ROLE OF SERINE AND ICE-LIKE PROTEASES IN INDUCTION OF APOPTOSIS BY ETOPOSIDE IN HUMAN LEUKEMIA HL-60 CELLS

This study was undertaken to examine the role of proteases in etoposid e-induced apoptosis of human leukemia HL-60 cells. We found the potent activity to produce internucleosomal DNA fragmentation in a 150 000 g supernatant of cell lysate which was prepared from etoposide-treated HL-60 cells undergoing apoptosis. This nuclear-DNA fragmenting activit y could be detected when the supernatant was incubated with isolated n uclei under Mg2+-dependent conditions. On the other hand, we could not detect such activity in the supernatant of cell lysate from non-treat ed HL-60 cells. Treatment of the supernatant with a serine protease in hibitor, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK), abolished t he DNA fragmenting activity. An inhibitor of interleukin 1-beta-conver ting enzyme (ICE), Z-Val-Ala-Asp-fluoromethyl ketone (VAD-FMK), had no effect on this DNA fragmenting activity in vitro. However, when the c ells were incubated with etoposide in the presence of VAD-FMK, the for mation of TPCK-sensitive DNA fragmenting activity was blocked. Our dat a indicate that serine and ICE-like proteases may be involved in etopo side-induced apoptosis at the different stages, and especially a serin e protease may be closely associated with the final step for induction of internucleosomal DNA fragmentation during apoptosis in HL-60 cells .