Mechanism of apoptosis: Balance between suicidal and protecting factors determines survival of CTLL2 cells

The morphological hallmark of apoptosis is the condensation of nuclear chromatin, accompanied in many cases by the fragmentation of genomic DNA, prior to the disruption of the cell membrane and intracellular organelles. In studies summarized in this dissertation, various experimental approaches were explored to investigate the mechanism of apoptosis. The model selected for most of the studies is CTLL2, an Interleukin-2 (IL-2) dependent T lymphocyte cell line. IL-2 deprivation causes DNA fragmentation and apoptosis in CTLL2. The following hypotheses were tested. The control of apoptosis in CTLL2 by IL-2 is achieved by the balance between suicidal factors and protecting factors.Since transcriptional blockers abolished the IL-2 rescue of CTLL2, it appears that IL-2 mediated gene transcription is necessary for survival. Apoptosis induced by either the deprivation of IL-2 or the inhibition of IL-2 mediated gene transcription was blocked by translational blockers, indicating the presence of suicidal genes. The suicidal gene was pursued by (1) searching for the mammalian genes homologous to ced-4, a known cell death gene in Caenorhabditis elegans; (2) isolation of genes differentially expressed in apoptotic cells was undertaken; (3) characterization and partial purification of an endonuclease activity detected in apoptotic CTLL2 cells.The candidate protecting genes were searched for by differential screening of cDNA clones specifically expressed in IL-2 stimulated cells. Proto-oncogene bcl-2, which is known to protect a number of cells from apoptosis, was tested in CTLL2 cells. Deregulated expression of bcl-2 prolongs the survival of IL-2 deprived CTLL2 cells. The expression of endogenous bcl-2 was rapidly down-regulated upon IL-2 withdrawal within 8 h and to a non-detectable level after three days. Addition of IL-2 induced endogenous bcl-2 transcription. In conclusion, the survival of death prone CTLL2 can be viewed as IL-2 dependent suppression of suicide, probably by the IL-2-induced expression of the cellular bcl-2 gene