Strand-scission of HeLa cell deoxyribonucleic acid by bleomycin in vitro and in vivo

Abstract

The ability of bleomycin (BLM) to cause strand breaks of DNA in vitro has been confirmed by means of alkaline sucrose sedimentation. BLM-induced breakage of extracted HeLa S3 cell DNA occurs extensively after dialysis following reaction with 50 μg/ml of BLM. 2-Mercaptoethanol (0·003 M) is not obligatory for BLM-induced degradation of DNA in vitro, but enhances it. The presence of 0·025 M EDTA in the reaction mixture completely prevents single-strand breaks of DNA by BLM. In good agreement with the in vitro results, the DNA from the HeLa cell lysate prepared in a lysing medium containing 0·015 M of EDTA is fragmented a little after a 30-min treatment with 25 μg/ml of BLM, while nonspecific and extensive degradation of DNA occurs when the cells are lysed in the absence of EDTA. A 30-min treatment with BLM also provokes a small amount of unscheduled incorporation of 3H-thymidine into non-S phase cells, indicating that a small number of single-strand breaks induced are repair-patched. Moreover, 25 μg/ml of BLM exerts a somewhat inhibitory effect on the joining of short segments of replicating DNA after a 30-min 3H-thymidine pulse, but the joining ability is soon resumed. These data suggest that BLM may either hardly enter HeLa S3 cells or may be readily inactivated.