Quantitative analysis of DNA-cleaving activities of macromomycin, auromomycin, and their free chromophores, and some DNA-binding characteristics.
The in vitro DNA-cutting activities of macromomycin (MCR), auromomycin (AUR), and their free chromophores were quantitatively analyzed by agarose gel electrophoresis of radioactive supercoiled pBR322 DNA. Methanol extract (chromophore) of MCR inhibited growth of cultured L5178Y cells and caused DNA strand scission without addition of reducing agents, whereas MCR required dithiothreitol (DTT) for its DNA cleavage. In the absence of DTT, concentrations of MCR chromophore, AUR and AUR chromophore to induce 50% degradation of form I DNA were 320 micrograms eq./ml, 44 micrograms/ml and 60 micrograms eq./ml, respectively. DTT stimulated DNA breakage by the drugs to a similar degree (ca. 4-fold). Since both chromophores showed the same biological activity, further studies were performed with AUR and its free chromophore. DNA strand breakage by AUR occurred rapidly and completed in 5 minutes. The rate of strand scission did not significantly change in a range of pH 7.0 to 10.5, but gradually declined below pH 7.0. Of 4 sulfhydryl compounds tested, DTT and cysteine exhibited potent stimulation, but glutathione and 2-mercaptoethanol weak or no enhancement. The DNA-cleaving activity of AUR and its free chromophore is blocked by addition of ethidium bromide, suggesting that the chromophore binds to DNA by an intercalation mechanism. Addition of poly[dG], poly [dG] X poly[dC], poly [dG-dC] X poly[dG-dC] or poly[dA-dT] X poly[dA-dT] protected DNA against degradation by AUR. Differential protecting effects of synthetic polynucleotides suggest that the chromophore shows higher affinity for guanine than for adenine, cytosine and thymine, and binds preferentially to a purine-pyrimidine neighboring sequence.
D000818:Animals; D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D002863:Chromogenic Compounds / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004996:Ethidium / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051379:Mice; D010455:Peptides / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013997:Time Factors
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D002863:Chromogenic Compounds; D010455:Peptides; D013438:Sulfhydryl Compounds; C001666:auromomycin; D004247:DNA; D004996:Ethidium; D004229:Dithiothreitol