Recent studies have highlighted the significant role of 5-hydroxymethylcytosine (5hmC) in carcinogenesis. However, the specific role of 5hmC in osteosarcoma (OS) remains largely unexplored. The-re-fore, this study aimed to investigate the function of 5hmC and TET3 in OS. In this study, we found a decreased total level of 5hmC in OS tissues. The expression of the TET3 protein was also decreased in OS. Importantly, the decreased levels of TET3 were associated with a decreased disease-free survival (DFS) rate in patients. To investigate the role of TET3 and 5hmC in OS, we manipulated the levels of TET3 in MG-63 cells. Silencing TET3 in these cells resulted in a twofold increase in proliferation. Additio-nally, the level of 5hmC decreased in these cells. Con-versely, over-expression of TET3 in MG-63 cells led to the expected inhibition of proliferation and invasion, accompanied by an increase in 5hmC levels. In conclusion, both 5hmC and TET3 protein levels were decreased in OS. Additionally, the over-expression of TET3 inhibited the proliferation of MG-63 cells, while the suppression of TET3 had the opposite effect. These findings suggest that decreased levels of 5hmC and TET3 may serve as potential markers for OS.
- MeSH
- 5-Methylcytosine * analogs & derivatives metabolism MeSH
- DNA Demethylation * MeSH
- Dioxygenases * metabolism MeSH
- Epigenesis, Genetic * MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Bone Neoplasms genetics metabolism pathology MeSH
- Osteosarcoma genetics metabolism pathology MeSH
- Cell Proliferation * MeSH
- Proto-Oncogene Proteins metabolism genetics MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
- MeSH
- Chromosomes, Plant genetics MeSH
- Cytosine * metabolism MeSH
- Epigenesis, Genetic MeSH
- Transcription, Genetic MeSH
- DNA Methylation * MeSH
- Gene Expression Regulation, Plant MeSH
- DNA, Ribosomal * genetics MeSH
- Gene Silencing * MeSH
- DNA Copy Number Variations MeSH
- Publication type
- Journal Article MeSH
Cryptococcus neoformans is an encapsulated yeast that can cause cryptococcosis and cryptococcal meningitis, which conventional treatment involves antifungal drugs such as polyenes, flucytosine, azoles, and their combinations. However, the high cost, toxicity, and increase in fungi resistance to antifungal agents stimulate the search for therapeutic strategies such as drug repurposing and combination therapy. This study evaluated the activity of the antihypertensive verapamil (VEH) alone and combined with amphotericin B (AmB) against C. neoformans. VEH exhibited antifungal activity against C. neoformans with minimum inhibitory concentration and minimum fungicidal concentration of 118 μg per mL. The combination of VEH and AmB exhibited synergism, reducing at least eightfold both drugs' concentrations. Moreover, the combination decreased the size and glucuronoxylomannnan content of C. neoformans capsule. However, no difference was observed in ergosterol levels of C. neoformans after treatment with VEH and AmB in combination. Altogether, VEH in combination with AmB exhibits potential as a candidate as for the development of anti-cryptococcal drug.
- MeSH
- Amphotericin B pharmacology therapeutic use MeSH
- Antifungal Agents pharmacology therapeutic use MeSH
- Cryptococcus neoformans * MeSH
- Flucytosine pharmacology therapeutic use MeSH
- Cryptococcosis * drug therapy microbiology MeSH
- Microbial Sensitivity Tests MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: The pathogenesis of psoriasis vulgaris involves changes in DNA molecules, genomic instability, telomere attrition, and epigenetic alterations among them. These changes are also considered important mechanisms of aging in cells and tissues. OBJECTIVE: This study dealt with oxidation damage, telomere length and methylation status in DNA originating from peripheral blood of 41 psoriatic patients and 30 healthy controls. METHODS: Oxidative damage of serum DNA/RNA was determined immunochemically. Real-time PCR was used for the analysis of the telomere length. ELISA technique determined levels of 5-methylcytosine in blood cells' DNA. RESULTS: Oxidative damage of serum DNA/RNA was higher in patients than in controls (median, 3758 vs. 2286pg/mL, p<0.001). A higher length of telomeres per chromosome was found in patients whole-cell DNA than in controls (3.57 vs. 3.04 kilobases, p=0.011). A negative correlation of the length of telomeres with an age of the control subjects was revealed (Spearman's rho=-0.420, p=0.028). Insignificantly different levels of 5-methylcytosine in patients and controls were observed (33.20 vs. 23.35%, p=0.234). No influences of sex, smoking, BMI, PASI score, and metabolic syndrome on the methylation status were found. STUDY LIMITATIONS: i) A relatively small number of the participants, particularly for reliable subgroup analyses, ii) the Caucasian origin of the participants possibly influencing the results of the parameters determined, and iii) Telomerase activity was not directly measured in serum or blood cells. CONCLUSION: The study demonstrated increased levels of oxidized DNA/RNA molecules in the serum of patients with exacerbated psoriasis vulgaris. The results were minimally influenced by sex, the presence of metabolic syndrome, or cigarette smoking. In the psoriatic blood cells' DNA, the authors observed longer telomeres compared to healthy controls, particularly in females. Insignificantly higher global DNA methylation in psoriasis cases compared to the controls indicated marginal clinical importance of this epigenetic test performed in the blood cells' DNA.
- MeSH
- 5-Methylcytosine MeSH
- DNA metabolism MeSH
- Epigenesis, Genetic MeSH
- Humans MeSH
- Metabolic Syndrome * MeSH
- Oxidative Stress genetics MeSH
- Psoriasis * genetics MeSH
- RNA metabolism MeSH
- Telomere genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Acquired Immunodeficiency Syndrome * drug therapy prevention & control MeSH
- Antiviral Agents history MeSH
- Cidofovir MeSH
- History, 20th Century MeSH
- History, 21st Century MeSH
- Emtricitabine, Tenofovir Disoproxil Fumarate Drug Combination MeSH
- Chemistry, Organic MeSH
- Drug Approval history MeSH
- Tenofovir MeSH
- Drug Development history MeSH
- Check Tag
- History, 20th Century MeSH
- History, 21st Century MeSH
- Publication type
- Historical Article MeSH
Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.
- MeSH
- 5-Methylcytosine * MeSH
- Deoxyribonucleosides MeSH
- DNA-Directed DNA Polymerase metabolism MeSH
- DNA metabolism MeSH
- Epigenesis, Genetic MeSH
- Nucleotides metabolism MeSH
- Pyrimidine Nucleotides * MeSH
- Pyrimidines MeSH
- DNA Restriction Enzymes metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.
- MeSH
- Chromatography, Liquid MeSH
- Cytosine Deaminase MeSH
- Flucytosine * MeSH
- Fluorouracil MeSH
- Mass Spectrometry MeSH
- Prodrugs * MeSH
- Publication type
- Journal Article MeSH
DNA methylation, i.e., addition of methyl group to 5'-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.
- MeSH
- 5-Methylcytosine metabolism MeSH
- Biosensing Techniques methods MeSH
- Epigenesis, Genetic genetics MeSH
- Humans MeSH
- DNA Methylation genetics physiology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
- MeSH
- 5-Methylcytosine metabolism MeSH
- Cell Line MeSH
- DNA biosynthesis MeSH
- DNA Breaks, Single-Stranded * MeSH
- Humans MeSH
- Methylation MeSH
- Neurons metabolism MeSH
- DNA Repair * MeSH
- Poly(ADP-ribose) Polymerases metabolism MeSH
- DNA Replication MeSH
- Sequence Analysis, DNA MeSH
- Enhancer Elements, Genetic genetics MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Intramural MeSH
Polyomavirus infections occur commonly in humans and are normally nonfatal. However, in immunocompromised individuals, they are intractable and frequently fatal. Due to a lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections, has been repurposed as an antipolyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as a broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as antipolyomavirus agents are still under research. Here, polyomavirus large tumor antigen (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system utilizing only purified proteins. We present evidence that DNA helicase and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl ester derivatives of cidofovir, suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) binding activities of Tags. These findings show that the alkoxyalkyl ester derivatives of cidofovir are effective in vitro without undergoing further conversions, and we conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.
