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

Abstract

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...

DNA damage-inducible transcripts in mammalian cells.

“…The 254-nm UV radiation and the AAAF have been grouped together, since DNA damage by both these agents is repaired by nucleotide-excision repair (17). The dose of UV radiation used (14 J m-2) reduced survival to 4-10% of that of untreated cells, while the dose of AAAF used (20 uM) would be expected to reduce cell survival to 1-10% of untreated cells (18). Both MMS and H202 produce predominantly nonbulky base damage in DNA which is repaired by base-excision repair mechanisms (17).…”

Differentiation of the mammary gland and susceptibility to carcinogenesis

“…This fact demonstrates the effectiveness of HU and DM BA in the inhibition of scheduled DNA synthesis. In a separate but related experiment, the effect of HU and DMBA on scheduled DNA synthesis was examined, and the results obtained are summarized cinogen from DNA of cultured cells (321,(328)(329)(330)(331)(332)(333)(334)(335). No studies, however, have dealt with the removal of adducts of DMBA from the rat mammary gland, which is a target organ for the carcinogenic action of DMBA.…”

Different rate-limiting steps in excision repair of ultraviolet- and N-acetoxy-2-acetylaminofluorene-damaged DNA in normal human fibroblasts.

“…An accompanying paper by Amacher et al (30) shows by direct measurement of excision of dimers and loss of AAAF products from DNA in cells of several species that the two types of damage are removed at very different rates and hence presumably by different enzymatic pathways.…”