Chikova_2011_PLoS.One_6_e27978

Isolation of polyadenilated mRNA from human immortalized bronchial epithelial cell line BEP2D revealed the presence of multiple isoforms of RNA coded by the CHRNA9 gene for alpha9 nicotinic acetylcholine receptor (nAChR). BEP2D cells were homozygous for the rs10009228 polymorphism encoding for N442S amino acid substitution, and also contained mRNA coding for several truncated isoforms of alpha9 protein. To elucidate the biologic significance of the naturally occurring variants of alpha9 nAChR, we compared the biologic effects of overexpression of full-length alpha9 N442 and S442 proteins, and the truncated alpha9 variant occurring due to a loss of the exon 4 sequence that causes frame shift and early termination of the translation. These as well as control vector were overexpressed in the BEP2D cells that were used in the assays of proliferation rate, spontaneous vs. tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced cellular transformation, and tumorigenicity in cell culture and mice. Overexpression of the S442 variant significantly increased cellular proliferation, and spontaneous and NNK-induced transformation. The N442 variant significantly decreased cellular transformation, without affecting proliferation rate. Overexpression of the truncated alpha9 significantly decreased proliferation and suppressed cellular transformation. These results suggested that alpha9 nAChR plays important roles in regulation of bronchial cell growth by endogenous acetylcholine and exogenous nicotine, and susceptibility to NNK-induced carcinogenic transformation. The biologic activities of alpha9 nAChR may be regulated at the splicing level, and genetic polymorphisms in CHRNA9 affecting protein levels, amino acid sequence and RNA splicing may influence the risk for lung cancer.