) In recent years the dogma of the central role of caspases in apoptosis has been challenged by the realization that cells induced to undergo apoptosis under conditions in which caspases are not activated often die nevertheless, albeit by a nonapoptotic mechanism. We have shown that anti-apoptotic oncogenes (Bcl-2, Bcr-Abl) block this caspase- independent death pathway, and thus this is not simply an effect of damage or lack of repair. We have called this process """"""""commitment"""""""" to indicate that it is part of the normal apoptotic pathway but that it provides an alternative route to death when caspase activation is impaired. Since cell death is an important check on the oncogenic process, this caspase-independent commitment is very likely to play a role in blocking transformation Since almost nothing is known about the mechanisms or regulation of caspase-independent commitment, we will approach this problem using three interconnected approaches. These are: 1. What are the cellular and molecular events that correspond to commitment and caspase-independent death? Here we will extend our studies to correlate caspase-independent commitment for death with events that correspond to the time at which this occurs. The changes in mitochondrial function that correlate with commitment, including release of cytochrome c, loss of transmembrane potential, and generation of reactive oxygen species (ROS) will be evaluated using a variety of in vivo imaging methods. Alternatively we will examine possible nuclear involvement in this process. 2. How does caspase- independent commitment contribute to the control of oncogenesis? We will test whether apoptotic signals produced by some oncogenes and tumor suppressor genes promote caspase-independent cell death, and examine the contribution of defects in the downstream apoptotic pathway to cancer. We will generate models for co-transformation in vitro, employing Apaf-1 null fibroblasts and examine the co-transforming activity of a naturally occurring dominant negative form of caspase-9. We will then determine whether human cancers ever accumulate mutations in these genes. 3. What mechanisms for bypassing commitment contribute to transformation? Most models of oncogenesis currently include anti-apoptotic components, but our studies suggest that it is commitment (and caspase independent death) and not only apoptosis, which acts as a check on transformation. Here we will employ a variety of retroviral libraries and specific screening techniques to identify molecular mechanisms for evading commitment that are independent of anti-apoptotic mechanisms per se. This will provide new insights not only into oncogenesis but into the commitment process itself.
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