Date of Award

6-2023

Document Type

Union College Only

Department

Biology

First Advisor

Robert Lauzon

Language

English

Keywords

Apoptosis, Anastasis, Programmed Cell Death

Abstract

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 was to determine whether anastasis represented 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 following camptothecin treatment and IL2 deprivation and that HT2 cells gradually regained normal cell cycle distribution during the 72-hour rescue window. However, cells induced with staurosporine could not be rescued from apoptosis. Interestingly, sustained caspase activity was observed throughout the rescue period for cells treated with camptothecin for 9 hours. Our findings suggest that anastasis may be a normal physiological process in animal cells independent of malignancy but dependent on the inducer of apoptosis.

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