DETECTION OF APOPTOSIS-ASSOCIATED DNA FRAGMENTATION USING A RAPID AND QUANTITATIVE FILTER ELUTION ASSAY

DNA degradation to oligonucleosome size fragments is one of the cellular markers of apoptosis. Based on DNA filter elution methodology, we have designed a simple assay to monitor apoptosis-associated DNA fragmentation from cultured cells or from a reconstituted cell-free system. The cells are prelabeled with [C-14]-thymidine for one cell cycle and chased in nonradioactive medium for a few hours in order to allow incorporation of the radiolabeled thymidine in high molecular weight DNA. After the apoptosis-inducing treatment, cells (or subcellular fractions of the cell-free system) are deposited onto a protein-adsorbant filter and washed with physiological saline. Lysis is then performed with a mild detergent (sarkosyl) and 2 M salt. The lysis fraction is collected, counted, and computed to calculate the fraction of DNA in the lysis fraction. In normal cells more than 90% of the DNA remains on the filter, while in apoptotic cells the kinetics of DNA fragmentation can be monitored and more than 80% of the counts can be found in the lysis. The DNA filter elution assay is sensitive, quantitative, and rapid. By using different types of filters and lysis solution (+/- proteinase), the protein-bonding to the DNA fragments can be determined. Some applications of this assay to study the effects and mechanisms of action of new therapeutic drugs are presented and discussed. (C) 1995 Wiley-Liss, Inc.