Measurement of bleomycin, neocarzinostatin, and auromomycin cleavage of cell-free and intracellular simian virus 40 DNA and chromatin.
Simian virus 40 (SV40) was used as a model for eukaryotic chromatin to allow analysis of strand scission drug action on DNA of the same nucleotide sequence in different environments. Cleavage of purified DNA, cell-free chromatin, and intracellular chromatin by the antitumor drugs bleomycin, neocarzinostatin, and auromomycin was assayed. Comparison of total (single-and double-stranded) drug-induced cutting of purified SV40 DNA and isolated viral minichromosomes, as measured by the conversion of supercoiled DNA to the nicked circular form, revealed that a 2-3-fold higher concentration of all three drugs is required to cut minichromosomes as extensively as naked DNA. In addition, differences in drug activity on cell-free nuclear viral chromatin and intracellular SV40 chromatin were measured. Three times more auromomycin was needed to cut intracellular SV40 DNA as extensively as the viral DNA in isolated nuclei, whereas, surprisingly, the increases in bleomycin and neocarzinostatin concentrations were 200-fold and 1000-fold respectively. When the drugs were used to treat SV40-infected cells, no repair of strand scissions was detected. Formation of double-stranded breaks by bleomycin, neocarzinostatin, and automomycin, measured at equivalent levels of total cleavage, was also examined to provide information on the distribution of strand scissions in different environments. Nucleoproteins were found to cause a 3-fold higher level of neocarzinostatin-induced double-stranded breaks to be made on isolated minichromosomes than on purified DNA, but they had no effect on double-stranded break formation by either bleomycin or auromomycin. In contrast, auromomycin made twice as many double-stranded breaks on intracellular SV40 DNA as on minichromosomes in isolated nuclei. However, neither bleomycin- nor neocarzinostatin-induced double-stranded breaks on intracellular SV40 DNA were significantly different from those made on SV40 in isolated nuclei.
D000818:Animals; D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001761:Bleomycin / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000187:drug effects*; D004249:DNA Damage; D004260:DNA Repair; D004278:DNA, Superhelical / Q000187:drug effects; D004279:DNA, Viral / Q000187:drug effects*; D007668:Kidney; D007700:Kinetics; D010455:Peptides / Q000494:pharmacology; D013539:Simian virus 40 / Q000187:drug effects / Q000235:genetics*; D009353:Zinostatin / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D002843:Chromatin; D004278:DNA, Superhelical; D004279:DNA, Viral; D010455:Peptides; C001666:auromomycin; D001761:Bleomycin; D009353:Zinostatin