Drug-specific sites of topoisomerase II DNA cleavage in Drosophila chromatin : Heterogeneous localization and reversibility

• Published in: Cancer research (Chicago, Ill.) Vol. 56; no. 8; pp. 1855 - 1862
• Main Authors: BORGNETTO, M. E, ZUNINO, F, TINELLI, S, KÄS, E, CAPRANICO, G
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 15-04-1996
• Subjects: Amsacrine - pharmacology; Animals; Antineoplastic agents; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Chromatin - drug effects; Chromatin - metabolism; DNA - drug effects; DNA - metabolism; DNA Primers; DNA Topoisomerases, Type II - metabolism; DNA, Satellite - genetics; Doxorubicin - analogs & derivatives; Doxorubicin - pharmacology; Drosophila melanogaster; Enzyme Inhibitors - pharmacology; General aspects; Genes, Insect; Histones - genetics; Kinetics; Medical sciences; Molecular Sequence Data; Pharmacology. Drug treatments; Promoter Regions, Genetic; Substrate Specificity; Teniposide - pharmacology; Topoisomerase II Inhibitors; Antineoplastic agent; DNA topoisomerase (ATP-hydrolysing); Chromatin; Nucleotide sequence; Enzyme; Insecta; Acridine derivatives; Drosophila; Enzyme inhibitor; Podophyllotoxine derivative; In vitro; Site of action; Drosophilidae; Specificity; Isomerases; Arthropoda; DNA; Cleavage; Anthracyclins; Invertebrata; Diptera; Comparative study
• ISSN: 0008-5472; 1538-7445

DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells.