Apoptosis is a form of programmed cell death that occurs in multicellular organisms in which cells are dismantled and removed in a controlled manner, avoiding harm to surrounding tissues. Apoptotic cell death involves a series of characteristic morphological and biochemical changes that occur in specific temporal fashion, and include early/intermediate (i.e.: caspase activation and phosphatidylserine externalization) and late (i.e.: internucleosomal DNA fragmentation) events. Previous studies carried out in cancer cell lines treated with various apoptotic inducers revealed that surprisingly, apoptosis was reversible and that cells could recover and resume growth following removal of the inducer. In this study, we have developed a mammalian model of apoptosis rescuability and reversibility using cultured mouse HT-2 T lymphocytes deprived of the essential growth and survival factor interleukin 2 (IL2). Using quantitative flow cytometry analysis, we demonstrate that HT-2 cells accumulate in the G1 phase of the cell cycle by 24 hours following IL2 deprivation prior to undergoing DNA fragmentation and death within 36-48 hours. Our findings further indicate that IL2-deprived HT-2 lymphocytes can be rescued from apoptosis by IL2 re-addition to the culture medium. We show that cellular morphology and cell cycle re-entry is restored within 24-36 hours. Experiments are currently underway to determine which events along the apoptotic pathway (caspase activity, internucleosomal DNA fragmentation/TUNEL assay, and phosphatidylserine externalization/Annexin V binding) are congruent with reversibility.
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