A fast and sensitive technique to study the kinetics and the concentrationdependencies of DNA fragmentation during drug-induced apoptosis

Apoptotic cell death with its characteristic coordinated cellular breakdown can be triggered by cytotoxic drugs. One prominent feature that differenti ates apoptotic from necrotic cell death is the caspase-mediated activation of an endonuclease that internucleosomally cleaves DNA resulting in the so- called apoptotic DNA ladder. Here we report a new rapid, sensitive and inex pensive column separation technique to study drug-induced DNA fragmentation from 10(6) or less cells. This technique, which is based on a modified pla smid spin column kit, avoids the use of hazardous chemicals. With this proc edure and subsequent densitometric analysis it was possible to study the co ncentration dependencies and the kinetics of drug-induced DNA fragmentation .. The applicability of this technique is shown for okadaic-acid- and canth aridic-acid-treated pituitary GH(3) cells as well as highly okadaic acid-re sistant sublines. These studies allowed us to compare as well as to differe ntiate the effects and potencies of these structurally different but functi onally quite similar inhibitors of ser/thr phosphatases 1 and 2A. (C) 2000 Elsevier Science Inc. All rights reserved.