Reversal of Apoptosis (Anastasis) as a Homeostatic Mechanism in Mammalian T-lymphocytes

Programmed cell death by apoptosis is a vital feature of multicellular life. It is characterized by a common set of morphological and biochemical changes that include chromatin condensation, caspase activation, DNA cleavage, membrane blebbing, and cellular fragmentation. Apoptosis can be reversed by a process called anastasis, which has been previously documented as a contributing mechanism in cancer recurrence following chemotherapy. The goal of our current work has been to determine whether anastasis is a more generalized cellular process independent of cancer. We used cultured mouse HT2 T-lymphocytes as a model system, a non-cancerous cell line that is dependent on the cytokine Interleukin 2 (IL2) for survival. Apoptosis was induced in HT2 cells with three different methods: IL2 deprivation (starvation), treatment with staurosporine, or camptothecin. We have previously shown that apoptosis induced by IL2 deprivation for 24 hours was reversible following IL2 re-addition, as cells were observed to gradually resume their normal physiological processes within 72 hours of rescue. Here, HT2 cells were also treated with staurosporine and camptothecin, washed free of each inducer, recovered in IL2, and analyzed by flow cytometry at 12, 24, 48, and 72 hours following the removal of the chemical inducer. Cell viability and executioner caspase 3/7 activity were determined jointly using Sytox Red vital dye and NucView 488 substrate, respectively, whereas cell cycle distribution was monitored post-fixation using propidium iodide to determine DNA content per cell. Our findings revealed that apoptosis in HT2 cells was reversible in all three treatments and that HT2 cells gradually regained normal cell cycle distribution during the 72-hour rescue window. Interestingly, sustained caspase activity was observed throughout the rescue period for cells treated with camptothecin. Our findings suggest that anastasis may be a normal physiological process in animal cells, independent of malignancy.