Analysis of protein phosphorylation in cisplatin-treated human cells following annexin V-based separation and multi-antibody screening

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.