FORMATION OF HIGH-MOLECULAR-MASS DNA FRAGMENTS IS A MARKER OF APOPTOSIS IN THE HUMAN LEUKEMIC-CELL LINE, U937

Inhibitors of macromolecular synthesis and topoisomerases induce apopt osis in the human leukaemic cell line, U937. In this study, U937 cells were treated with the RNA synthesis inhibitor, actinomycin D (1 mu M) , the protein synthesis inhibitors, emetine (1 mu M) and cycloheximide (100 mu M), the topoisomerase II inhibitor, teniposide (5 mu M), or t he topoisomerase I inhibitor, camptothecin (1 mu M) Apoptotic cell dea th was assessed both by flow cytometry and agarose gel electrophoresis , and was correlated to the appearance of large (20 to greater than or equal to 580 kilobase pairs) DNA fragments, as assessed by field inve rsion gel electrophoresis. In all cases, the appearance of DNA fragmen ts of 20-50 kilobase pairs accompanied the appearance of an apoptotic population and of internucleosomal cleavage. However, teniposide addit ionally induced a marked increase in fragmentation to greater than or equal to 580 kilobase pairs. The cotreatment of cells with zinc (1 mM) inhibited the formation of all large DNA fragments, internucleosomal cleavage and the appearance of an apoptotic population. We conclude th at the generation of large DNA fragments is characteristic of apoptosi s induced by various stimuli in U937, as has been found previously in rat thymocytes. However, unlike what occurs in rat thymocytes, zinc tr eatment does not dissociate the formation of large fragments from conv entional markers of apoptosis.