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 pr ostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells de tached from the monolayer; untreated or VP-16-treated attached cells r etained a normal morphology. We describe a radiolabeled alu-l sequence -based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecu lar weight DNA fragments in detached (apoptotic) and attached (preapop totic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were greater than or equal to 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that t hese DNA fragments increased 9 +/- 2-, 8 +/- 2-, and 25 +/- 11-fold ve rsus control, respectively, with a 24-hr exposure to 30 mu M 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 grea ter than or equal to 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weigh t DNA fragmentation was inhibited by cycloheximide treatment and was c ommon to other apoptotic agents, including melphalan and bleomycin. Th ese findings suggest that the greater than or equal to 1-mbp and 450-6 00-kbp DNA fragments are products of endonuclease activation and are n ot topoisomerase II/DNA interactions. Finally, the generation of the 3 0-50-kbp DNA fragments may mediate chromatin condensation, which chara cterizes apoptosis.