Our long-term objective is to understand molecular mechanisms of pulmonary neuroendocrine cell (PNEC) differentiation, which plays an important role in fetal lung growth and maturation. These studies may also clarify mechanisms of disorderly differentiation in lung carcinogenesis, especially for small cell lung cancer, which is derived from PNECS. We will employ a new animal model for induced neuroendocrine (NE) lung tumors, which provides a unique opportunity to analyze PNEC differentiation. Within 7 weeks, 75% of hamsters treated with both diethylnitrosamine (DEN) and hyperoxia (70% 02) develop multifocal lung PNEC tumorlets. First, we will characterize this hamster model by mapping kinetics of gene expression in vivo by in situ hybridization, immunostaining and northern blot analyses for known PNEC markers. We will also study gene expression in tissue culture of primary and/or transformed PNECS. Second, we will identify and characterize new genes involved in the process of PNEC differentiation by constructing subtraction CDNA libraries from hamster lung in the early phase of DEN/O(2)-induced neoplasia, with selected cDNAs absent from control adult lung by slot blot MRNA analyses. MRNA & peptide processing in specific tissues will be determined. Potential PNEC-specific inducer genes will be sequenced and transfected into undifferentiated cell lines to assess their ability to induce NE differentiation. If these cDNAs do not induce differentiation, we will work backwards using new PNEC marker genes to identify other earlier expressed cDNAs. We will also screen hamster tumor and human fetal lung libraries with probes for other genes implicated in cell differentiation, including MyoDl and homeobox genes. Third, we will analyze pathogenetic mechanisms of PNEC tumor development. We will compare expression of new genes during DEN/02-induced tumorigenesis to that occurring during lung development. In years 4 and 5, we will construct transgenic mice over-expressing new mRNAs to compare and contrast the kinetics of normal lung development as well as the induction of NEC hyperplasia/neoplasia. Factors regulating PNEC gene expression during tumorigenesis and lung development will be determined. A transient gene expression assay will be developed using PNEC cultures to dissect promoter sequences involved in this regulation. These analyses of PNEC gene expression in the hamster tumor model will be critical in clarifying molecular pathways of PNEC differentiation in normal lung development
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