Doxorubicin requires the sequential activation of caspase-2, protein kinase Cδ, and c-Jun NH2-terminal kinase to induce apoptosis

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 PKC delta as a novel caspase-2 substrate. PKC delta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKC delta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKC delta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKC delta and JNK inhibition show that PKC delta 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 PKC5 and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKC delta, 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, PKC delta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.