and Specific Aims.) Pulmonary neuroendocrine cells (PNECs) are the first differentiated epithelial cells to appear in fetal lung, in which they are abundant and play a role in promoting fetal lung growth and maturation. PNECs are also thought to be precursors for neuroendocrine (NE) lung tumors [carcinoids and small cell lung cancers (SCLCs)]. A hamster model of preneoplastic lung injury has been characterized in which intense PNEC differentiation is induced after a critical period (about 8 wks) of combination treatment with nitrosamines and 60 percent O2 (NNK/O2). This PNEC hyperplasia is transient and ultimately only non-NE tumors develop in NNK/O2-treated hamsters. The long-term objective is to understand molecular mechanisms of PNEC differentiation.
Specific Aim 1 is to identify new PNEC-specific genes involved in PNEC differentiation. Genes potentially involved in triggering the NE phenotype may be induced in hamster lung after 8 weeks of NNK/O2 treatment and may also be expressed in well-differentiated SCLCs but silent in normal hamster lung and undifferentiated SCLCs. Selection of novel differentially expressed cDNAs for further study will include partial cDNA sequencing, antisense blockade of NE differentiation in cell lines, and confirmation of PNEC specificity by in situ hybridization.
Specific Aim 2 is to use several in vitro models to test potential roles of selected genes in the regulation of PNEC differentiation, including tumor necrosis factor-alpha (TNFa), CD10/neutral endopeptidase 24.11 (CD10/NEP), and novel PNEC-specific cDNAs. TNFa and CD10/NEP mRNAs peak in hamster lung after 4 to 8 wks of NNK/O2 treatment. TNFa, a species-specific cytokine may induce PNEC differentiation. CD10/NEP could modulate PNEC differentiation by hydrolyzing specific peptide mediators. Novel PNEC- specific genes may be capable of inducing NE features when transfected into undifferentiated cells. SCLC cell lines and fetal lung explants will be treated with: (a) TNFa, TNF a blocking antibodies, and/or CD10/NEP inhibitors; (b) transfection of sense or antisense expression cDNAs. PNEC differentiation will be assessed by RNA analyses, EM, NE-specific enzymatic assays, and immunostaining for NE markers. These approaches may elucidate pathways of PNEC differentiation during the pathogenesis of PNEC hyperplasia in chronically injured lungs, and potentially also during normal fetal lung development and lung epithelial neoplasia.
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