Pleural mesothelial cells, the progenitor of the asbestos-induced tumor mesothelioma, undergo apoptosis when exposed to asbestos. Important mechanisms may include oxygen radical generation, DNA damage and fiber internalization. Because an inability to undergo apoptosis may allow cells with DNA damage to survive, a loss of apoptosis may be a major step in the multistep process leading to neoplasia. Therefore, mechanisms promoting or inhibiting apoptosis may play a dynamic process determining whether the toxic effects of asbestos eventually result in neoplasia. We propose to investigate the role of apoptosis in the pathology of asbestos to the mesothelial cells and pleura. Initially, Dr. Broaddus aims to delineate the major mechanisms inducing asbestos-induced apoptosis in both rabbit and human pleural mesothelial cells and rabbit pleura. By measures of DNA damage, oxidant stress and stress-related signaling (ceramide, jun kinase, MAP kinase) in vitro and in vivo, she propose to show the pathways essential to mesothelial apoptosis. Then, she will determine the mechanisms by which certain signals, namely those initiated by vitronectin receptors, inhibit asbestos-induced apoptosis. Using vitronectin-coated particles or anti-integrin antibodies to activate the integrins, she will confirm the role of vitronectin receptors as anti-apoptotic, and identify whether the integrins are altering the balance of the apoptotic pathways such as ceramide and JNK with proliferative pathways such as MAP kinase. In in vivo studies in rabbits after activation or inhibition of vitronectin receptors or in genetically-engineered mice without vitronectin or vitronectin receptors, she will learn the importance of this anti-apoptotic effect in vivo. Finally, she intends to use inhibitors of asbestos-induced apoptosis (anti-oxidant B-carotene, inhibition of PARP, vitronectin-coated beads) to determine whether longterm inhibition of apoptosis will lead to accumulation of DNA damage in asbestos-exposed mesothelial cells. We will assess chronically asbestos-exposed cells for the presence of DNA structural damage by the presence of micronuclei and for changes in gene copy number using comparative genomic hybridization. From these studies, Dr. Broaddus will proceed to long term studies in genetically-engineered mice without vitronectin, the B5 subunit of a major vitronectin receptor, or p53 to confirm that this inhibition of apoptosis leads to an increased sensitivity to asbestos-induced abnormalities, including mesothelioma. These studies should provide important information about the mechanisms by which asbestos induces apoptosis in mesothelial cells and the role of asbestos-induced apoptosis in asbestos-induced pathology.
