title: Actinomycin D-associated lesions mimicking DNA-DNA interstrand crosslinks detected by alkaline elution in cultured mammalian cells.
abstract: Cultured human fibroblasts were exposed to various concentrations of actinomycin D, a DNA intercalating agent, and studied by various alkaline elution techniques for the presence of DNA lesions. DNA-protein crosslinks increased proportionately with increasing actinomycin D, with 1.53 crosslinks/10(6) nucleotides after 5 micrograms/ml exposure. However, in the single-strand DNA break assay, elution of DNA initially increased as expected with increasing actinomycin D but thereafter decreased, with only 0.09 breaks/10(6) nucleotides detected after 25 micrograms/ml exposure, suggesting the presence of DNA crosslinking. A standard alkaline elution assay for DNA-DNA crosslinking was performed, and lesions which mimicked such crosslinks were detected, with a relative crosslink frequency of 2.30 after 5 micrograms/ml exposure. These actinomycin D-associated lesions disappeared when the alkaline elution procedure was modified to include additional proteinase digestion and use of the detergent sodium dodecyl sulfate (SDS) in the elution buffer, suggesting that they represented undigested DNA-protein crosslinks or nonspecific protein on the filters inhibiting DNA elution. Greater than ten times as many DNA-protein crosslinks were detected in fibroblasts than the number of single-strand DNA breaks after cellular exposure to actinomycin, even after determining breaks using the modified methodology for decreasing cellular protein interference. The data suggest that the actinomycin-DNA complex is associated with the formation of DNA-protein crosslinks which represent lesions other than endonuclease-associated DNA strand scission.
mesh_terms: D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D003609:Dactinomycin / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007364:Intercalating Agents / Q000633:toxicity*; D007930:Leucine / Q000378:metabolism; D011506:Proteins / Q000378:metabolism
chemical_list: D007364:Intercalating Agents; D011506:Proteins; D003609:Dactinomycin; D004247:DNA; D007930:Leucine