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 etoposide-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 activity could be detected when the supernatant was incubated with isolated nuclei under Mg2+-dependent conditions. On the other hand, we could not detect such activity in the supernatant of cell lysate from non-treated HL-60 cells. Treatment of the supernatant with a serine protease inhibitor, N-tosyl-L-phenylala-nylchloromethyl ketone (TPCK), abolished the DNA fragmenting activity. An inhibitor of interleukin 1-beta-converting enzyme (ICE), Z-Val-Ala-Asp-fluoromethyl ketone (VAD-FMK), had no effect on this DNA fragmenting activity in vitro. However, when the cells were incubated with etoposide in the presence of VAD-FMK, the formation of TPCK-sensitive DNA fragmenting activity was blocked. Our data indicate that serine and ICE-like proteases may be involved in etoposide-induced apoptosis at the different stages, and especially a serine protease may be closely associated with the final step for induction of internucleosomal DNA fragmentation during apoptosis in HL-60 cells.
Yoshida
A
A
First Department of Internal Medicine, Fukui Medical School, Japan.
Takauji
R
R
Inuzuka
M
M
Ueda
T
T
Nakamura
T
T
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D020170:Caspase 1; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D002474:Cell-Free System; D017077:Culture Media, Conditioned / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000502:physiology*; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000032:analysis; D005047:Etoposide / Q000494:pharmacology*; D018922:HL-60 Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000502:physiology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D017077:Culture Media, Conditioned; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D005047:Etoposide; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D020170:Caspase 1