Camptothecin induces G(2)/M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species

In the present study, we report that camptothecin (CPT) caused irreversible cell cycle arrest at the G(2)/M phase, and was associated with decreased levels of cell division cycle 25C (Cdc25C) and increased levels of cyclin B1, p21, and phospho-H3. Interestingly, the reactive oxygen species (ROS) inhibitor, glutathione, decreased CPT-induced G(2)/M phase arrest and moderately induced S phase arrest, indicating that the ROS is required for the regulation of CPT-induced G(2)/M phase arrest. Furthermore, transient knockdown of nuclear factor-erythroid 2-related factor 2 (Nrf2), in the presence of CPT, increased the ROS’ level and further shifted the cell cycle from early S phase to the G(2)/M phase, indicating that Nrf2 delayed the S phase in response to CPT. We also found that CPT-induced G(2)/M phase arrest increased, along with the ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2)-Cdc25C axis. Additionally, the proteasome inhibitor, MG132, restored the decrease in Cdc25C levels in response to CPT, and significantly downregulated CPT-induced G(2)/M phase arrest, suggesting that CPT enhances G(2)/M phase arrest through proteasome-mediated Cdc25C degradation. Our data also indicated that inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibited CPT-induced p21 and cyclin B1 levels; however, inhibition of ERK blocked CPT-induced G(2)/M phase arrest, and inhibition of JNK enhanced apoptosis in response to CPT. Finally, we found that CPT-induced G(2)/M phase arrest circumvented apoptosis by activating autophagy through ATM activation. These findings suggest that CPT-induced G(2)/M phase arrest through the ROS-ATM-Chk2-Cdc25C axis is accompanied by the activation of autophagy.