Extrachromosomal chromatin: novel target for bleomycin cleavage in cells and solid tumors

The preference of bleomycin, a DNA strand scission antitumor agent, to damage extrachromosomal (episomal) DNA was investigated. These episomes contain transcriptional promoters, replication origins, and oncogenes from MMTV, BPV, and v-Ha-ras and confer a neoplastic phenotype to a mouse fibroblast cell line. We found that bleomycin induces dose-dependent single- and double-stranded cleavage of intracellular episomes as measured by topological forms conversion. Bleomycin scission of episomes occurs within 1 min, and upon drug removal, damaged episomes are as rapidly repaired. By expressing the episomal and genomic damage as breaks per nucleotide, bleomycin has a 30-50-fold cleavage preference for episomal chromatin compared to genomic DNA. The episomes have preferred regions of the bleomycin-induced damage, particularly within the MMTV LTR and BPV origin of replication. Also, it is possible to assess bleomycin action on episomes in solid tumors in mice. Single intravenous injections of BLM into tumor-bearing mice result in single- and double-stranded cleavage of episomes that are dose related and occur within 1 min. Specific double-stranded breaks occur in the same regulatory regions of episomes in solid tumors and in cultured cells. Finally, we observe that damage to the episomal drug target occurs at therapeutic doses in mice.