Indirubin regulates MPL and TNF expression in peripheral blood mononuclear cells from patients with primary immune thrombocytopenia.

Indirubin, a traditional Chinese medicine, is currently used to treat certain autoimmune diseases such as primary immune thrombocytopenia (ITP) in clinics. However, the effects of indirubin on expression of related genes in peripheral blood mononuclear cells (PBMCs) from ITP patients have not been investigated. In the present study, PBMCs were isolated from 19 adult patients with well-characterized active ITP and 20 healthy controls (HCs) and then treated with increasing concentrations of indirubin. The mRNA expression levels of thrombopoietin receptor (MPL), GATA binding protein 3 (GATA3), DNA methyltransferase 3B (DNMT3B), interleukin-6 (IL6), tumor necrosis factor (TNF), and interferon gamma (IFN-γ) were determined by quantitative real-time polymerase chain reaction (PCR). We found that indirubin had no cytotoxic effect on PBMC viability. Significantly lower MPL (p < 0.05) and GATA3 (p < 0.05) expression together with markedly higher IL6 (p < 0.05), TNF (p < 0.0001), and IFN-γ (p < 0.001) mRNA levels were observed in ITP patients compared with HCs. Notably, indirubin significantly enhanced MPL expression and inhibited TNF expression in PBMCs from ITP patients (p < 0.05). In summary, indirubin may play a direct role in thrombopoiesis by activating cellular MPL and normalizing TNF expression to suppress inflammation in ITP. This study may thus improve our understanding of indirubin and provide important information for optimizing therapeutic strategies for ITP patients.

Effects of age and sex on epigenetic modification induced by an acute physical exercise.

It has been observed that, after 2 hours of aerobic exercise, plasma interleukin-6 (IL-6) increases whereas nuclear concentrations of enzyme DNA methyltransferase (DNMT) 3B significantly decreased in peripheral blood mononuclear cells (PBMCs), with no change observed in DNMT3A. The aim of the present study was to detect differences in these changes induced by exercise in plasma IL-6 levels as well as in PBMC nuclear concentrations of DNMT3A and DNMT3B, in relation to age and sex. Four groups were studied: 12 young men (24.8 ±â€Š1.77 years old), 12 young women (23.8 ±â€Š1.81 years old), 12 adult men (45.8 ±â€Š1.82 years old), 12 adult women (mean 44.5 ±â€Š2.07 years old). Participants had to run at 60% of maximal oxygen consumption (VO2max) for 120 minutes, interspersed with sprints at 90% of VO2max for the last 30 seconds of every 10 minutes. About 250 µL of PBMCs (1 × 10 cells) were treated with 100 µL of either pre-exercise plasma or post-exercise plasma and nuclear DNMT3A and DNMT3B concentrations were quantified. No change in nuclear concentration of DNMT3A following the exercise was observed. Conversely, nuclear concentrations of DNMT3B significantly decreased, with a reduction of about 78% in young men, 72% in young women, 61% in adult men, and 53% in adult women. Moreover, a strong positive correlation between the nuclear concentration of DNMT3B in PBMC following stimulation with post-exercise plasma and post-exercise plasma concentrations of IL-6 was observed in all the 4 studied groups. This study confirms that a single bout of endurance exercise is sufficient to decrease nuclear concentrations of DNMT3B and thus protein upregulation. Moreover, the epigenetic mechanisms induced by exercise apparently cause more intense changes in men than in women and that, in both of them, this effect seems to decrease with age.

MTHFR GENE C677T POLYMORPHISM AND LEVELS OF DNA METHYLTRASFERASES IN SUBCLINICAL HYPOTHYROIDISM.

The aim of our study was to investigate the link between MTHFR gene C677T polymorphism and DNMTs levels in patients with Subclinical Hypothyroidism (SCH). In this study 19 adult patients with subclinical hypothyroidism and 19 healthy controls (mean age 31±5.5 and 33±5.1 years respectively) were recruited. All patients were diagnosed based on serum levels of TSH, FT4, anti-TG and anti-TPO antibodies. Written informed consents were obtained from all study subjects. Genomic DNA was extracted using Quick-DNA Universal Kit (Zymo Research, USA). The MTHFR C677T polymorphism was genotyped by PCR-RFLP method. Levels of DNMT1 and 3a were measured in nuclear extracts of PBMC using DNMTs assay kits (Abcam). Our data indicates that the frequency of genotypes and alleles were different among the patient and the control group. There is a significant increase in CC genotype distribution in the control group when compared to the SCH patient group, while the CT as well as TT genotype distribution were not increased significantly in SCH group versus control group. However the C allele is significantly prevalent in the control group compared to the SCH group, while T allele is prevalent in patients compared to the control group with a statically significant difference. In addition, individuals with TT and CT genotypes and hypothyroidism showed elevated amount of DNMT3a in nuclear extracts of PBMC compared with controls, while no significant difference in DNMT1 levels was observed. This study indicates the MTHFR C677T variant may contribute in alteration of epigenetic regulation such as DNA methylation mediated by DNA methyltransferases in patients with subclinical hypothyroidism and also, carriers of the T allele might have an increasing risk of developing SCH.

A STUDY OF THE RELATIONSHIP BETWEEN LEVELS OF METHYLTRANSFERASES IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND CHARACTERISTICS OF TUMOR IN PATIENTS WITH DUCTAL INVASIVE CARCINOMA OF BREAST.

The role of epigenetics in tumor development and progression is actively being studied. The aim of our current pilot study is to analyze correlation of changes in the levels of methyltransferases in nuclear extracts of blood cells with some morphological and phenotypic characteristics of breast cancer. The levels of DNMT1, DNMT3a and H3K4 methyltransferase were measured. The results showed that the level of DNMT1 was highest in the control group but correlation with the tumor grade was just moderate. DNMT3a was found in highest level in Grade III cancer group, followed by Grade II and Grade I groups. Correlation of DNMT1 level with tumor grade was moderate. An opposite pattern was seen for H3K4 methyltransferase. DNMT3a level was higher in larger tumors, while the level of H3K4 methyltransferase was lowest in large tumors with significant negative correlation with the tumor size. This primary study shows that there are some changes in methyltransferase levels in PBMC from breast cancer patients. These changes are most probably attributed to modification of initiation as well as sustainment of methylated status of products.

Methylation analysis of multiple genes in blood DNA of Alzheimer's disease and healthy individuals.

We collected blood DNA from 120 late-onset Alzheimer's disease (AD) patients and 115 healthy matched controls and analysed the methylation levels of genes involved in amyloid-beta peptide production (PSEN1 and BACE1), in DNA methylation (DNMT1, DNMT3A and DNMT3B), and in one-carbon metabolism (MTHFR), searching for correlation with age and gender, with biomarkers of one-carbon metabolism (plasma homocysteine, and serum folate and vitamin B12 levels), and with disease status (being healthy or having AD). We also evaluated the contribution of the APOE ϵ4 allele, the major late-onset AD genetic risk factor, to the studied gene methylation levels. All the genes showed low mean methylation levels (<5%) in both AD and control DNA, no difference between groups, and no correlation with the studied biomarkers, except for MTHFR that showed methylation levels ranging from 5% to 75%, and correlation with circulating biomarkers of one-carbon metabolism. However, mean MTHFR methylation levels were similar between groups (31.1% in AD and 30.7% in controls, P=0.58). Overall, present data suggest that none of the studied regions is differently methylated in blood DNA between AD and control subjects.

Abnormal Epigenetic Modifications in Peripheral T Cells from Patients with Abdominal Aortic Aneurysm Are Correlated with Disease Development.

BACKGROUND: Increasing evidence suggests that abdominal aortic aneurysm (AAA) is a T-cell-mediated autoimmune condition. This study investigates the epigenetic modifications that occur in the T cells of AAA patients and evaluates the correlation of these modifications with disease development. METHODS AND RESULTS: Peripheral T cells were collected from 101 AAA patients and 102 healthy controls (HCs). DNA methylation and histone acetylation levels were measured by ELISA. Methyl-CpG-binding domain, DNA methyltransferase (DNMT) and histone deacetylase (HDAC) mRNA levels were determined by real-time PCR. DNA from the T cells of the AAA patients exhibited significant hypomethylation compared with the HCs (1.6-fold, p < 0.0001). Expression of DNMT1 at the mRNA level in the T cells of the AAA patients was 1.52-fold lower than that of the HCs (p < 0.0001). The extent of DNA methylation in the AAA patients was negatively correlated with the corresponding aortic diameter (r = -0.498, p < 0.0001). H3 (1.59-fold, p < 0.0001) and H3K14 (2.15-fold, p < 0.0001) acetylation levels in the T cells of the AAA patients were higher than those of the HCs, but the HDAC1 mRNA level was 2.33-fold lower than that of the HCs (p < 0.0001). CONCLUSIONS: DNA methylation and the histone modification status are significantly altered in the T cells of AAA patients. These changes could play a pivotal role in the activation of pathological immune responses and may influence AAA development.

Anxiety is associated with higher levels of global DNA methylation and altered expression of epigenetic and interleukin-6 genes.

OBJECTIVES: Anxiety is associated with elevated levels of the inflammatory cytokine interleukin-6 (IL-6) and an increased risk for diseases with an inflammatory aetiology. In cancer, higher levels of IL-6 have been associated with increased expression of the epigenetic enzymes DNMT1 and Enhancer of Zeste Homolog 2 (EZH2). However, the relationship between IL-6 and DNA methyltransferases (DNMTs) and EZH2 expression has not previously been examined in anxious individuals. METHODS: Global DNA methylation levels were measured using the Methylflash Methylated DNA Quantification Kit and gene expression levels of the DNMT and EZH2 genes in anxious (n=25) and nonanxious individuals (n=22) were compared using quantitative real-time PCR. Specifically, we investigated whether global DNA methylation or aberrant expression of these genes was correlated with IL-6 mRNA and protein serum levels in anxious individuals. RESULTS: Anxious participants had significantly higher levels of global DNA methylation compared with controls (P=0.001). There were no differences in the mean mRNA expression levels of the DNMT1/3A/3B, EZH2 and IL-6 genes in anxious individuals compared with controls. However, the expression of DNMT1/3A, EZH2 and IL-6 genes increases with increasing Hospital Anxiety and Depression Scale-Anxiety scores in the anxious cohort only. Interestingly, IL-6 gene expression was correlated strongly with DNMT1/3A/3B and EZH2 expression, highlighting a potential relationship between IL-6 and important epigenetic regulatory enzymes. CONCLUSION: This study provides novel insight into the relationship between anxiety, epigenetics and IL-6. Moreover, our findings support the hypothesis that changes in DNA methylation profiles may contribute to the biology of anxiety.

[Protein expressions of HDAC1 and DNMT1 in non-small-cell lung cancer and its clinical significance].

OBJECTIVE: To detect the serum protein expressions of histone deacetylase 1 (HDAC1) and DNA methyltransferase 1 (DNMT1) in patients with non-small-cell lung cancer and explore the correlation between their expressions and clinicopathological features. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to measure the protein expressions of HDAC1 and DNMT1 in 136 lung cancer patients hospitalized at Department of Respiratory Diseases, First Affiliated Hospital,Zhengzhou University from September 2012 to June 2013 (cancer group). And 147 healthy physical examination subjects from Sixth People's Hospital of Zhengzhou were selected as controls and their clinicopathological features analyzed. RESULTS: The protein expressions of HDAC1 and DNMT1 in patients with lung cancer (145 ± 53, 50 ± 11) were higher than those in control group (78 ± 56, 27 ± 6). And there was statistical significance (t = 596.16, 152.64, both P = 0.000) . The protein expressions of HDAC1 and DNMT1 were correlated with gender, smoking history and age (among P < 0.05). There was a positive association between the protein expressions of HDAC1 and DNMT1 in non-small-cell lung cancer (r = 0.525, P = 0.000). Statistical significance existed between the protein expressions of HDAC1 and DNMT1 and histological types or different stages (all P < 0.01). CONCLUSION: The higher protein expressions of HDAC1 and DNMT1 may play an important role in the early process of lung cancer.

[Expression and significance of DNA methyltransferase in sera of patients with lung cancer].

OBJECTIVE: To explore the protein expressions of DNMT1, DNMT3a and DNMT3b in sera of lung cancer patients. METHODS: Enzyme-linked immunosorbent assay (ELISA) method was used to measure the protein expressions of DNMT1, DNMT3a and DNMT3b in 136 lung cancer patients hospitalized at Department of Respiratory Diseases, First Affiliated Hospital, Zhengzhou University during September 2012 to June 2013. And 147 healthy controls were selected from a population of physical examination at Sixth People's Hospital of Zhengzhou. And the relationship was analyzed between protein expressions of DNMT1, DNMT3a and DNMT3b and clinic characteristics of lung cancer. RESULTS: The protein expressions of DNMT1, DNMT3a and DNMT3b in patients with lung cancer (15 ± 10, 997 ± 76 , 302 ± 25) were higher than those of the controls (13 ± 10, 344 ± 93, 108 ± 22). And there were statistical significance (t = 3.28, 62.51, 37.27; P = 0.021, 0.000, 0.000). The results of Logistic regression show that the protein expressions DNMT1, DNMT3a and DNMT3b increased morbidity for lung cancer (χ(2) = 14.811, 26.768, 12.057; P = 0.000, 0.000 0.001), especially so for DNMT1 (OR = 1.545, 95%CI: 1.238-1.928). No correlation existed between the protein expressions of DNMT1, DNMT3a, DNMT3b and histological types or stages (P > 0.05). CONCLUSIONS: The high protein expressions of serum DNMT1, DNMT3a and DNMT3b increase morbidity for lung cancer. And these markers may predict the early occurrence of lung cancer.

Abnormal DNA methylation in peripheral blood mononuclear cells from patients with vitiligo.

BACKGROUND: Vitiligo is a skin disorder characterized by the destruction of melanocytes by autoreactive lymphocytes. The genetic and environmental factors that trigger the autoimmune response are poorly understood. However, alterations to epigenetic DNA methylation patterns contribute to many other autoimmune diseases. OBJECTIVES: To investigate genomic and gene-specific DNA methylation levels in peripheral blood mononuclear cells (PBMCs) of patients with vitiligo and to relate any changes to the expression of genes that regulate methylation, as well as the autoimmune-related gene IL10. METHODS: We quantified global methylcytosine levels in PBMCs from 20 patients with vitiligo and 20 healthy controls. mRNA levels of DNA methyltransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and interleukin (IL)-10 were measured by real-time reverse transcriptase-polymerase chain reaction. Methylation of an IL10 regulatory element domain was determined by bisulphite genomic sequencing. RESULTS: Genomic DNA methylation in PBMCs of patients with vitiligo was increased relative to healthy controls (P = 0·012). DNMT1, MBD1, MBD3, MBD4 and MeCP2 expression was significantly higher than in control PBMCs (P = 0·013, 0·001, 0·005, 0·001 and 0·001, respectively). MBD1 and MBD3 expression correlated positively with global DNA methylation in vitiligo PBMCs (MBD1: r = 0·519, P = 0·019; MBD3: r = 0·529, P = 0·016). IL10 expression was significantly decreased (P = 0·030), and an IL-10 enhancer region was hypermethylated in vitiligo PBMCs compared with controls (P = 0·014). CONCLUSIONS: These data show that levels of DNA methylation are altered in PBMCs of patients with vitiligo, and this may contribute to disease activity by affecting the expression of autoimmunity-related genes.

Decreased DNA methyltransferase 3A and 3B mRNA expression in peripheral blood mononuclear cells and increased plasma SAH concentration in adult patients with idiopathic thrombocytopenic purpura.

OBJECTIVE: DNA methylation is known to play an important role in gene transcription and alterations of methylation contribute to the development of certain disorders such as cancer and immunodeficiency. Recent years have found an increasing interest in the role of epigenetic modifications in the etiology of human autoimmune diseases, such as systemic lupus erythromatosus (SLE) and rheumatoid arthritis (RA). DNA methyltransferases (DNMTs) are involved in the epigenetic control of DNA methylation processes. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, have important indicator action of cellular methylation status. The aim of this study is to explore if DNA methylation plays a role in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: DNMT1, DNMT3A, and DNMT3B mRNA expression in peripheral blood mononuclear cells (PBMCs) of adult ITP patients were analyzed by real-time quantitative polymerase chain reaction. Plasma SAM and SAH levels were assayed with reversed-phase high performance liquid chromatography (HPLC). RESULTS: DNMT3A and DNMT3B mRNA expressions were significantly lower in ITP patients than in healthy controls (p < 0.001), while DNMT1 mRNA expression was not significantly different between the two groups (p = 0.774). Plasma SAH concentration was significantly elevated in ITP patients than in healthy controls (p < 0.05), while the plasma SAM and SAM/SAH were not significantly different between the two groups (p = 0.133, p = 0.624 respectively). CONCLUSIONS: Our observations suggest that aberrant DNA methylation status reflected by increased plasma SAH concentration and decreased mRNA expression levels of DNMT3A and 3B are possibly involved in the pathogenesis of ITP although the precise mechanisms need further study.

Phase I and pharmacologic study of the human DNA methyltransferase antisense oligodeoxynucleotide MG98 given as a 21-day continuous infusion every 4 weeks.

PURPOSE: MG98 is a second generation phosphorothioate antisense oligodeoxynucleotide which is a highly specific inhibitor of translation of the mRNA for human DNA MeTase I (DNMT 1). This phase I study examined the toxicity and pharmacologic profile of MG98 administered as a continuous 21-day intravenous infusion every 4 weeks. PATIENTS AND METHODS: Fourteen patients with solid cancers received a total of 25 cycles of MG98 at doses ranging from 40 to 240 mg/m2/day. Steady-state concentrations of MG98 were measured as were several pharmacodynamic assessments including mRNA of the target gene, DNMT1, in PBMC. In addition, other potential surrogate markers of drug effects were explored, including hemoglobin F, Vimentin and GADD45. RESULTS: Dose limiting effects were drug-related reversible transaminase elevation and fatigue seen at doses of 240, 200 and 160 mg/m2/day. The dose level of 80 mg/m2/day was felt to be safe and tolerable when delivered on this schedule. No evidence of antitumor activity was observed. Although pharmacokinetic analysis revealed that at the higher dose levels, mean Css values of MG98 were approximately 10-fold times the IC50 values associated with target inhibition in vitro, the extent of MG98 penetration into target tumors in this trial was not determined. No consistent, dose-related changes in correlative markers including DNMT1 mRNA, hemoglobin F, Vimentin and GADD45, were observed. CONCLUSIONS: This schedule of MG98 given as a 21-day continuous intravenous infusion every 4 weeks was poorly tolerated in the highest doses; therefore, further disease-site specific evaluation of the efficacy of this agent will utilize a more favorable, intermittent dosing schedule. Pharmacodynamic evaluations undertaken in an attempt to explore and validate the biological mechanisms of MG98 did not show dose-related effects.

[Properties and specificity of action of DNA methylases from blood lymphocytes of healthy cattle and cattle with chronic lymphoid leukemia]. / Svoistva i spetsifichnost' deistviia DNK-metilaz limfotsitov krovi zdorovykh i bol'nykh khronicheskim limfoleikozom korov.

Using cell extract fractionation with ammonium sulfate and subsequent chromatography on DEAE- and DNA-cellulose and Blue Sepharose, two cytosine DNA-methylases were isolated from blood lymphocytes of cows suffering from lympholeukosis; one cytosine DNA-methylase was isolated from blood lymphocytes of healthy animals. The DNA-methylases from normal lymphocytes was purified 383-fold; the enzyme has a specific activity of 2.3 u./mg, Mr of 114 000 Da and a pH optimum of 7.6. The molecular mass of DNA-methylases from leukemic lymphocytes is about 130,000 Da. The enzymes isolated from leukemic lymphocytes, i.e., DNA-methylase I (568-fold purification, specific activity 14.2 u./mg) and DNA-methylase II (524-fold purification, specific activity 13.1 u./mg) possess different action optima at pH 7.8 and 6.7, respectively. The total DNA-methylase activity of leukemic lymphocytes is about 4 times that of normal lymphocytes. All the DNA-methylases under study methylate in vitro bacterial and animal DNAs of different base composition; bacterial DNAs are the best (GC content is approximately 70%), while homologous DNAs- the worst acceptors of CH3-groups. Heat-denaturated DNAs are methylated more intensively than initial DNAs. The optimal NaCl concentration in the reaction mixture is approximately 50 mM; EDTA (greater than 10 mM) inhibits the reaction. DNA-methylase I of leukemic and normal lymphocytes show the same pH optimum and specificity of action. In vitro methylation of bacterial DNA by these DNA-methylases in the presence of [3H-methyl]S-adenosylmethionine results in the similar type of label distribution among pyrimidine isopliths in the DNA. DNA-methylase II from leukemic lymphocytes methylates about two times more Pu-C-Pu sequences in th same DNA than does DNA-methylase I from leukemic and normal lymphocytes and thus reveals a different specificity. The changes in the type of DNA methylation as well as the appearance of an additional DNA-methylase possessing a different specificity of action in leukemic lymphocytes may be responsible for cell transformation and transcriptional changes in chronic lympholeukosis.

Enzymatic methylation of chicken erythrocyte DNA modified by two carcinogens, 2-(N-acetoxyacetylamino) fluorene and methylnitrosourea.

Both DNA-AAF and MNU-alkylated DNA are methylated less than nonmodified DNA by rat brain nuclei cytosine 5-methyltransferase purified either by chromatography on DEAE cellulose or by Dyematrex. The inhibition of methylation is proportional to the modification of the DNA, and DNA having a given percentage of bases modified with MNU is less methylated than DNA modified to the same extent with AAF. Moreover, DNA-AAF irreversibly inhibits the methylation of native DNA, whereas MNU-alkylated DNA does not inhibit the methylation of native DNA. The AAF-substituted DNA has a higher affinity for the enzyme than native DNA. However, this is probably not due to the AAF-induced local destabilization of the DNA helix, since heat-denatured DNA shows a lower affinity for the enzyme than double-stranded DNA. Addition of DNA-AAF to the enzyme preincubated with native DNA inhibits methylation, but only after a lag period. This agrees with the model in which the methylase walks along the strand to methylate cytosine residues before being detached from the DNA. AAF bound to guanine residues may block the movement of the enzyme along the helix. The in vitro hypomethylation of DNA, caused by carcinogens, could explain the in vivo observations made by several authors and could have significance in gene activity, cellular differentiation, and oncogenesis.