Cleavage of cellular DNA by calicheamicin γ1

It is assumed that the efficient antitumor activity of calicheamicin gamma1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calichearnicin gamma1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin gamma1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0degreesC as compared to 37degrees due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calichearnicin gamma1 cleaved purified plasmid DNA. DNA strand breaks introduced by calichearnicin gamma1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin gamma1-induced DSBs, were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin gamma. The data indicate that DSBs is the crucial damage after calichearnicin gamma1 and that calicheamicin gamma1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin gamma1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease. (C) 2002 Elsevier Science B.V. All rights reserved.