Here, we identified
caspase-2,
protein kinase C (PKC)delta, and c-Jun NH2-terminal
kinase (JNK) as key components of the
doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for
caspase-2 (AS2) as well as a chemical
caspase-2 inhibitor, we demonstrate that
caspase-2 is required in
doxorubicin-induced apoptosis. We also identified PKCdelta as a novel
caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after
doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient
doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a
kinase dead (KD) mutant of PKCdelta severely attenuated
doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in
doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to
doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with
SP600125, further supporting the importance of JNK in
doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in
doxorubicin-treated AS2 cells.
Caspase-2, PKCdelta, and JNK were furthermore implicated in
doxorubicin-induced apoptosis of primary
acute lymphoblastic leukemia blasts. The data thus support a sequential model involving
caspase-2, PKCdelta, and JNK signaling in response to
doxorubicin, leading to the activation of Bak and execution of apoptosis.