S. Gamen et al., Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes Delta Psi(m) loss, caspase-9 activity, and apoptosis in Jurkat cells, EXP CELL RE, 258(1), 2000, pp. 223-235
Doxorubicin induces caspase-3 activation and apoptosis in Jurkat cells but
inhibition of this enzyme did not prevent cell death, suggesting that anoth
er caspase(s) is critically implicated. Western blot analysis of cell extra
cts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by d
oxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-CHO, Z
-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpr
ession of CrmA, prevented many morphological features of apoptosis but not
loss of mitochondrial membrane potential (Delta Psi(m)), phospatidilserine
exposure, and cell death, Western blot analysis of cells treated with doxor
ubicin in the presence of inhibitors allowed elucidation of the sequential
order of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspas
e-9 activity, blocked the activation of all caspases studied, lamin B degra
dation, and the development of apoptotic morphology, but not cell death. AU
morphological and biochemical features of apoptosis, as well as cell. deat
h, were prevented by cotreatment of cells with the general caspase inhibito
r Z-VAD-fmk or by overexpression of Bcl-2, Doxorubicin cytotoxicity was als
o blocked by the protein synthesis inhibitor cycloheximide. Delayed additio
n of Z-VAD-fmk after doxorubicin treatment, but prior to the appearance of
cells displaying a low Delta Psi(m), prevented cell death. These results, t
aken together, suggest that the key mediator of doxorubicin-induced apoptos
is in Jurkat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X)
, which would cause Delta Psi(m) loss, release of apoptogenic factors from
mitochondria, and cell death. (C) 2000 Academic Press.