A fast and sensitive technique to study the kinetics and the concentration dependencies 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 differentiates 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 inexpensive column separation technique to study drug-induced DNA fragmentation from 10(6) or less cells. This technique, which is based on a modified plasmid spin column kit, avoids the use of hazardous chemicals. With this procedure and subsequent densitometric analysis it was possible to study the concentration dependencies and the kinetics of drug-induced DNA fragmentation. The applicability of this technique is shown for okadaic acid- and cantharidic-acid-treated pituitary GH(3) cells as well as highly okadaic-acid-resistant sublines. These studies allowed us to compare as well as to differentiate the effects and potencies of these structurally different but functionally quite similar inhibitors of ser/thr phosphatases 1 and 2A.
Steinfelder
H J
HJ
Institute of Pharmacology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. hsteinfe@med.uni-goettingen.de
Quentin
I
I
Ritz
V
V
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002193:Cantharidin / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects; D051381:Rats; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D019319:Okadaic Acid; D004247:DNA; D010749:Phosphoprotein Phosphatases; D002193:Cantharidin