Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis

✍ Scribed by Yoshiko Yokoyama; Kazunari Nohara; Tomoko Okubo; Itsu Kano; Keisuke Akagawa; Kazutaka Kano

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 296 KB
• Volume: 100
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.21024

✦ Synopsis

Abstract

The mechanism of induction of apoptosis by dolichyl phosphate (Dol‐P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 µg/ml of Dol‐P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol‐P, but nearly 50% of activity still remained at 20 µg/ml. Dol‐P induced release of cytochrome‐c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time‐dependent manner. Flow cytometric analysis also indicated that Dol‐P caused loss of mitochondrial membrane potential (Δψ~m~) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol‐P‐induced ROS generation and activation of caspase‐3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol‐P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol‐P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal‐regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol‐P‐induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol‐P‐induced apoptosis. J. Cell. Biochem. 100: 349–361, 2007. © 2006 Wiley‐Liss, Inc.