In Vivo Methylation of DNA in Human Leukocytes

MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP–bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.

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In vivo Imaging Flow Cytometry of Human Leukocytes

Optics in the Life Sciences Congress ◽

10.1364/boda.2017.bow3a.2 ◽

2017 ◽

Author(s):

Cheng-Ham Wu ◽

Chia-Hung Hsieh ◽

Shih-Hung Huang ◽

Jong-Wei Lin ◽

Tzung-Dau Wang ◽

...

Keyword(s):

Flow Cytometry ◽

In Vivo Imaging ◽

Human Leukocytes ◽

Imaging Flow Cytometry

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Latency of Viral Expression In Vivo Is Not Related to CpG Methylation in the U3 Region and Part of the R Region of the Long Terminal Repeat of Bovine Leukemia Virus

Journal of Virology ◽

10.1128/jvi.77.7.4423-4430.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4423-4430 ◽

Cited By ~ 13

Author(s):

Shigeru Tajima ◽

Masako Tsukamoto ◽

Yoko Aida

Keyword(s):

Long Terminal Repeat ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Cpg Methylation ◽

Terminal Repeat ◽

Luciferase Reporter ◽

Reporter System ◽

Methylation Of Dna ◽

R Region

ABSTRACT Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the host's immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.

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The potential toxicological insights about the anti-HIV drug azidothymidine-derived monoselenides in human leukocytes: Toxicological insights of new selenium-azidothymidine analogs

Human & Experimental Toxicology ◽

10.1177/0960327116674529 ◽

2016 ◽

Vol 36 (9) ◽

pp. 910-918 ◽

Cited By ~ 4

Author(s):

DOC Mariano ◽

D de Souza ◽

DF Meinerz ◽

J Allebrandt ◽

AF de Bem ◽

...

Keyword(s):

Antioxidant Properties ◽

Damage Index ◽

Human Leukocyte ◽

In Vitro Toxicity ◽

Organoselenium Compounds ◽

In Vivo Models ◽

Human Leukocytes ◽

Anti Hiv

Acquired immunodeficiency syndrome (AIDS) is a worldwide disease characterized by impairments of immune function. AIDS can be associated with oxidative stress (OS) that can be linked to selenium (Se) deficiency. Se is fundamental for the synthesis of selenoproteins, such as glutathione peroxidase and thioredoxin reductase. These enzymes catalyze the decomposition of reactive oxygen species and contribute to maintain equilibrium in cell redox status. Literature data indicate that organoselenium compounds, such as ebselen and diphenyl diselenide, have antioxidant properties in vitro and in vivo models associated with OS. Nevertheless, selenocompounds can also react and oxidize thiols groups, inducing toxicity in mammals. Here, we tested the potential cytotoxic and genotoxic properties of six analogs of the prototypal anti-HIV drug azidothymidine (AZT) containing Se (5′-Se-(phenyl)zidovudine; 5′-Se-(1,3,5-trimethylphenyl)zidovudine; 5′-Se-(1-naphtyl)zidovudine; 5′-Se-(4-chlorophenyl)zidovudine) (C4); 5′-Se-(4-methylphenyl)zidovudine (C5); and 5′-(4-methylbenzoselenoate)zidovudine). C5 increased the rate of dithiothreitol oxidation (thiol oxidase activity) and C2-C4 and C6 (at 100 µM) increased DNA damage index (DI) in human leukocytes. Moreover, C5 (200 µM) decreased human leukocyte viability to about 50%. Taken together, these results indicated the low in vitro toxicity in human leukocytes of some Se-containing analogs of AZT.

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Studies on Agranulocytosis. II. The Effect of Chlorpromazine upon the Influx of S35 L-Cystine and L-Methionine into Human Leukocytes

Blood ◽

10.1182/blood.v16.4.1456.1456 ◽

1960 ◽

Vol 16 (4) ◽

pp. 1456-1468 ◽

Cited By ~ 1

Author(s):

ANTHONY V. PISCIOTTA ◽

SHIRLEY N. EBBE ◽

MARY DALY ◽

MONA RUWALDT ◽

MILTON GLASER ◽

...

Keyword(s):

Whole Blood ◽

Blood Cells ◽

The Other ◽

In Vivo Studies ◽

Human Leukocytes ◽

In Vivo Administration

Abstract 1. When whole blood was incubated in vitro with S-35 L-cystine and L-methionine, the blood cells became radioactive. 2. Preincubation of whole blood from normals and from patients susceptible to agranulocytosis with chlorpromazine showed no effect upon uptake of S-35 L-cystine and L-methionine by leukocytes. 3. The in vivo administration of S-35 L-cystine was followed by the appearance of radioactive leukocytes. Peak radioactivity occurred in leukocytes in 5 to 12 days. 4. Pretreatment of test subjects with large doses of chlorpromazine did not block the uptake of S-35 L-cystine by leukocytes in vivo. Leukocytes of women showed an increase in the incorporation of S-35 L-cystine, in vivo. Studies performed in vivo on two persons during recovery from agranulocytosis showed enhanced uptake of L-cystine in one and a normal uptake in the other.

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