Programmed cell death (PCD) often occurs via apoptosis, a physiological form of cellular demise common during embryogenesis, turnover of tissues and selection of cell populations. Several genes have been recently identified that activate, execute or inhibit the cell death pathway. The bcl-2 family of apoptosis-regulatory genes modulates cell death induced by a wide array of signals. Two members of the family, bcl-2 and bcl-x are expressed in many tissues and their importance is underscored by the finding that mutant mice deficient in bcl-2 or bcl-x exhibit lethal phenotypes due to abnormal apoptosis. Moreover, deregulated expression of Bcl-2 and Bcl-x proteins play an important role in cancer development and resistance of tumor cells to therapy- induced apoptosis. Yet, the biochemical mechanism by which Bcl-2 family members regulate apoptosis is poorly understood. We have identified a novel gene, harakiri, that functions as a regulator of Bcl-2 and Bcl-X/L and apoptotic cell death in mammalian cells. The harakiri product (HRK) does not exhibit significant homology to Bcl-2 or Bcl-X/L and lacks conserved BH1 and BH2 domains which are shared by BCL-2 family members. HRK contains an 8 amino acid BH3 motif which is found in proteins of the BCL-2 family. Significantly, HRK physically interacts with anti- apoptosis proteins Bcl-2 and Bcl-X/L but not with death-promoting Bcl-2 members such as Bax and Bak. Expression of HRK induces rapid onset of cell death in mammalian cells. Importantly the death-promoting activity of HRK is repressed by Bcl-2 and Bcl-X/L suggesting that HRK is a common target for the anti-apoptosis proteins Bcl-2 and Bcl-XL. In this proposal we propose experiments (i) to further characterize the molecular interaction between HRK and Bcl-2/Bclk-X/L and identify additional targets of HRK; (ii) to examine the expression, regulation, subcellular localization; (iii) develop recombinant adenoviruses to study cellular targets of HRK and its ability to activate cell death in normal and cancer cells, and (iv) to generate mutant mice deficient in HRK to determine its physiological role in vivo. The studies outlined in this proposal should provide novel sight into the apoptosis pathway which could lead to alternative therapeutic strategies for treatment of cancer.

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