5-Azacytidine accelerates yeast-mycelium conversion in Candida albicans

The effect of the DNA demethylating agent 5-azacytidine (5-azaC) on the morphological development of Candida albicans blastospores has been investigated by microscopic observations. It was found that this compound does not produce any morphogenetic effect when the cells are not induced to mycelial form. By contrast, on the induced cells, 5-azaC markedly accelerates the process of germ tube formation. In addition in the treated cells, yeast-mycelium conversion develops synchronously, whereas it is asynchronous and slow in the normal cells. These data indicate that, together with phenotypic modifications, modulation of gene activity by DNA demethylation occurs in Candida albicans during morphogenetic changes.     

Impact of DNA methyltransferase inhibitor 5-azacytidine on cardiac development of zebrafish in vivo and cardiomyocyte proliferation,apoptosis, and the homeostasis of gene expression in vitro

Cardiac development is a peculiar process involving coordinated cellular differentiation, migration, proliferation, and apoptosis. DNA methylation plays a key role in genomic stability, tissue-specific gene expression, cell proliferation, and apoptosis. Hypomethylation in the global genome has been reported in cardiovascular diseases. However, little is known about the impact and specific mechanism of global hypomethylation on cardiomyocytes. In the present study, we explored the impact of DNA methyltransferase inhibitors 5-azacytidine on cardiac development. In vivo experiment showed that hypomethylation of zebrafish embryos with 5-azacytidine exposure significantly reduced survival, induced malformations, and delayed general development process. Furthermore, zebrafish embryos injected with 5-azacytidine developed pericardial edema, ventricular volume reduction, looping deformity, and reduction in heart rate and ventricular shortening fraction. Cardiomyocytes treated with 5-azacytidine in vitro decreased proliferation and induced apoptosis in a concentration-dependent manner. Furthermore, 5-azacytidine treatment in cardiomyocytes resulted in 20 downregulated genes expression and two upregulated genes expression in 45 candidate genes, which indicated that DNA methylation functions as a bidirectional modulator in regulating gene expression. In conclusion, these results show the regulative effects of the epigenetic modifier 5-azacytidine in cardiac development of zebrafish embryos in vivo and cardiomyocyte proliferation and apoptosis and the homeostasis of gene expression in vitro, which offer a novel understanding of aberrant DNA methylation in the etiology of cardiovascular disease.     

5-Azacytidine facilitates osteogenic gene expression and differentiation of mesenchymal stem cells by alteration in DNA methylation

Mesenchymal stem cells (MSCs) are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages. The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage. In this study, we investigated the effect of 5-azacytidine, a DNA demethylating agent, on osteogenic differentiation of MSCs. The cells were exposed to 5-azacytidine in culture medium for 24 h prior to osteogenic induction. Osteogenic differentiation was determined by several the appearance of a number of osteogenesis characteristics, including gene expression, ALP activity, and calcium mineralization. Pretreatment of MSCs with 5-azacytidine significantly facilitated osteogenic differentiation and was accompanied by hypomethylation of genomic DNA and increased osteogenic gene expression. Taking dlx5 as a representative, methylation alterations of the “CpG island shore” in the promoter caused by 5-azacytidine appeared to contribute to osteogenic differentiation.     

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39 degrees C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39 degrees C. Restoration of Ras activity at 33 degrees C leads to the rapid loss of TgTF1 and G418 resistance.     

Circulating cell-free DNA release in vitro: kinetics,size profiling,and cancer-related gene methylation

Circulating cell-free DNA (ccfDNA) is a biological entity of great interest due to its potential as liquid biopsy biomaterial carrying clinically valuable information. To better understand its nature, we studied ccfDNA in vitro in two human cancer cell lines MCF-7 and HeLa. Normalized indexes of ccfDNA per cell population decreased over time of culture but were significantly elevated after exposure to IC₅₀ doses of the demethylating/apoptotic agent 5-azacytidine (5-AZA-CR). Fragment-size profiling was indicative of active release, whereas exposure to 5-AZA-CR induced the release of additional shorter fragments, indicative of apoptosis. Finally, the methylation profile of a panel of cancer-specific genes as assessed by quantitative methylation analysis in ccfDNA was identical to the corresponding genomic DNA and followed accurately changes caused by 5-AZA-CR. Overall, our in vitro findings support that ccfDNA can be a reliable biosource of clinically relevant information that can be further studied in these cell culture models.     

Growth and differentiation of cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions

Cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.     

Proteomic analysis of a lymphoma-derived cell line (DG75) following treatment with a demethylating drug: modification of membrane-associated proteins

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.     

Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells

A human diploid lung fibroblast cell strain, TIG-7, has a heteromorphic chromosome 15 with an extra short arm carrying a homogeneously staining region (15p+hsr). We demonstrated previously that the 15p+hsr consists of an inactive and G+C-rich rDNA cluster characterized by fluorescence in situ hybridization (FISH) and various chromosome banding techniques. Thus, it was suggested that the region could contain highly methylated DNA. To observe methylation status on the target region directly under the microscope, we used a demethylating agent, 5-azacytidine (5-azaC), to induce decondensation of the chromatin containing methylated DNA. At 24 h after treatment with 0.5 microM 5-azaC, marked decondensation of the 15p+hsr was observed in almost all of the metaphases. Furthermore, we observed micronuclei, which were equivalent to the rDNA of the 15p+hsr demonstrated by FISH in the same preparation. In contrast, the DNA cross-linking agent mitomycin C (MMC) preferentially induced 15p+hsr-negative micronuclei. These findings indicated that chromatin decondensation and subsequent DNA strand breakage induced by the demethylating effect of 5-azaC led specifically to 15p+hsr-positive micronuclei.     

Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum

Summary Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved growth in the presence of kanamycin.     

Hypomethylation enhances Ia antigen expression by the FRTL5 rat thyroid cell line

The effect of DNA methylation on Ia antigen expression by FRTL5 rat thyroid cells was assessed using 5-azacytidine. Hypomethylation with this agent did not alone induce Ia antigen expression but did increase the expression of Ia antigen induced by suboptimal concentrations of rat gamma-interferon, present in a T cell conditioned medium or in recombinant form. These results suggest that DNA methylation does not account for the constitutive lack of Ia expression by thyroid cells, but methylation of gamma-interferon response regions may confer partial resistance to the Ia-inducing effect of this lymphokine.     

Growth and differentiation of cell hybrids obtained by fusing mouse PCC4aza1 teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions

Cell hybrids obtained by fusing mouse PCC4aza1 teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.     

Flowering induced by 5-azacytidine, a DNA demethylating reagent in a short-day plant, Perilla frutescens var. crispa

Treatment with 5-azacytidine, a DNA demethylating reagent, induced flowering in Perilla frutescens (L.) Britton var. crispa (Thunb. ex Murray) Decne. ex L. H. Bailey, an absolute short-day plant under long days. The 5-azacytidine treatment induced slight suppression of vegetative growth but had no obvious effect on any other phenotypes. The Southern hybridization analysis of the genomic DNA isolated from the leaves of 5-azacytidine-treated plants and digested with restriction enzyme, methylation-insensitive Msp I or methylation-sensitive Hpa II with P. frutescens 25S-18S rDNA intergenic spacer probe indicated that the 5-azacytidine treatment caused demethylation of the genomic DNA. The 5-azacytidine-induced flowering was delayed as compared with the short day-induced flowering. Flowers were formed even at the lower nodes which had not been directly treated with 5-azacytidine. The results suggest that DNA demethylation induced flowering by inducing the production of a transmissible flowering stimulus in P. frutescens .     

Induction of alpha-fetoprotein synthesis in differentiating F9 teratocarcinoma cells is accompanied by a genome-wide loss of DNA methylation.

F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.     

Induction of class II MHC antigen expression on the murine placenta by 5-azacytidine correlates with fetal abortion.

During the gestational cycle the placental tissue does not express class II MHC antigens and whether this phenomenon is important to fetal survival has not yet been evoked. It has been reported that class II antigen expression precedes renal and cardiac graft rejection, which may also be the case in fetal abortion. In a recent report we showed that placental cells can be induced to express class II antigens in vitro and that these cells undergo different regulatory mechanisms depending on their anatomical position in the placenta. Thus, spongiotrophoblast-derived cells express these antigens after interferon-gamma treatment, whereas labyrinthine trophoblast-derived cells are induced by 5-azacytidine. In the present study we examined the effect of 5-azacytidine on class II antigen expression in the placenta and fetal abortion in vivo. We report that 5-azacytidine, when given to pregnant females before the ectoplacental cone formation, dramatically increases fetal loss, which correlates with class II antigen expression in the labyrinthine trophoblast zone. No site effects of 5-azacytidine on placental cell proliferation, splenic T and B cell responses, or reproductive capability of treated females were observed. However, after treatment with 5-azacytidine placental cells can stimulate maternal spleen cells to proliferate in a mixed cell reaction, whereas untreated controls cannot. Furthermore, the abortive effect of 5-azacytidine can be rescued in allogeneic pregnancy by anti-paternal class II monoclonal antibody injection into the animals during the 5-azacytidine treatment. These results suggest that the maintenance of the class II antigen-negative expression on the placenta is indeed necessary to avoid maternal immune attack and ensure fetal survival.     

Induction of a step in carcinogenesis that is normally associated with mutagenesis by nonmutagenic concentrations of 5-azacytidine.

The permanent cell line BHK-21/cl 13 can be transformed by mutagenic carcinogens as the result of the induction of a recessive somatic mutation. Yet when these cells were treated with 5-azacytidine under conditions in which no mutants resistant to either ouabain or 6-thioguanine could be detected, they were transformed efficiently. These transformants were induced, not selected. 6-Azacytidine was ineffective at transforming BHK cells; 2'-deoxy-5-azacytidine was exceptionally effective. When tested by cell fusion, transformants induced by 5-azacytidine fell into the same complementation group as those induced by highly mutagenic carcinogens, but they were phenotypically distinct in that they were unstable during prolonged passage and rarely displayed the temperature-limited phenotypes so common among BHK transformants induced by strongly mutagenic carcinogens. These results raise the possibility that a cell can be induced by either genetic or epigenetic means to traverse the same single step in carcinogenesis.     

Can 5-azacytidine convert the adult stem cells into cardiomyocytes? A brief overview

From the first report that bone marrow cells (BMC) have stem cells characteristics, several studies have debated the possibility of intervening in myocardial remodeling after injury, for example myocardial infarction, by using BMC. The goal of this paper is to review the concept of whether the demethylating agent 5-azacytidine influences the myogenic differentiation of bone marrow-derived cells. The existing data seem to indicate that in vitro treatment with 5-azacytidine, even if not enough to generate mature CMC, promotes the in vivo and in vitro commitment of BMC into cells that express muscle-specific proteins and genes and, at a very low rate, show spontaneous contractions. It is probable this treatment makes the cells less responsive to other inductive factors secreted by the microenvironment that might modulate the differentiation. These data suggest that this approach may be used to prime cells prior to their transplantation in an injury area in the heart.     

Methylation and expression of the Myo D1 determination gene

Mouse embryo cells induced to differentiate with the demethylating agent 5-azacytidine represent an excellent model system to investigate the molecular control of development. Clonal derivatives of 10T1/2 cells that have become determined to the myogenic or adipogenic lineages can be isolated from the multipotential parental line after drug treatment. These determined derivatives can be cultured indefinitely and will differentiate into end-stage phenotypes on appropriate stimulation. A gene called Myo D1, recently isolated from such a myoblast line, will confer myogenesis when expressed in 10T1/2 or other cell types (Davis et al. 1987). The cDNA for Myo D1 contains a large number of CpG sequences and the gene is relatively methylated in 10T1/2 cells and an adipocyte derivative, but is demethylated in myogenic derivatives. Myo D1 may therefore be subject to methylation control in vitro. On the other hand, preliminary observations suggest that Myo D1 is not methylated at CCGG sites in vivo so that a de novo methylation event may have occurred in vitro. These observations may have significance in the establishment of immortal cell lines and tumours.     

Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation

The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria , whose flowering state is stable and the short day plant Pharbitis nil , whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens . On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state.