Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells

The rate and extent of disappearance of two DNA lesions (pyrimidine dimers and covalently bound acetylaminofluorene), both thought to be removed by the socalled wide-patch (approximately 100 nucleotides) repair process were studied in a variety of cultured mammalian cells. With the exception of mouse cells, dimers were removed more rapidly and extensively than covalently bound acetylaminofluorene. In human cells, for example, about 50% of the dimers were excised from DNA in 1 hr while only 25-50% of the chemically induced lesions were excised from DNA after 48 hr. Surprisingly mouse cells, which remove few dimers, were about as competent as control human fibroblasts at removing acetylaminofluorene lesions; however, xeroderma pigmentosum cells (group D) removed fewer N-acetoxy-2-acetylaminofluo- N-acetoxy-2-acetylaminofluorene (NA-AAF) is frequently used experimentally as a chemical carcinogen that directly attacks DNA, and the damage it produces in DNA is thought to be repaired by a series of steps similar or identical to those involved in the repair of pyrimidine dimers (6,9,(16)(17)(18)(19). In spite of the many similarities, however, short-term experiments fail to elicit as much repair synthesis with NA-AAF (even at high concentrations) as that seen with low to moderate fluences of cells were grown in antibiotic-free diploid growth medium supplemented with 10% fetal-calf serum at 370 in a 5% CO2 atmosphere. All other cell lines were plated in antibiotic-free Dulbecco's modified Eagle's medium containing 4.5 g/liter of glucose and 110 mg/liter of sodium pyruvate supplemented with either 10% fetal-calf serum or 10% calf serum, and grown in a 10% CO2 atmosphere. The cells were free of mycoplasma contamination. To facilitate comparisons among cell types, eliminate any possible effects of the cell cycle on removal, and obviate the necessity to correct for the effect of DNA synthesis on specific activity, we performed all the studies on removal of lesions with confluent cells.Measurement of Thymine-Containing Pyrimidine Dimers. Cells in the logarithmic phase of growth were labeled with[3H]thymidine (New England Nuclear), 1 ;jCi 3H/ml of medium (specific activity, 50-60 Ci/mmol) for 24 hr. The medium was changed, and the cells were grown to confluence. Before irradiation, the medium was temporarily removed, and the cells were washed twice with Dulbecco's phosphate-buffered saline (370). The cells were irradiated with a 15 W germicidal lamp (254 nm) at a flux of 2.5 j/m2 per sec (total dose, 10-20 J/m2), and, after exposure, the same medium at 37°-was replaced when appropriate. Incidental exposure to room lighting was minimized throughout the procedure. Pyrimidine dimers were isolated from total nuclei (13, 18) and detected by the methods of Carrier and Setlow (24) and Goldmann and Friedberg (25) with some modification: glass backed thin-layer chromatography plates were used (MN-300-cellulose; Brinkman) and hydrolyzed, irradiated polyd(T) was used as a standard (26).Measurement of Binding and Removal of N...

Section: Resultsmentioning

confidence: 92%

mentioning

confidence: 99%

Differences in removal of acetylaminofluorene and pyrimidine dimers from the DNA of cultured mammalian cells.

Amacher¹,

Elliott²,

Lieberman³

1977

“…Cellular repair processes for structurally modified DNA (15,16) involve the elimination of altered bases from DNA. The question, therefore, arose as to whether the tissue-specific carcinogenic effect of EtNU might be related to tissue differences in the elimination rates of ethylated bases from DNA.…”

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confidence: 99%

Persistence of O 6-Ethylguanine in Rat-Brain DNA: Correlation with Nervous System-Specific Carcinogenesis by Ethylnitrosourea

Goth¹,

Rajewsky²

1974

414

120

The nervous system specificity of the carcinogenic effect of N-ethyl-N-nitrosourea Present evidence suggests that the probability of neoplastic transformation is not a simple function of the degree of cellular interaction with an oncogenic agent, but is also strongly dependent on the proliferative and functional state of the target cell (6, 1). In the search for cellular determinants of malignant transformation the tissue specificity of carcinogens may be a factor of considerable heuristic value, provided it is not primarily due to activation of the compounds by enzymes particularly active in the tissues in question.The carcinogenic (8) and mutagenic (9) acylalkylnitrosamide N-ethyl-N-nitrosourea (EtNU) yields an electrophilic ethyl cation via nonenzymatic, heterolytic decomposition, occurring with a half-life of about 8 min under physiological conditions (7). A single pulse of EtNU, applied to fetal or newborn rats up to an age of 10-20 days, specifically results in a high incidence of malignant neuroectodermal tumors associated with the central and peripheral nervous system (refs.

“…DNA repair in response to both radiation and chemical damage has been studied in various cultured mammalian cells (1)(2)(3)(4)(5)(6)(7) and tissues (8)(9)(10). To date, interest has focused primarily upon differences in the responsiveness of various cell lines (1,5,11,12), evaluation of several repair deficient lines (2,3,11,13,14), and classification of response patterns to various agents (7,(15)(16)(17)(18).…”

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confidence: 99%

“…To date, interest has focused primarily upon differences in the responsiveness of various cell lines (1,5,11,12), evaluation of several repair deficient lines (2,3,11,13,14), and classification of response patterns to various agents (7,(15)(16)(17)(18). One important aspect of this problem that has not been extensively studied is the distribution of repair within the genome.…”

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confidence: 99%

Intragenomal Distribution of DNA Repair Synthesis: Repair in Satellite and Mainband DNA in Cultured Mouse Cells

Lieberman

Poirier

1974

DNA repair synthesis was examined in mouse satellite and mainband DNA derived from confluent Balb/c 3T3 cells damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluorene. Two different approaches were used: (i) Contact-inhibited cells were treated with hydroxyurea to reduce replicative synthesis to low levels; and (ii) bromodeoxyuridine was used to label newly replicated DNA in cells that had escaped contact inhibition. DNA was separated into mainband and satellite fractions in Ag+-Cs2SO4 gradients. After treatment with either ultraviolet radiation or N-acetoxy-2-acetylaminofluorene, repair synthesis occurred to the same extent in mainband and satellite DNA. Repair synthesis increased over an ultraviolet radiation dose range of 30-200 erg/mm, and the extent of repair in the two DNA species was similar at each dose level. An analysis of the separated strands of satellite DNA from ultraviolet-irradiated cells indicated that the extent of repair is closely correlated with the availability of pyrimidines for cyclobutyl dimer formation and provided evidence that repair synthesis occurs at the site of damage. Within the precision of our experiments the results suggest that at least one group of highly repetitive, nontranslated DNA sequences is repaired to about the same extent as the rest of the genome.