The objective of this research is to understand the molecular genetic mechanisms responsible for regulating radiation-induced apoptotic cell death in tumor cells. This proposal is predicated on the hypothesis that apotosis is an important mode of cell death in some irradiated tumors and that tumor cell resistance to radiation may be due to the failure of the cell to undergo apoptosis. To examine this hypothesis we have developed a mouse lymphoma cell system that consists of two cell lines, one of which is sensitive to radiation-induced apoptosis and another that is resistant to radiation-induced apoptosis. The resistant line grew out of the sensitive line after long-term culture. We propose the following specific aims to illuminate these important mechanisms: 1. It is known that apoptosis is regulated in a number of normal processes by signal transduction and second messenger pathways. While less is known about radiation-induced apoptosis, we propose to investigate the role of these regulatory mechanisms in irradiated cells undergoing apoptosis using a new well-established battery of inhibitors of various steps in these pathways using our lymphoma system. This will initially focus on Ca++, calmodulin, protein kinase C and cAMP dependent protein kinase. 2. In a number of instances cell undergoing apoptosis are known to require nascent mRNA and protein synthesis. Several genes have been identified which appear to mediate or regulate apoptosis. We intend to examine the potential role of these genes in radiation-induced apoptosis. These genes include but are not limited to, SGP-2, transglutaminase, p53, fos, myc, jun, calmodulin and Bcl-2. Some of the effect of these genes is known to be mediated by cytokines (IL-2, IL-3, IL-6). Modulation of those genes involved in radiation-induced apoptosis by cytokines will also be examined. 3. We also propose to identify new genes associated with radiation-induced apoptosis by the method of substractive hybridization. Our apoptosis resistant and sensitive lymphoma model is well suited for this aspect. It is increasingly clear that apoptosis is highly regulated at the molecular level. By knowing what these mechanisms are and how they are influenced by exogenous agents such as growth factors and cytokines, we may ultimately be able to specifically enhance apoptosis in human tumors to overcome radioresistance in radiotherapy.
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