A novel filter binding assay to measure internucleosomal DNA fragmentation

We have developed a double filter binding assay that measures internucleosomal DNA fragmentation associated with apoptosis. The assay is based on a novel principle that consists of using simultaneously two types of glass fibre filters to harvest [³H]thymidine-labeled cell lysates. One filter is neutral and traps chromatin-associated DNA. The other filter is positively charged with DEAE active groups and traps low-molecular-weight DNA fragments. DNA fragmentation is quantified by measuring the amount of radioactivity retained by each of the filters. The assay was tested using the histiocytic lymphoma cell line U937 and the topoisomerase inhibitors camptothecin, etoposide and doxorubicin. Each of these drugs caused a dosedependent decrease of the radioactivity retained by the neutral filter and a corresponding increase in the radioactivity trapped by the DEAE filter. Consistent with the induction of internucleosomal DNA fragmentation, these effects were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using the filter binding assay correlated with visual observation of DNA ladders on agarose gel electrophoresis. Thus, the double filter binding assay is a "bona fide" measure of apoptotic internucleosomal DNA fragmentation. Because the introduction of DEAE filter increases the sensitivity of the assay and also eliminates false positives, the assay is amenable to high-throughput screening, and may be used to search for modulators of apoptosis within large libraries of compounds.