Progression through G1 and entry into the S phase are carefully regulated during the eukaryotic cell cycle. Induction of cell proliferation involves several sequential steps that include: 1) activation of mitogen-activated protein kinases (MAPK), 2) expression of early response genes such as c- fos and c-jun, 3) transcriptional licensing and S phase entry. Asbestos, reactive oxygen, and reactive nitrogen species (ROS/RNS) influence progression through G1 and entry into the S phase by activating or perturbing MAPK cascades, altering the activity of G1 cyclins or alt4ering the activation of E2F. Here, we propose specific aims to examine progression through G1 and S phase entry in models of allergic airway hyperresponsiveness/fibrosis and asbestosis. First, we will document the relationship between cell cycle progression and patterns of MAPK activation, expression of cyclin D1, and origin licensing by Cdc6 in synchronized murine alveolar type II (C1) cells in response to asbestos, RO2 and cationic proteins. Second, we will use homologous recombination of bacterial artificial chromosomes (BACs) to generate alleles of cyclin D1 and cfdc6 that contain internal ribosome entry sites (IRES)-enhanced green fluorescent protein (EGFP) expression cassettes in the 3' untranslated regions of the genes. Using a novel gene transfer technique, these alleles will be transferred into cells in cultured, and regulated expression during the cell cycle of dicistonic mRNAs from the BAC alleles will be assessed. Third, those BAC alleles displaying proper expression of dicistonic mRNAs encoding cyclin D1-IRES-EGFP and Cdc6-IRES-EGFP will be used to generate transgenic reporter mice. Mice bearing these transgenic BAC alleles will be used to study progression through G1 and commitment to S phase in epithelial cells in inhalation models used in projects 1-3. Finally, backcrossing of BAC transgenic mice with mice expressing dominant negative MEK1 will be used to define the role of ERKs in governing progression through G1 and S phase entry, as well as their relationship to the development of epithelial cell proliferation and fibrosis.
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