Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） ind...Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） induced formation of topoisomerase I cleavable complex （TOPlcc）, degradation of TOP1 and activation of DNA damage responses （DDR）. Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPTo indneed TOPl degradation, while none of above three processing activities affected TOPlcc formation. Replication- and transcription-initiated proeessing （RIP and TIP） of TOPlee were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOPlec triggered the CPT-induced RPA pbosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chkl/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOPlcc-activated, but not ionization radiationactivated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respec- tively. Together, our results support that both RIP and TIP pathways of TOPlcc are required for the activation of CPT-induced DDR and cytotoxicity.展开更多
文摘Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） induced formation of topoisomerase I cleavable complex （TOPlcc）, degradation of TOP1 and activation of DNA damage responses （DDR）. Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPTo indneed TOPl degradation, while none of above three processing activities affected TOPlcc formation. Replication- and transcription-initiated proeessing （RIP and TIP） of TOPlee were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOPlec triggered the CPT-induced RPA pbosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chkl/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOPlcc-activated, but not ionization radiationactivated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respec- tively. Together, our results support that both RIP and TIP pathways of TOPlcc are required for the activation of CPT-induced DDR and cytotoxicity.
