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Updated: Jun 8, 2026
Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Analysis of protein phosphorylation in cisplatin-treated human cells following annexin V-based separation and
Elaine O'Meara1, Séverine Cruet-Hennequart, Michael P Carty
1DNA Damage Response Laboratory, Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland.
Abstract:
Cancer chemotherapy relies heavily on DNA damaging agents such as cisplatin to induce tumour cell death. The response of cells to genotoxic insult, including cell cycle arrest, DNA repair and cell death, is mediated by the DNA damage response (DDR). To address the relationship between the DDR and the outcome of exposure, this study utilised a magnetic-activated cell sorting (MACS®)-based approach to isolate apoptotic and non-apoptotic cells from a DNA polymerase eta-deficient human cell line. The pattern of phosphorylation of the key DNA damage response protein RPA2 on serine 4/8 was altered in apoptotic cells isolated following cisplatin treatment. By combining MACS® with multi-antibody screening for phosphorylated proteins, apoptosis-associated changes were characterized in a number of key signalling pathways. Phosphorylation of Erk1 on Thr202/Tyr204, and Erk2 on Thr185/Tyr187 was increased in apoptotic cells. This approach provides novel insights into the relationship between cisplatin-induced protein phosphorylation and the cellular consequences of exposure to this chemotherapeutic agent.
Insights
Chemotherapy drug cisplatin triggers DNA damage response (DDR) pathways. Researchers identified altered protein phosphorylation in apoptotic cells, revealing new insights into cancer cell death mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cancer chemotherapy utilizes DNA-damaging agents like cisplatin to induce tumor cell death.
- The DNA damage response (DDR) orchestrates cellular reactions to genotoxic stress, including cell cycle arrest, DNA repair, and apoptosis.
Purpose of the Study:
- To investigate the relationship between DDR signaling and cellular outcomes following cisplatin exposure.
- To characterize apoptosis-associated protein phosphorylation changes in response to cisplatin.
Main Methods:
- Utilized magnetic-activated cell sorting (MACS®) to isolate apoptotic and non-apoptotic cells from a DNA polymerase eta-deficient human cell line.
- Employed multi-antibody screening to identify changes in protein phosphorylation patterns.
Main Results:
- Observed altered phosphorylation of RPA2 (Replication Protein A 2) on serine 4/8 in apoptotic cells after cisplatin treatment.
- Detected increased phosphorylation of Erk1 (Extracellular signal-regulated kinase 1) and Erk2 (Extracellular signal-regulated kinase 2) in apoptotic cells.
Conclusions:
- The study provides novel insights into the specific protein phosphorylation events linked to cisplatin-induced apoptosis.
- Characterization of DDR signaling in apoptotic cells offers a deeper understanding of chemotherapeutic agent efficacy.
