rg108 and pyrimidin-2-one-beta-ribofuranoside

Current considerations suggest that the mechanisms of long term memory are based on the changes of the neuronal genetic algorithms. Process of the enzyme DNA methylation have great importance to the differential genes expression, and is likely to be one of the key mechanisms of the consolidation and memory storage. The present study aimed to investigate the DNA methylation processes role in the mechanisms of the conditioned food aversion memory reconsolidation and also in the amnesia development mechanisms, caused by disturbances of the reconsolidation in Helix lucorum. It was found that DNA-methyltransferase inhibitors zebularine and RG108 had no influence on the memory reconsolidation. We investigated the effects of the DNA-methyltransferase inhibitors on different amnesia stage. The amnesia was induced by NMDA glutamate receptors antagonists. At the early stage of amnesia (3rd day after its induction) injections of the DNA-methyltransferase inhibitors in combination with reminder by conditioned food stimuli led to memory recall. Memory was recovered by the DNA-methyltransferase inhibitors injections before the reminder and also 3h, but not 9h after the reminder. In case when enzyme inhibitors or reminders were applied separately, no effect on memory was revealed. At the late stage of amnesia (10th day) the DNA-methyltransferase inhibitors didn't affects amnesia development. It might be hypothesized that presentation of reminding stimuli result in the reactivation/reconsolidation of molecular processes, involved in amnesia development, one of the key mechanisms of which could be the DNA methylation/demethylation of neural cells.

Topics: Amnesia; Animals; Avoidance Learning; Cytidine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Helix, Snails; Memory, Long-Term; Phthalimides; Tryptophan

The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in female rats. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

Topics: Animals; Brain; Copulation; CpG Islands; Cytidine; Disorders of Sex Development; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; DNA, Intergenic; Estradiol; Female; Gene Expression Regulation, Developmental; Male; Mice; Microfilament Proteins; Nerve Tissue Proteins; Phthalimides; Preoptic Area; Protein Isoforms; Rats; Rats, Sprague-Dawley; Sex Characteristics; Sex Differentiation; Testosterone; Tryptophan

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee.

Topics: Animals; Bees; Cyclic AMP Response Element-Binding Protein; Cytidine; DNA; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Epigenesis, Genetic; Memory, Long-Term; Phthalimides; Smell; Tryptophan; Up-Regulation

We studied the role of DNA methylation in the mechanisms of amnesia in edible snails, which was induced by impairment of conditioned food aversion memory reconsolidation with NMDA glutamate receptor antagonist. The effects of DNA methyltransferase inhibitors were shown to depend on the stage of amnesia. At the early stage of amnesia (day 3 after induction), injections of methyltransferase inhibitors in combination with conditioned food stimulus (reminder) were followed by memory recovery. Application of inhibitors in the absence of the reminder was ineffective. Methyltransferase inhibitors were ineffective at the late stage of amnesia (day 10). Our results suggest that the presentation of reminding conditioned stimuli is followed by reactivation of amnesia. Methylation or demethylation of DNA in nerve cells serves as one of the key mechanisms for amnesia.

Topics: Amnesia; Animals; Conditioning, Psychological; Cytidine; Dizocilpine Maleate; DNA Methylation; DNA-Cytosine Methylases; Helix, Snails; Learning; Phthalimides; Receptors, N-Methyl-D-Aspartate; Tryptophan

The p53 protein ensures cellular fidelity by suppressing or killing cells under stresses that enhance the mutation rate. Evidence suggests that the p53 protein may also ensure the fidelity of the epigenome. In this study a group of drugs that alter the deoxycytosine methylation patterns in cellular DNA are shown to preferentially kill human and mouse cells that contain p53 mutations or deficiencies. These observations are extended to mice that contain p53 deficiencies or missense mutations in their genome, which are preferentially killed when compared to mice with a wild type p53 gene. This is also the case for human cancer cell xenografts containing p53 mutations, which preferentially are killed by these drugs when compared to similar tumors with wild type p53. The loss of p53 function enhances a synthetic lethality with drugs that block or alter the patterns of deoxycytidine methylation in the genome.

Topics: Animals; Anticarcinogenic Agents; Azacitidine; Catechin; Cell Line, Tumor; Cell Proliferation; Cytidine; Decitabine; DNA Methylation; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Neoplasm Transplantation; Phthalimides; Tryptophan; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50nM) for 1h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5nM 5-aza-dC for 1h and subsequently treated with 50nM TSA for another 1h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were significantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5nM 5-aza-dC and 50nM TSA for 1h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos.

Topics: Animals; Cloning, Organism; Cytidine; DNA Methylation; Embryo Culture Techniques; Embryonic Development; Epigenomics; Fibroblasts; Histone Deacetylase Inhibitors; Nuclear Transfer Techniques; Phthalimides; Swine; Tryptophan

DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.

Topics: Acetylation; Cadherins; CD11b Antigen; Cell Differentiation; Cell Proliferation; CpG Islands; Cytidine; Enzyme Inhibitors; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Indoles; Leukemia, Promyelocytic, Acute; Methylation; Phthalimides; Promoter Regions, Genetic; Propionates; Tretinoin; Tryptophan

DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine, Vidaza) has recently been approved as an antitumor agent, and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems, which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR), 5-aza-2'-deoxycytidine (5-aza-CdR), zebularine, procaine, (-)-epigallocatechin-3-gallate (EGCG), and RG108. Of these, 5-aza-CR, 5-aza-CdR, zebularine, and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic, as evidenced by the induction of micronuclei. In addition, 5-aza-CR, 5-aza-CdR, zebularine, and RG108 caused concentration-dependent demethylation of genomic DNA, whereas procaine and EGCG failed to induce significant effects. Finally, the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase, which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs.

Topics: Azacitidine; Catechin; Cell Line, Tumor; Cytidine; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Humans; Indoles; Jurkat Cells; Phthalimides; Procaine; Propionates; Tryptophan