The value of hydroxyurea in assessing repair synthesis of DNA in HeLa cells

Brandt, William; Flamm, W.G.; Bernheim, Naomi J.

doi:10.1016/0009-2797(72)90072-5

Cited by 76 publications

(10 citation statements)

References 18 publications

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“…We also investigated the effect of hydroxyurea and aphidi- colin, agents known to inhibit normal DNA replication (19,20), on the replicative synthesis occurring after UV damage. At all UV doses examined, hydroxyurea completely inhibited incorporation of nucleotides into replicative density DNA ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Cohn¹,

Krawisz²,

Dresler³

et al. 1984

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

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A marked induction of DNA replication was observed in confluent human diploid fibroblast cultures treated with low relatively nontoxic doses of UV radiation, NV-methyl-N-nitrosourea (MNU), and N-acetoxy-2-acetylaminofluorene (AAAF). Isopycnic CsCl density gradient analysis of newly synthesized DNA labeled with BrdUrd indicated that most of the synthesis was semiconservative. The rate of semiconservative DNA synthesis was maximal 24 hr after damage. This induction of DNA replication was greatest at 3 J/m2 UV, 0.5 mM MNU, or 1.0 ,uM AAAF; was inhibited by hydroxyurea and aphidicolin; and also occurred in repair-deficient xeroderma pigmentosum fibroblasts. Autoradiographic examination of both confluent cultures and serum-arrested cultures showed a large increase in the fraction of densely labeled (S phase) cells after UV treatment. These densely labeled cells retain the capacity for cell division and subsequent proliferation. We conclude that low doses of at least three different DNA damaging agents are capable of recruiting quiescent cells into a state of DNA replication similar to that observed in the normal cell cycle.DNA replication occurring soon after DNA damage is an important factor in mutagenesis, cell survival, and in vitro transformation (e.g., see refs. 1-5). Furthermore, DNA replication after carcinogen treatment in vivo appears to be a necessary step in the initial stages of carcinogenesis in many tissues (reviewed in refs. 6 and 7). Although most studies have indicated that DNA-damaging agents inhibit DNA replication in actively growing cultured cells (e.g., see refs. 8-10) and in regenerating tissues in vivo (reviewed in ref. 6), a few papers suggest that carcinogenic agents may also induce or stimulate DNA replication (11)(12)(13). We have investigated this possibility in detail and have found that at least three agents that damage DNA can also induce semiconservative DNA synthesis in quiescent human fibroblasts. MATERIALS AND METHODSCell Culture. Normal human diploid fibroblasts (AG1518; Human Genetic Mutant Cell Repository, Camden, NJ), xeroderma pigmentosum group G (GM3021A; Human Genetic Mutant Cell Repository), and xeroderma pigmentosum group A (CRL1223; American Type Culture Collection) were cultured, labeled with [14C]thymidine and grown to confluence as described (14). Cells were used between passages 12 and 17 within 7 days of reaching confluence. For some experiments, growth was arrested at low cell density by incubating cultures in medium with 0.05% fetal calf serum.UV and Chemical Damage and Labeling of Damaged Cells. Thirty minutes prior to damage, 50 uM BrdUrd (final concentration) was added to the culture medium. Some cultures were irradiated with UV light as described (15) (17) and ethanol-precipitated. For alkaline CsCl gradients, the precipitated DNA was dissolved in 4.5 ml of CsCl, pH 13 (0.1 M KOH; p = 1.799) and centrifuged at 50,000 rpm for 8 hr at 20'C in a Sorvall TV865 rotor. For neutral CsCl gradients, the precipitated DNA was dissolved in 4.5 ml of CsCl, pH 7.5 (p...

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Section: Resultsmentioning

confidence: 99%

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Cohn¹,

Krawisz²,

Dresler³

et al. 1984

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

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“…More recently, attempts have been made to develop, refine, and apply a procedure wherein it is unnecessary to resolve the two types of DNA synthesis. This approach involves the total suppression of normal DNA replication, thereby providing assurance that incorporation of all precursors into DNA reflects repair synthesis only, uncomplicated by normal replication (41)(42)(43). Hydroxyurea has been used the most extensively for the purpose of inhibiting normal replication, though recently an artifact associated with its use in studying repair synthesis has been observed (41).…”

Section: Methods Of Assessing Repair In Mammalsmentioning

confidence: 99%

“…Certain N-hydroxy compounds which appeared to induce repair (i.e., hydroxylamine and N-hydroxycyclohexylamine) were found to interfere with the ability of hydroxyurea to suppress normal replication (41). Thus, the stimulation of DNA synthesis produced by exposure of these cells to N-hydroxy compounds was an apparent consequence of their interference with the inhibitory effect of hydroxyurea on semiconservative replication and not due to the ability of these N-hydroxy compounds to induce repair synthesis per se.…”

Section: Methods Of Assessing Repair In Mammalsmentioning

confidence: 99%

The role of repair in environmental mutagenesis.

Flamm¹

1973

Environ Health Perspect

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“…For measurement of DNA repair synthesis, an inhibitor of replicative synthesis, hydroxyurea (HU, Brandt et al, 1972) was present in the incubation mixture at 10-2 M. This chemical inhibits normal semiconservative DNA replication, but at the concentration used does not inhibit repair synthesis.…”

Section: Dna Replication and Repair Synthesis In The Lungmentioning

confidence: 99%

Studies in a Rat Lung Tumor Model: Cellular Biochemistry and Cytogenetics

Rasmussen¹

1982

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The value of hydroxyurea in assessing repair synthesis of DNA in HeLa cells

Cited by 76 publications

References 18 publications

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

The role of repair in environmental mutagenesis.

Studies in a Rat Lung Tumor Model: Cellular Biochemistry and Cytogenetics

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