Investigating the Kinetics of Anastasis in HT2 Cells Following Camptothecin-Induced Apoptosis

Apoptosis, or programmed cell death, is a highly regulated biological process essential for tissue homeostasis, immune system regulation, and cell turnover. This process ensures the orderly removal of damaged or unnecessary cells without damage to the host. Apoptotic cell death is characterized by distinct morphological and biochemical changes that occur in a sequential order, including cytoplasmic caspase activation and phosphatidylserine externalization (PS) in the cell's plasma membrane. In addition, cells have the ability to reverse the apoptotic process through a process called anastasis. Anastasis occurs when cells that have entered the early to intermediate stages of apoptosis, characterized by caspase activation and PS externalization, manage to recover and survive, following exposure to environmental stress, which includes growth factor deprivation or treatment with chemical inducers of apoptosis. In this study, we used cultured mouse HT2 cells, to investigate the kinetics of anastasis (e.g.: apoptosis reversibility), caspase activation and PS externalization, following apoptosis induction with the chemotherapeutic drug camptothecin. Using quantitative flow cytometry, we determined that a 9-hour induction period with camptothecin was optimal to induce apoptosis reversibility in HT2 cells. Caspase activity was measured using the NucView 488 fluorogenic substrate, whereas PS externalization was monitored using Annexin CF 640R binding. Our findings indicate that HT2 cells undergoing anastasis gradually lose PS externalization and caspase activity over a 96 hour rescue period.