dolichol-monophosphate and herbimycin

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 microg/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 microg/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 (Deltapsi(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.

Topics: Apoptosis; Benzoquinones; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Respiration; Cell Shape; Cytochromes c; Dolichol Phosphates; Dolichols; Enzyme Activation; Enzyme Inhibitors; Humans; Lactams, Macrocyclic; Microscopy, Electron, Transmission; Mitochondria; Mitogen-Activated Protein Kinases; Pyrrolidines; Reactive Oxygen Species; Rifabutin; Thiocarbamates; Time Factors

Exogenous dolichyl phosphate (Dol-P) induced apoptosis in the human monoblastic leukemia cell line U937 within 4 hours. Phosphorylation of p42 mitogen-activated protein kinase (MAP kinase) increased prior to DNA fragmentation. MAP kinase activation occurred within 5 min, and the maximum response was observed at 30 min. Inhibition of tyrosine phosphorylation of MAP kinase by herbimycin A resulted in complete inhibition of DNA fragmentation and partial inhibition of cell death. These results suggested that Dol-P-induced apoptosis is mediated by the MAP kinase cascade.

Topics: Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Survival; DNA Damage; DNA, Neoplasm; Dolichol Phosphates; Enzyme Activation; Humans; Kinetics; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Quinones; Rifabutin; Time Factors; Tumor Cells, Cultured