Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Survival and mutagenic effects of 5-azacytidine in Escherichia coli

Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli. Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase. The dcm, MspI, and EcoRII methyltransferase genes all decreased survival. There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine. Survival was not affected by uvrA, uvrB or umuCD mutations. Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase. Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system. The mechanism of cell death in these situations is therefore different. Mutation of the rpoB gene by 5-azacytidine was studied. The mutation rate was decreased by the presence of recA and lexA mutations. Mutation in umuCD had little effect on the mutation rate. The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis. The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment. The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase. The former could be due to the opening of the 5-azacytosine ring in DNA. Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.     

Unnatural enantiomers of 5-azacytidine analogues: syntheses and enzymatic properties

2'-Deoxy-beta-L-5-azacytidine(L-Decitabine), beta-L-5-azacytidine, and derivatives were stereospecifically prepared starting from L-ribose or L-xylose. D- and L-enantiomers of 2'-deoxy-beta-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK), whereas both enantiomers of beta-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. None of the reported derivatives of beta-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA).     

The effect of 5-azacytidine on the growth and thymidine kinase activity of coleoptile sections ofTriticum

The growth of coleoptile sections ofTriticum and its stimulation by indole-3-acetic acid (IAA) are inhibited by 5-azacytidine added into the cultivation medium. 50 per cent depression of the elongation was observed at 2×10^?3M 5-azacytidine concentration. Thymidine kinase activity in cell-free extracts prepared from coleoptile sections treated with 5-azacytidine, and caleulated per 10 mg of their wet weight, is increased while IAA administration resulted in its depression. The observed changes in thymidine kinase activity can be explained assuming the different uptake of water due to 5-azacytidine and IAA treatment.     

Genetic effects of 5-azacytidine in Saccharomyces cerevisiae

The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in different organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.     

Properties of phenylalanine transfer ribonucleic acid with modified 3''-terminal end in protein biosynthesis using a rabbit reticulocyte cell-free system: effect of the replacement of cytidine residues from the CpCpA end of tRNA by 5-iodocytidine or 2-thiocytidine.

Phe-tRNA Phe from yeast containing 2-thiocytidine or 5-iodocytidine in position 75 of the polynucleotide chain or Phe-tRNA Phe in which both positions 74 and 75 were substituted by 5-iodocytidine were investigated in the poly U-dependent polyphenylalanine synthesis on ribosomes from rabbit reticulocytes. Phe-tRNA Phe-Cps2CpA was nearly as active as the native Phe-tRNA Phe-CpCpA in the overall process. Phe-tRNA Phe-Cpi 5CpA as well as Phe-tRNA Phe-i5Cpi 5CpA were considerably less active than the native species. Investigation of individual steps of protein biosynthesis with these modified substrates revealed that the donor activity of peptidyl-tRNAs which contain 5-iodocytidine in their 3'-terminus is strongly imparied suggesting exacting structural requirements for the interaction of the CpCpA end of tRNA with the ribosomal P-site.     

5-Azacytidine increases the total cellular copper content and basal level metallothionein mRNA accumulation of human Hep G2 cells.

In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.     

Thymus involution induced by 5-azacytidine

Hypomethylation of DNA, which can be achieved by incorporation of 5-azacytidine, has been correlated with derepression of genes. In order to examine thein vivo effects of 5-azacytidine on organ development and differentiation, young rats were treated with the drug. There was an almost complete reduction of thymus and a marked reduction of spleen weight, while other organs, including testes were only marginally affected. Control experiments with cytosine-arabinoside suggest that treatment with an inhibitor of DNA replicationper se is not responsible for the very rapid thymus involution triggered by 5-azacytidine in rats. In spite of the drastic reduction of thymus and spleen weight, lymphocytes of these organs were not impaired in their response to the T cell mitogen Concanavalin A.     

Effect of 5-azacytidine on rat liver alpha-fetoprotein gene expression

Neonatal rats given 5-azacytidine intraperitoneally (30 micrograms/animal/day) on days 1-5 postpartum had 55% lower serum alpha-fetoprotein levels on day 6 compared to saline injected controls. On day 14, alpha-fetoprotein levels were 4-fold lower in 5-azacytidine treated animals. Cytosol alpha-fetoprotein was proportionately reduced. There were no significant changes in liver to body weight ratio, total serum protein, and both serum and cytosol albumin levels. The molecular basis for decreased serum alpha-fetoprotein levels was found to be a reduced concentration of alpha-fetoprotein mRNA in the livers of 5-azacytidine injected animals. These results are discussed with respect to the effects of 5-azacytidine on DNA methylation and cell differentiation.     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

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.     

Changes in the placenta and in the rat embryo caused by the demethylating agent 5-azacytidine

DNA methylation is an important mechanism for regulation of gene expression during vertebrate development. 5-azacytidine is used as an experimental tool for demethylation. In this work, a single dose of 5-azacytidine (5 mg/kg body weight) was administered to rats at different stages of development. After 5-azacytidine administration on the first or third day of pregnancy, no changes were detected. After administration on the fourth day of pregnancy or later, a reduction in growth was observed. After treatment on day five and on any other day till day eleven of pregnancy, no living fetuses were found. Of those treated on day twelve, 24% of fetuses survived, but forelimb and hindlimb malformations were present. Administered on day thirteen, 5-azacytidine did not interfere with survival, but malformations were still present. From day fourteen on, 5-azacytidine caused no gross external malformations. Placentas were also influenced by 5-azacytidine. They were significantly smaller and histological evaluation showed the labyrinthine part to be severely reduced. In contrast, trophoblast giant cells were more abundant than in controls.     

Mechanism of resistance to 5-azacytidine inBacillus Subtilis

Spontaneous mutants resistant to 5-azacytidine (5-AzCyd-r) and to 5-aza-2′-deoxycytidine (5-dAzCyd-r) were isolated inBacillus subtilis. There is no cross resistance between the two markers. Genetic transfer by a transformation process was possible with a low efficiency only with the 5-AzCyd-r marker. Independently isolated 5-AzCyd-r mutants did not show any differences in the level of resistance.     

Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells

Bone marrow stromal cells (BMSC) can differentiate into various cell types including myocytes, which may be valuable in cellular therapy of myocardial infarction. In an attempt to increase the myogenic commitment of BMSC, we investigated the extent of conversion induced by the demethylation agent 5-azacytidine. BMSC isolated from the adult rat tibia were exposed in culture to 5microM 5-azacytidine for 24h, 1 day after seeding. The treatment was repeated at weekly intervals and the expression of muscle-specific proteins and genes was assessed. The results revealed that cultured cells lost the native expression of osteocalcin and alkaline phosphatase as a function of time and began to express connexin 43. Exposure to 5-azacytidine of BMSC induced, at 14 days, a myocyte-resembling phenotype that included the expression of muscle-specific proteins (sarcomeric alpha-actin, troponin T, desmin, alpha-actinin, and GATA-4) and genes (GATA-4, myoD, desmin, and alpha-actinin), numerous mitochondria and myofilaments; however, the latter did not form sarcomeres. Although some of these myogenic markers also appeared in untreated cells, exposure to 5-azacytidine induced an enhanced response of calcium channels, as well as a threefold increase in desmin and myoD gene expression and a twofold increase in alpha-actinin gene and protein expression above the control values. In conclusion, the results demonstrate a promoting effect of 5-azacytidine on the expression of muscle-specific proteins and genes in BMSC in culture. Notably, the myogenic differentiation takes place over a short period of time. Priming of mesenchymal cells to cardiomyogenic differentiation may have significant applications in cellular approaches to ameliorate muscle loss after myocardial ischemia.     

Genotoxicity of 5-azacytidine in somatic cells of Drosophila

The newly developed somatic mutation and recombination test, utilizing the wing-hair mutations mwh and flr3, was used to evaluate the genotoxicity of the base analog 5-azacytidine in larvae of Drosophila melanogaster. Third instar larvae were fed media wetted with various concentrations of the compound, and wings of surviving adults were removed and scored for the presence of clones of cells possessing malformed hairs. Wings of exposed flies trans-dihybrid for mwh and flr3 had significantly increased frequencies of twin spots, small single spots and large single spots. Significant linear regression of twin-spot frequencies upon concentration was also obtained. Induction of twin spots by 5-azacytidine unambiguously demonstrates its recombinogenic activity in somatic cells of Drosophila. Significantly increased frequencies of large single spots on wings of inversion-heterozygous flies were also observed and suggest that 5-azacytidine may also be inducing somatic gene mutations (or deletions).     

Differential inhibition of mitogen induced T cell proliferation by 5-azacytidine and cytosine-arabinoside

The cytotoxic drugs 5-azacytidine and cytosine-arabinoside influence the enzymatic methylation of DNA in opposite ways (1,2). The in vitro effects of these two drugs on Con A induced proliferation of thymic and splenic rat lymphocytes were investigated. Cytosine-arabinoside was found to inhibit mitogen induced proliferation already at a concentration of 0.001 microM, whereas 5-azacytidine was inhibitory only above concentrations of 1 microM. A stimulation of mitogen induced T cell proliferation was consistently seen with 5-azacytidine, but not with cytosine-arabinoside, at concentrations lower than the cytotoxic concentration. The results show that 5-azacytidine and cytosine-arabinoside interfere with mitogen stimulated lymphocyte proliferation by different mechanisms and suggest that hypomethylated DNA plays a role in the proliferation of T cells.     

The effect of 5-azacytidine on the root meristem ofVicia faba

The primary roots ofVicia faba seedlings were placed in a solution of 5-azacytidine and their further growth was observed after being replaced in running tap water. No inhibition of elongation occurred during the action of the 10^?5 M solution of 5-azacytidine for 24 hours, but during subsequent cultivation in water in the absence of inhibitor, further growth was blocked. This inhibition could be overcome by cytidine, uridine, sodium azide, 5-azidomethyluracil and simultaneously with the 5-azacytidine solution. Inhibition was accompanied by a high incidence of chromosome stickiness and to a less extent by an incidence of chromosome aberrations. The occurrence of stickiness and chromosome aberrations was prevented by adding excess cytidine to the 5-azacytidine solution.     

Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.