Participation of various kinases in staurosporine-induced apoptosis of RAW 264.7 cells

Abstract

Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage‐like cell line, as determined by DNA fragmentation, the increase of annexin V‐stained cells, and the cleavage of poly(ADP‐ ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub‐G₁ cells revealed that the DNA fragmentation occurred in a time‐ and concentration‐dependent manner at 0.021–2.1 μm of staurosporine. Staurosporine induced phosphorylation of p38 mitogen‐activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal‐regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3‐kinase (P13K) inhibitor LY294002 potentiated the staurosporine‐induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H‐89 potentiated the staurosporine‐induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro‐31–8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine‐induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and P13K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation.