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Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA
Ting-Hsiang Huang1, Hsiang-Chin Chen, Shang-Min Chou
1Department and Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 10018.
Abstract:
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.
Insights
Camptothecin (CPT) triggers DNA damage responses by forming topoisomerase I cleavable complexes (TOP1cc). Two pathways, replication-initiated processing (RIP) and transcription-initiated processing (TIP), distinctly activate cellular responses and contribute to CPT-induced cell killing.
Area of Science:
- Molecular Biology
- Cellular Biology
- Pharmacology
Background:
- Topoisomerase-targeting drugs like camptothecin (CPT) are crucial in cancer therapy.
- Cellular responses to CPT involve complex processing pathways.
- Understanding these pathways is key to optimizing CPT efficacy.
Purpose of the Study:
- To elucidate the distinct roles of replication-initiated processing (RIP) and transcription-initiated processing (TIP) of topoisomerase I cleavable complexes (TOP1cc) in CPT-induced DNA damage responses (DDR).
- To investigate the mechanisms of TOP1 degradation and its impact on DDR.
- To determine how RIP and TIP pathways influence CPT-induced cytotoxicity.
Main Methods:
- Cellular exposure to CPT and analysis of TOP1cc formation, TOP1 degradation, and DDR activation.
- Investigating the involvement of transcription, proteasome, and replication in TOP1 degradation.
- Utilizing nonreplicating cell models to assess S-phase independence of TIP.
- Employing proteasome inhibitors and pharmacological interference with RIP and TIP pathways.
Main Results:
- CPT induced TOP1cc formation and TOP1 degradation, with transcription and proteasome-dependent proteolysis involved in degradation, but not replication.
- RIP and TIP were identified as independent pathways processing TOP1cc, contributing distinctly to DDR.
- RIP triggered RPA phosphorylation in cycling cells, while TIP was required for ATM, p53, and Chk1/2 phosphorylation, particularly at higher CPT doses and was S-phase independent.
- TIP was implicated in TOP1cc-activated DDR, distinct from radiation-activated DDR, and proteasome inhibition mimicked transcription inhibition effects.
Conclusions:
- Both RIP and TIP pathways of TOP1cc are essential for activating CPT-induced DDR and cytotoxicity.
- Distinct contributions of RIP and TIP pathways to DDR activation and cell killing offer potential therapeutic targets.
- Understanding the differential roles of RIP and TIP can guide CPT dosage and combination therapies.
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