtime PCR was performed using Applied Biosystems, TaqMan gene expression assay. ITGA4 gene-specific CpG methylation was investigated in real time using pyrosequencing technology. Results: ITGA4 was differentially expressed in CLL patients. The CpG sites-1, 2 and 3 showed significantly higher mean levels than healthy controls (p = <0.001, 0.007 and 0.009). Significant association between CpG site-1 and CLL has been detected using age-adjusted logistic regression (p < 0.001). Conclusion: Hypermethylation at ITGA4 gene CpG sites (1,2,3) is a characteristic feature in CLL.

The Changes of Expression and Methylation of Genes Involved in Oxidative Stress in Course of Chronic Mild Stress and Antidepressant Therapy with Agomelatine

Preclinical studies conducted so far suggest that oxidative stress processes may be associated with the mechanism of depression development. This study shows the effects of chronic administration of agomelatine on expression and the methylation status of Sod1, Sod2, Gpx1, Gpx4, Cat, Nos1, and Nos2 in the brain stricture and blood in the chronic mild stress (CMS) animal model of depression. The animals were exposed to the CMS procedure and treatment with agomelatine (10 mg/kg/day, IP) for five weeks and then were sacrificed. TaqMan Gene Expression Assay, Western blot, and methylation-sensitive high-resolution melting techniques were used to evaluate mRNA and protein expression of the genes, and the methylation status of their promoters. Gpx1, Gpx4, and Sod2 expression in the PBMCs and Sod1 and Sod2 expression in the brain were reduced in the stressed group after agomelatine administration. CMS caused an increase in the methylation of the third Gpx4 promoter in peripheral blood mononuclear cells and Gpx1 promoter in the cerebral cortex. Additionally, stressed rats treated with agomelatine displayed a significantly lower Gpx4 level in the hypothalamus. The results confirm the hypothesis that the CMS procedure and agomelatine administration change the expression level and methylation status of the promoter region of genes involved in oxidative and nitrosative stress.

Expression Analysis of Cytokine and Chemokine Genes during the Natural Course of Murine Experimental Autoimmune Uveoretinitis

C57BL/6 mice were immunized with human interphotoreceptor retinoid-binding protein peptides to induce experimental autoimmune uveoretinitis (EAU). From the day of immunization to 30 days later, RNA was isolated daily from the mouse eyes. Dynamic changes in gene expression during the pathogenesis of EAU were analyzed by TaqMan gene expression assay that contained most chemokines/cytokines and their receptors, and signal transducer and activator of transcription (STAT) family genes, using beta-actin as the endogenous control. Gene clusters based on their expression profiles were analyzed to determine the candidate genes for the pathogenesis of inflammation. Hierarchical cluster analysis showed gene expression during EAU development in seven clustering patterns. Hierarchical cluster analysis also identified four distinct phases in daily expression: entrance, acceleration, deceleration, and remission. Gene expression changes in the EAU active phase showed synergetic upregulation of Th1-type genes (IFN-gamma and CXCL10/IP-10) with elevated Th2-type genes (CCL17/TARC and IL-5). Sequential expression changes of STAT1, STAT6, and STAT3 genes represented the dynamic changes of Th1, Th2, and Th17-type inflammatory genes, respectively. The expression pattern of STAT1 was representative of many gene movements. Our results suggested that coordinated action of Th1, Th2, and Th17 genes and STAT family genes are involved in EAU development and resolution.

Effect of Hypoxia on the Expression of BRIT1 in K562 Cell Line: Implication for Resistance to Imatinib.

BRIT1 (Microcephalin, MCPH1) is a chromatin binding protein that forms ionizing irradiation-induced nuclear foci (IRIF); it is a crucial DNA damage regulator in the ATM/ATR pathways. Here we have analyzed BRIT1 expression on K562 cell line after treatment with imatinib 0,1-1 μM, UV irradiation (12 μJ/cm2) and after exposure to an hypoxic condition (3% O2). K562 in standard culture conditions (21% O2) were used to establish BRIT1 basal expression. Total RNA from K562 cultures was prepared at 24 and 48 hs. 1 μg total RNA was reverse transcribed from each sample. TaqMan Gene expression assay (Applied Biosystems) in Real-Time PCR was employed to examine the expression of BRIT1. Cellular count was assayed by trypan blue. Treatment with IM (0,1 and 1 μM) and UV induced a 2–3 fold increased expression of BRIT1 at 24 h and a minor increase at 48 h. Cell mortality at 24 h was 30% for IM and 20% for UV while at 48 h cells started to grow again, possibly as a consequence of the DNA damage repair by BRIT1. Exposure of K562 cells to hypoxia increased BRIT1 expression about 5- and 6-folds at 24 and 48 h respectively. Cell mortality at 24 h was 30% while after 48 h it was 20%. We therefore concluded that BRIT1 expression increases in hypoxia more than after IM and UV exposure. Since we have found that K562 in hypoxic conditions are relatively resistant to imatinib, (K562 cell mortality after 24 h exposure to IM 1μM was 30% ± 0,3 in normoxia vs 9% ± 0,8 in hypoxia, P<0,005) we have hypothesized that BRIT1 could be involved in resistance to IM. Therefore, we exposed K562 to UV and, after 48 h we incubated the cells with imatinib 1 μM. Pre-exposure of K562 cells to UV reduced imatinib-induced mortality (9,7% ± 1,6 vs 21,3% ± 1,5 of control, p< 0,005) thus indicating that upregulation of BRIT1 (as we have observed especially in hypoxia) could contribute to resistance to imatinib in K562 cell line. Understanding of BRIT1 function may well contribute to novel therapeutic approaches for cancer.