Concentrations Of 5-azacytidine Research Articles

Combination of 5-azaytidine and hanging drop culture convert fat cell into cardiac cell.

One of the promising approaches for the treatment of cardiac disease is stem cell therapy. In this study, we compared the cardiomyogenic differentiation rate, from human adipose-derived stem cells (hADSCs) in a three-dimensional (3D) hanging drop (HD) spheroid culture system, versus a two-dimensional (2D) culture condition at different concentrations of 5-azacytidine (5-Aza). 5-Azaytidine (5-Aza) is a pyrimidine nucleoside analogue of cytidine that initiates cell differentiation programs through DNA demethylation. The hADSCs were isolated and cultured both in 2D and 3D HD conditions, with either 10 or 50 μM concentrations of 5-Aza. Then DNA content, gene expression, and protein content were analyzed. 3D HD culture resulted in a higher percentage of cells in G0/G1 and S phase in the cell division cycle, whereas 2D culture led to a greater percentage of cells in the G2/M phase. A significantly higher gene expression rate of HAND1, HAND2, cTnI, Cx43, βMHC, GATA4, NKX2.5, and MLC2V was observed in HD treated with 50 μM 5-Aza. This was confirmed by immunocytochemistry. These findings suggest that 50 μM concentration of 5-Aza can induce hADSCs to differentiate into cardiomyocytes. The differentiation rate was significantly higher when accompanied by the 3D HD culture system. This work provides a new culture system for cell differentiation for cardiovascular tissue engineering.

Effect of 5-azacytidine on in vitro biofilm formation of Streptococcus pneumoniae

Streptococcus pneumoniae is a gram-positive bacterium that causes otitis media, pneumonia, meningitis and sepsis in young children and the elderly. Previous studies reported that pneumococci in different diseases do not behave as planktonic cells, but predominantly show characteristics of a biofilm. In this study we examine the effect of 5-azacytidine on S. pneumoniae, particularly on biofilm formation and investigate the gene expression involved in synthesis of autoinducer-2, competence and DNA repair. The effect of 5-aza on in vitro biofilm formation was studied by the crystal violet microtiter plate method. The S. pneumoniae biofilms were grown with different concentration of 5-azacytidine (15–500 μm), at variable time intervals and the inhibition percentages were calculated. The effects of 5-aza on the morphology of biofilms were analyzed by scanning electron microscope. The relative quantification of 11 genes of biofilms grown with 5-aza involved in autoinducer-2 synthesis, competence and DNA repair was carried out by real-time RT-PCR with respect to biofilms grown without 5-aza. The crystal violet microtiter assay detected a significant inhibitory effect of 5-aza on in vitro biofilm formation, at concentration that did not inhibited planktonic cell growth. The SEM analysis demonstrated thin and disrupted biofilms, without micro-colonies in the samples treated with 5-aza, while these structures were present in the biofilms grown without 5-aza. The relative quantification of gene expression of 5-aza biofilms showed a significant down regulation of genes involved in the methionine and homocysteine recycling pathway which produces quorum sensing molecule autoinducer-2 as by-products. A significant decrease in the expressions of luxS, metK, pfs and cmK was detected. In conclusion, 5-aza inhibits in vitro biofilm formation and decreases the expression of luxS, pfs and metK, which are involved in the synthesis of autoinducer-2 as by-products of the methionine recycling pathway. The inhibitory effect of 5-aza may be either due to down regulation of pfs, luxS and metK or due to accumulation of the toxic substrate of pfs, luxS and metK genes.

Pharmacological genome demethylation increases radiosensitivity of head and neck squamous carcinoma cells

Aberrant inactivation of tumor suppressor genes by promoter hypermethylation has been recognized as a crucial step of tumor development and is related to aggressiveness and therapy resistance. To identify potential novel treatment strategies, we evaluated pharmacological genome demethylation for the increase of irradiation treatment effectiveness in head and neck squamous cell carcinoma (HNSCC) in this in vitro study. HNSCC cells were cultured with 2 different concentrations of 5-azacytidine (5-Aza) for 72 h, followed by a single fraction irradiation with 4 or 50 Gy, respectively. To show successful genome demethylation, the methylation status of the tumor suppressor gene hic1 (hypermethylated in cancer) promoter was analyzed by methylation specific PCR (MSP) as well as hic1 transcription by quantitative RT-PCR. Survival, apoptosis, viability, and migration of the tumor cells were analyzed as functional parameters of combined treatment response. After 5-Aza treatment the hic1 promoter was demethylated and gene transcription restored demonstrating genome demethylation. 5-Aza treated cells tended to be less viable and showed decreased survival indicated by lower colony numbers. Apoptosis and migration were not affected. The combined application of irradiation and 5-Aza significantly reduced survival compared to the single treatments. Accordingly, apoptosis was strongly increased after combined 4 Gy/5-Aza treatment. Viability was not additionally affected by combined treatment. Migration was affected weakly by combined high dosage irradiation/5‑Aza treatment. Our data show that the combined application of 5-Aza and irradiation is effective in vitro. A demethylating concept prior to irradiation should be further evaluated for its potential to reduce irradiation resistance.

Increase in susceptibility to EcoRII restriction of bacteriophage lambda produced by propagation on host cells growing in 5-azacytidine: a new in-vivo method for demonstration of DNA-methylation inhibition.

The efficiency of plating on EcoRII-restricting cells of bacteriophage lambda vir propagated on an Escherichia coli K-12 dcm+ host decreased with increase in concentration of 5-azacytidine (5-azaC) in the propagating medium. This illustrates, in-vivo, the inhibition of DNA-cytosine methylation induced by 5-azaC and provides a simple system for the detection of DNA-methylation inhibitors.

DNA demethylation in PD-1 gene promoter induced by 5-azacytidine activates PD-1 expression on Molt-4 cells

The expression of the programmed death 1 (PD-1) gene is an indicator of exhausted T-cells with decreased activation and function. It remains unknown, however, whether the methylation status of the PD-1 gene promoter is associated with PD-1 expression level. This study shows the changes of PD-1 expression levels and the demethylation status of the PD-1 promoter region in Molt-4 cells under different concentrations of 5-azacytidine (5-Zac). The result demonstrated that DNA demethylation at PD-1 promoter may contribute to PD-1 overexpression.

RUNX3 gene methylation in a cutaneous malignant melanoma cell line A375

Objective To investigate the relationship between the methylation of CpG island of RUNX3 gene promoter and its expression in a human cutaneous malignant melanoma cell line A375, and to assess the role of RUNX3 gene methylation in the pathogenesis of human cutaneous malignant melanoma. Methods Cultured A375 cells were treated with various concentrations (0, 1, 5, 10, 20 μmol/l) of 5-azacyti-dine for 24 or 72 hours followed by another 5 days of culture. Then, methylation-specific PCR (MSP) was performed to evaluate the methylation status of RUNX3 promoter region, and Western-blot analysis to detect the protein expression of RUNX3 in A375 cells. Results The RUNX3 gene promoter region was hypermethylated in untreated A375 cells, along with the absence of protein expression of RUNX3. However, after the treatment with 5-azacytidine, the promoter region of RUNX3 gene was demethylated partly, and the expression of RUNX3 protein was restored in A375 cells. Further, the expression intensity was directly correlated with the concentration of 5-azacytidine. Conclusions The promoter hypermethylation of RUNX3 gene may be related to the silencing of RUNX3 gene expression in A375 cells, whereas 5-azacytidine can cause the demethylation of RUNX3 gene, reactivate the gene which has been inactivated by the promoter hypermethylation, and finally induce the re-expression of RUNX3 protein. Key words: Cell line, tumor, Melanoma; DNA methylation; Azacitidine; RUNX3 gene

38 EFFECTS OF 5-AZACYTIDINE ON DNA HYPERMETHYLATION STATUS OF CLONED MINIATURE PIG EMBRYO

During somatic cell nuclear transfer, somatic cells should undergo epigenetic reprogramming in order to attain successful totipotency. Although there are many reasons of low efficiency of cloning, the aberrant DNA methylation status in donor cells is thought to reduce efficiency and increase abnormalities in cloned embryos. The methylation level of cloned embryos is higher than that of in vivo-produced or in vitro-fertilized embryos before occurring de novo methylation. This hyper DNA methylation status has been considered as a reason for the abnormal development of cloned embryos and the related decrease in term number. The aim of this research is to validate the DNA methylation pattern in cloned porcine embryos and to confirm whether reduction of hyper DNA methylation levels results in an improvement in cloning efficiency. First, immunostaining was used to evaluate the stage-specific changing pattern of DNA methylation of cloned embryos. In this result, we demonstrated that the 1-cell stage embryos and blastocyst had the highest level of methylation compared to 2- or 4-cell stage embryos. Second, this methylation level was higher in SCNT-derived embryos compared to parthenogenic embryos, suggesting that the methylation level was associated with the nucleus of donor cells. Third, cloned embryos were treated with various concentration of 5-azacytidine, which is a demethylating agent, to find adequate concentration. In results, 500 nm of 5-azacytidin group from 0 to 24 h after activation produced significantly more blastocysts compared to control group (control; 5.4 ± 2.3, 500 nm/0 24-h treatment; 12.7 ± 2.1, P < 0.05) and also showed low DNA methylation level of inner cell mass different from that of control group. In conclusion, our results suggest that cloned porcine embryos show typical demethylation. But, they are generally on a hyper level of DNA methylation. This high DNA methylation level is associated with somatic cells and affects on blastocyst development. We also suggest that 5-azacytidine could improve blastocyst development rate of cloned porcine embryos by aiding weak and abnormal demethylation process.

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.

Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes

Background Myocardial infarction results in the death of cardiomyocytes, which are replaced by scar tissue. Cardiomyocytes cannot regenerate because they are terminally differentiated. Mesenchymal cells are pluripotent cells, which have the potential to differentiate to specialized tissues under appropriate stimuli. The aim of this study was to direct differentiation of the adult mesenchymal stem cells isolated from fatty tissue into cardiomyocytes using 5-azacytidine. Methods Adult mesenchymal stem cells were isolated from the fatty tissue of New Zealand White rabbits and cultured in RPMI medium. Second-passaged mesenchymal cells were treated with various concentrations of 5-azacytidine and incubated for different intervals of time. The cells were plated in six-well dishes at 500, 5,000, and 50,000 cells/well. These cells were treated with 1-, 3-, 6-, 9-, and 12-μmol/L concentrations of 5-azacytidine and incubated for 12, 24, 48, and 72 hours. Later, the medium was replaced with fresh medium and incubated in a CO 2 incubator. The medium was changed once at 3 to 4 days. At 2 months, the cells were fixed with 0.4% glutaraldehyde for 2 hours and later washed with phosphate-buffered saline. The transformed cells were subjected to immunostaining for the myosin heavy chain, α actinin, and troponin-I. Results After treatment with 5-azacytidine, the adult mesenchymal stem cells were transformed into cardiomyocytes. At 1 week, some cells showed binucleation and extended cytoplasmic processes with adjacent cells. At 2 weeks, 20% to 30% of the cells increased in size and formed a ball-like appearance. At 3 weeks, these cells began to beat spontaneously in culture when observed under phase contrast microscope. Immunostaining of the transformed cells for myosin heavy chain, α actinin, and troponin-I was positive. The differentiated cells maintained the phenotype and did not dedifferentiate up to 2 months after treatment with 5-azacytidine. Conclusions These observations confirm that adult mesenchymal stem cells isolated from fatty tissue can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking Cry1Ab expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the cry1Ab gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of cry1Ab gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%-30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low concentration of 5-azacytidine (45 mg/L for 1 d and 2 d) could lead to the highest reactivation ratio and the highest expression level of the cry1Ab gene.

The influence of 5-azacytidine on the condensation of the short arm of rye chromosome 1R in Triticum aestivum L. root tip meristematic nuclei.

This paper describes the effects of 5-azacytidine on the condensation state of rye (Secale cereale L.) chromatin introduced into the wheat genome (Triticum aestivum L. cv. Beaver). The wheat cultivar Beaver carries a translocation between the short arm of rye chromosome 1R (1RS) and the long arm of wheat chromosome 1B (1BL/1RS). 1RS can be detected using genomic in situ hybridisation and carries a ribosomal DNA (rDNA) locus that can be simultaneously detected using multiple labelling strategies. The rDNA locus divides 1RS into a distal region that is gene rich and a proximal region that is gene poor and highly methylated. 1RS also carries a large block of subtelomeric heterochromatin. The drug, which acts to inhibit DNA methylation in plants, has three pronounced effects on interphase nuclei. (1) It induces aberrant condensation of the rye subtelomeric heterochromatin and in many cases induces sister chromatid separation in the subtelomeric heterochromatin of G2 nuclei. (2) Nuclei trisomic for 1RS are observed at low frequency in treated material and are probably a consequence of aberrant sister chromatid separation or condensation. (3) The drug alters normal condensation of 1RS euchromatin. However, contrary to expectation the effect is not simply to induce decondensation. The proximal region of the arm actually condenses at low levels of drug administration while the distal region remains unaltered or increases its decondensation state. Increasing the concentration of 5-azacytidine induces a biphasic response and at the highest concentration used all regions of the arm show signs of decondensation. Thus the influence of the drug on chromatin condensation depends on the genomic structure.

De novo methylation of an MHC class I transgene following transformation with human adenoviruses is not correlated with its altered expression.

The biological importance of class I histocompatibility antigens in a large variety of immune mechanisms is widely recognized, and their role in tumor rejection has been proven in several experimental tumor systems. Reduced expression of class I antigens, which is correlated with enhanced tumorigenicity, was shown in these systems to be mainly the result of transcriptional down-regulation. Mouse embryonal fibroblasts expressing H-2 antigens and the product of a miniature swine class I transgene, transformed by adenovirus 12, exhibit low levels of all class I antigens on the cell surface. Half of the cell lines demonstrate a suppressed level of class I mRNAs. Cell lines derived from transformation with the early region of adenovirus 5 express a high level of class I antigens. DNAs from adenovirus-transformed cells are extensively hypermethylated both in the 5' and the coding regions of the transgene compared to DNAs from immortalized cell lines and primary embryonal fibroblasts. Nevertheless, hypermethylation of these sequences is not correlated with mRNA level or cell-surface expression of the transgene product. Treatment of the transformed cells with high concentration of 5-azacytidine (5 Aza-C) induced merely a minor enhancement in the expression of class I mRNAs and class I antigens. Thus, this system is a perfect example of where viral transformation is associated with induced methylation of a class I gene, but hypermethylation does not affect its expression. The role of de novo methylation of genes in this system might be associated with transformation, or generation of mutations in CpG-rich sequences.

Differential Effect of the DNA Analogue, 5-Azacytidine, on the Constitutive Heterochromatic Blocks in B and T Lymphocytes

A selective under-condensation of the heterochromatic blocks 1qh, 9qh and 16qh was induced in normal peripheral blood lymphocyte cultures, using the DNA analogue 5-azacytidine. With 5-azacytidine at a concentration of 3.5 × 10-7 µM, phytohemagglutinin (PHA)-stimulated cultures (predominantly proliferating T lymphocytes) showed a significantly higher percentage of metaphases with under-condensation of heterochromatic blocks than pokeweed (PWM)-stimulated cultures (mainly activated B lymphocytes). When the concentration of 5-azacytidine in the culture medium was increased ten times, no significant difference was noticed between PHA- and PWM-stimulated cultures. At this higher dose, the response in PHA cultures remained the same while the response of PWM cultures increased dramatically. Thus, there is a differential in vitro relaxation of the heterochromatin condensation by 5-azacytidine, with T lymphocytes being much more sensitive than B lymphocytes.

Multiple new phenotypes induced in [formula omitted] and 3T3 cells treated with 5-azacytidine

Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T 1 2 CL8 mouse cells treated with 5-azacytidine or 5-aza-2′-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T 1 2 CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T 1 2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.