Kinetics of mitochondrial membrane potential and caspase activation in a reversible model of apoptosis in cultured mammalian T lymphocytes

Apoptosis, also known as programmed cell death, is a natural physiological process that helps maintain balance within the body, regulating cell numbers in tissues and organs. An early hallmark of this process is the loss of the mitochondrial membrane potential (MMP), followed by the activation of enzymes called caspases, which systematically dismantle the cell. Apoptosis can also be reversed by a process called anastasis, during which cells fully recover after the removal of the inducer. This study utilized cultured mouse HT2 lymphocytes, which are dependent on the growth factor interleukin 2 (IL2). Apoptosis was induced by treating HT2 cells with the chemotherapeutic drug camptothecin. Using a quantitative flow cytometry approach, we specifically investigated the optimal camptothecin exposure time and kinetics of apoptosis reversibility (e.g.: anastasis). Anastasis was initiated by washing the cells free of the drug followed by growth in culture medium supplemented with IL2. Our initial experiments demonstrated that a 9-hour treatment time was optimal to observe apoptosis reversibility. We next asked whether loss of MMP and caspase activation were also reversible events following camptothecin treatment. MMP was measured using the Mitoview fluorescent substrate, whereas caspase activity was monitored with the NucView 488 caspase substrate. Our findings indicated that caspase activity gradually decreased as MMP returned to control levels over a 96-hour rescue period. Experiments are currently underway to measure metabolic rate kinetics of HT2 cells during anastasis, using an MTT reduction assay.