: T lymphocytes undergo apoptosis in vivo by at least two distinct mechanisms. T cells activated by sub-optimal stimuli, or in the presence of inadequate survival factors, undergo passive cell death. This is believed to correspond to the intrathymic death by neglect of unselected cortical thymocytes. Conversely, cells that are fully activated display a variety of death receptors, and become susceptible to activation-induced cell death. This is believed to correspond to intrathymic death by negative selection. The E2F transcription factors, E2F-4 and E2F-1, control two distinct checkpoints that occur in G1 phase of the cell cycle. The E2F-4 checkpoint controls a cell fate decision in which the alternatives are cell cycle progression versus stable quiescence. Apoptosis may occur if this checkpoint is disrupted. In contrast, the E2F-1 checkpoint controls a cell fate decision in which the alternatives are DNA synthesis versus apoptosis. It is our hypothesis that the E2F-4 checkpoint is intimately involved in passive cell death of T cells, while the E2F-1 checkpoint is involved in activation-induced cell death. E2F-4 and E2F-1, and the pocket proteins p107 and pRb that regulate them, contain highly conserved caspase-3 cleavage sites. Cleavage of E2F-4 is predicted to result in loss of transcriptional activation, but not loss of DNA binding capacity. We predict this will disrupt the E2F-4 checkpoint, prevent E2F-4-dependent quiescence of T cells, and result in passive cell death. In contrast, the cleavage of E2F-1 is predicted to result in loss of its susceptibility to regulation by pRb, and to generate a fragment with constitutive E2F-1 activity. We propose that inappropriate E2F-1 activity will result in activation-induced cell death. In this proposal, these hypotheses will be tested in primary T cells and T cell lines, and through retroviral transduction of bone marrow stem cells. Dominant-interfering mutants of E2F will be used to enhance or block the effects of caspase-3 mediated E2F-4 and E2F-1 cleavage. If the hypothesis is substantiated, E2F-4 and E2F-1 will have been identified as specific vital targets, important in passive and activation-induced T cell death respectively.
