5-Hydroxymethyl-2′-deoxyuridine, a normal DNA constituent in certain Bacillus subtilis phages is cytostatic for mammalian cells

Waschke, S R; Reefschläger, J; Bärwolff, D.; Langen, P.

doi:10.1038/255629a0

Cited by 57 publications

(22 citation statements)

References 12 publications

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“…These experiments provide evidence that the toxicity of HmdUrd does not result from any intrinsic toxic property of HmUra as a substitute for thymine in DNA (25,32). Previously we showed that V79 cells could tolerate levels of HmdUrd substitution as high as 1 HmUra per 1,000 thymines; however, at higher levels of substitution, toxicity was seen (4).…”

Section: Discussionmentioning

confidence: 95%

“…The phenotype proved stable, remaining unchanged after 9 months of continuous culture in the presence or absence of continued selection. This V79mutl cell strain is phenotypically distinct from other HmdUrd-resistant cell lines, all of which are characterized by a deficiency in HmdUrd incorporation without an alteration in HmUra-DNA glycosylase activity (3,20,21,32).…”

Section: Discussionmentioning

confidence: 99%

“…However, the administration of the nucleoside 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) to cells grown in culture and to laboratory animals results in significant toxicity (18,20,25,32). Since HmdUrd is incorporated into DNA as a thymidine analog, its cytotoxicity suggested that the formation of HmUra opposite adenine is deleterious to * Corresponding author.…”

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confidence: 99%

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A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

Boorstein¹,

Chiu²,

Teebor³

1992

Mol. Cell. Biol.

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We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic reparability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

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A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

Boorstein¹,

Chiu²,

Teebor³

1992

Mol. Cell. Biol.

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“…Although HMdU replaces thymidine in the DNA of some phages of Bacillus subtilis (24)(25)(26), it was cytostatic when added to growth medium of baby hamster kidney, mouse L, and Ehrlich ascites cells and human embryonic fibroblasts (27). HeLa cells were resistant.…”

Section: Discussionmentioning

confidence: 99%

Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.

Teebor¹,

Frenkel²,

Goldstein³

1984

Proc. Natl. Acad. Sci. U.S.A.

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HeLa cells grown in the presence of [methyl-3H]thymidine contained large amounts of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in their DNA. When the cells were grown in [6-3H]thymidine and their DNA was labeled to the same specific activity, no HMdU was present. When such [6-3H]thymidine-labeled cells were exposed to increasing amounts of V-radiation, small but increasing amounts of HMdU were formed in their DNA. This indicates that HMdU can be formed in DNA by two distinct mechanisms. The first is the result of the transmutation of 3H to 3He (13 decay) in the methyl group of thymidine, leading to formation of a carbocation. This short-lived ion reacts with hydroxide ions of water, yielding the hydroxymethyl group. HMdU that is formed by this mechanism is formed at the rate of f3 decay of 3H. It appears only in [methyl-3H]thymidine residues and is present in the DNA of both nonirradiated and V-irradiated cells. The second mechanism is the result of the radiolysis of water caused by ionizing radiation. The resultant radical species, particularly hydroxyl radicals, may react with many sites on DNA.When the methyl group of thymine is attacked by hydroxyl radicals, the hydroxymethyl group is formed. The formation of HMdU by this mechanism was detected only when [6-3H]thymidine-labeled cells were used, since transmutation of 3H in position 6 of thymine cannot yield HMdU.Ionizing radiation causes the formation of strand breaks in DNA, release of bases from the DNA backbone, and modification of the bases themselves (1-5). This modification is thought to be effected primarily through the reaction of radical species derived from the radiolysis of water (6-10) with the bases, of which thymine (Thy) is the most reactive (9, 10). forming HMdU than was externally applied y-radiation. We report here that this discrepancy between radiation dose and formation of HMdU is due to its formation primarily through transmutation of 3H to 3He in the methyl group of thymine and not through ionizing radiation. This is a demonstration of a unique transmutation product of a 3H-labeled radionuclide in cellular DNA. However, in this report we show that HMdU can also be formed in cellular DNA through the action of externally applied radiation and we cite evidence that the formation of HMdU by this mechanism may contribute to the cytotoxic effects of ionizing radiation. [methyl-3H]thymidine it was diluted with unlabeled thymidine to achieve the same final thymidine concentration. The radioactive medium was removed and cells were grown for an additional 2 hr in nonradioactive thymidine (10 PM). MATERIALS AND METHODSVIrradiation of Cells and Isolation of DNA. Cells were washed once with cold phosphate-buffered saline, resuspended in phosphate-buffered saline, and divided into two tubes. One tube (control nonirradiated HeLa cells) was placed in an ice-water bath, while the other, also in an iceAbbreviations: HMdU, 5-hydroxymethyl-2'-deoxyuridine; HMUra, 5-hydroxymethyluracil; TG, thymine glycol (5,6-dihydroxy-5,6-dihydrothymine); dTG, thymi...

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“…We also have found that levels of H202 generated vary, not only among individuals but also among samples of PMNs from the same individual but obtained on different dates (34 (45)(46)(47)(48). In addition, HMdU is mutagenic in bacterial and mammalian cells, and its mutagenic effects are comparable in many ways to those caused by ionizing radiation (49).…”

Section: Tpa-induced Pmn Activationmentioning

confidence: 82%

Oxidation of DNA bases by tumor promoter-activated processes.

Frenkel

1989

Environ Health Perspect

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Evidence has accumulated showing that active oxygen species participate in at least one stage of tumor promotion. Tumor promoters can induce various types of cells to undergo processes that result in formation of active oxygen species. They stimulate polymorphonuclear leukocytes (PMNs) to undergo an oxidative burst that is characterized by rapid formation of * 0 -and H202. We find that in vitro formation of H202 by tumor promoter-activated PMNs correlates with their in vivo first-stage promoting activity. Moreover, two thymidine derivatives are formed in DNA coincubated with tumor promoter-stimulated PMNs: 5-hydroxymethyl-2 '-deoxyuridine (HMdU) and thymidine glycol (dTG). The amounts of HMdU and dGl formed correlate with the first-stage tumor-promoting potencies of the agents used for PMN stimulation and with the amount of H202 generated. We find that HMdU is also formed in the DNA of HeLa cells coincubated with 12-0-tetradecanoylphorbol-13-acetate (TPA)-activated PMNs, with the amount of HMdU being proportional to that of TPA used. Even in the absence of PMNs, HMdU is increasingly formed in cellular DNA with increased TPA concentration, although at much lower levels than in the presence of PMN&s When rat liver microsomes are incubated with benzo[a]pyrene (BaP), a complete carcinogen, H202 is also generated. Production of H202 increases linearly with increasing concentrations of BaP. Furthermore, HMdU is formed in DNA exposed to BaP-treated microsomes, and its formation is ithibited by catalase. These results suggest that carcinogen-induced processes generating H202 are associated with the first-stage promoting activity of complete carcinogens.

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5-Hydroxymethyl-2′-deoxyuridine, a normal DNA constituent in certain Bacillus subtilis phages is cytostatic for mammalian cells

Cited by 57 publications

References 12 publications

A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.

Oxidation of DNA bases by tumor promoter-activated processes.

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