Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage.
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 (TOP1cc), degradation of TOP1 and activation of DNA damage responses (DDR). Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPT-induced TOP1 degradation, while none of above three processing activities affected TOP1cc formation. Replication- and transcription-initiated processing (RIP and TIP) of TOP1cc were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOP1cc triggered the CPT-induced RPA phosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chk1/2 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 TOP1cc-activated, but not ionization radiation-activated 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, respectively. Together, our results support that both RIP and TIP pathways of TOP1cc are required for the activation of CPT-induced DDR and cytotoxicity.
Huang
Ting-Hsiang
TH
Department and Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 10018.
Chen
Hsiang-Chin
HC
Chou
Shang-Min
SM
Yang
Yu-Chen
YC
Fan
Jia-Rong
JR
Li
Tsai-Kun
TK
D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin / Q000633:toxicity; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D053960:DNA Breaks; D004260:DNA Repair; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C516794:RPA2 protein, human; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin