This project will focus on the molecular mechanisms by which crocidolite and chrysotile asbestos induce proliferation of rodent pleural mesothelial (RPM) and tracheobronchial epithelial (HTE) cells, the progenitor cells of mesotheliomas and bronchogenic carcinomas. Since increased cell proliferation is causally related and/or contributory to many cancers, we hypothesize that asbestos fibers provide a persistent mitogenic stimulus necessary for tumor development as is suggested by the persistent induction of c-fos and c-jun protooncogenes by asbestos. To determine if crocidolite and chrysotile induce proliferation by similar or unique processes in various cell types, we will examine the mechanisms of activation of c-fos and c-jun gene expression using inhibitors of protein kinase C (PKC) and ADP-ribose transferase. Scavengers of active oxygen species (AOS) and iron-chelated fibers will be used to test the hypothesis that asbestos-associated induction of c-fos and c-jun may be mediated by AOS. Moreover, comparative experiments using nonasbestos fibers [refractory ceramic (RCF) and glass (MMVF-10)] of similar dimension to asbestos, sized long (>10 microm) vs. short (<2 micro) chrysotile fibers, and particles (riebeckite, glass) of similar chemical composition to asbestos at a range of concentrations will determine the properties of fibers intrinsic to induction of c-fos and c-jun. Whether increased expression of these protooncogenes is causally related to cell proliferation by asbestos will be assessed using both transient transfection assays to assess the effects of crocidolite and chrysotile fibers on the expression of an AP-1 dependent reporter gene in HTE cells and immunolocalization of c-fos and c-jun proteins in cell cultures exposed to asbestos. In the latter experiments, we will use immunocytochemistry to determine if c-fos and c-jun proteins are expressed exclusively in proliferating mesothelial and tracheal epithelial cells as identified by the expression of proliferating cell nuclear antigen (PCNA) or the incorporation of 5'-bromo-deoxyuridine (BrdU) into DNA. Comparative experiments using cells after exposure to both proliferative and cytotoxic concentrations of fibers in vitro and in situ localization of c-fos and c-jun proteins after inhalation of fibers by rats will allow us to link expression of these gene products with cell proliferation and/or cytotoxicity. Knowledge of the mechanisms of induction of c-fos and c-jun by asbestos in target cells of asbesto- induced cancers will open up avenues for preventive and therapeutic approaches to disease. This project involves collaborations between Dr. Mossman, a cell biologist with expertise in asbestos and AOS, Dr. Heintz, a molecular biologist with expertise in cell replication, and Dr. Leslie, a lung pathologist with expertise in immunocytochemistry.
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