Genesis of discrete higher order DNA fragments in apoptotic human prostatic carcinoma cells

Higher order DNA fragmentation may be an essential signal in apoptosis. We found that etoposide (VP-16) induced apoptosis in human DU-145 prostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells detached from the monolayer; untreated or VP-16-treated attached cells retained a normal morphology. We describe a radiolabeled alu-I sequence-based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecular weight DNA fragments in detached (apoptotic) and attached (preapoptotic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were > or = 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that these DNA fragments increased 9 +/- 2-, 8 +/- 2-, and 25 +/- 11-fold versus control, respectively, with a 24-hr exposure to 30 microM VP-16 in attached cell populations. In detached VP-16-treated cells, there was accrual of 30-50-kbp DNA fragments with a concomitant loss of the > or = 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weight DNA fragmentation was inhibited by cycloheximide treatment and was common to other apoptotic agents, including melphalan and bleomycin. These findings suggest that the > or = 1-mbp and 450-600-kbp DNA fragments are products of endonuclease activation and are not topoisomerase II/DNA interactions. Finally, the generation of the 30-50-kbp DNA fragments may mediate chromatin condensation, which characterizes apoptosis.