A GLASS FIBER DIETHYLAMINOETHYL DOUBLE FILTER BINDING ASSAY THAT MEASURES APOPTOTIC INTERNUCLEOSOMAL DNA FRAGMENTATION/

A filter binding assay that measures internucleosomal DNA fragmentatio n associated with apoptosis is described. The assay is based on a nove l principle that consists of using simultaneously two kinds of glass f iber filters to harvest [H-3]thymidine-prelabeled cells following thei r incubation with inducers of apoptosis. One filter, which is neutral, traps intact chromatin and high-molecular-weight DNA. The other filte r, which is positively charged with DEAE active groups, traps low-mole cular-weight DNA fragments. DNA fragmentation is quantified by measuri ng the radioactivity retained by each of the filters. The assay was ev aluated with the histiocytic lymphoma cell line U937 and the topoisome rase inhibitors camptothecin, etoposide, and doxorubicin. These agents caused a dose-dependent decrease of radioactivity in the neutral filt er and a parallel increase of radioactivity in the DEAE filter. Irradi ation-induced single strand breaks and topoisomerase-mediated primary DNA damage were not detected by this method. Consistent with the detec tion of internucleosomal DNA fragmentation, the effects measured by th is assay were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using this assay were validated by observation of DNA ladders on agarose gels and by morphologic examination of apoptotic features. Evaluation of the a ssay in a mock screen demonstrated that the introduction of the DEAE f ilter increases the assay sensitivity and eliminates false positives. Thus, this assay may be used in high-throughput screening approaches t o discover novel modulators of apoptosis. (C) 1996 Academic Press, Inc .