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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES006499-03
Application #
2155347
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1993-09-01
Project End
1998-08-31
Budget Start
1995-09-01
Budget End
1996-08-31
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Pathology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Shukla, A; Timblin, C; BeruBe, K et al. (2000) Inhaled particulate matter causes expression of nuclear factor (NF)-kappaB-related genes and oxidant-dependent NF-kappaB activation in vitro. Am J Respir Cell Mol Biol 23:182-7
Jung, M; Davis, W P; Taatjes, D J et al. (2000) Asbestos and cigarette smoke cause increased DNA strand breaks and necrosis in bronchiolar epithelial cells in vivo. Free Radic Biol Med 28:1295-9
Robledo, R; Mossman, B (1999) Cellular and molecular mechanisms of asbestos-induced fibrosis. J Cell Physiol 180:158-66
Janssen-Heininger, Y M; Macara, I; Mossman, B T (1999) Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappaB: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappaB by oxidants. Am J Respir Cell Mol Biol 20:942-52
Zanella, C L; Timblin, C R; Cummins, A et al. (1999) Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis. Am J Physiol 277:L684-93
Timblin, C R; Guthrie, G D; Janssen, Y W et al. (1998) Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers. Toxicol Appl Pharmacol 151:88-97
Janssen, Y M; Driscoll, K E; Timblin, C R et al. (1998) Modulation of mitochondrial gene expression in pulmonary epithelial cells exposed to oxidants. Environ Health Perspect 106 Suppl 5:1191-5
Mossman, B T; Churg, A (1998) Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med 157:1666-80
Timblin, C R; Janssen, Y M; Goldberg, J L et al. (1998) GRP78, HSP72/73, and cJun stress protein levels in lung epithelial cells exposed to asbestos, cadmium, or H2O2. Free Radic Biol Med 24:632-42
Fung, H; Kow, Y W; Van Houten, B et al. (1998) Asbestos increases mammalian AP-endonuclease gene expression, protein levels, and enzyme activity in mesothelial cells. Cancer Res 58:189-94

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