Abstract
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 α9 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 α9 protein. To elucidate the biologic significance of the naturally occurring variants of α9 nAChR, we compared the biologic effects of overexpression of full-length α9 N442 and S442 proteins, and the truncated α9 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 α9 significantly decreased proliferation and suppressed cellular transformation. These results suggested that α9 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 α9 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.
Highlights
The functional components of cholinergic system are highly conserved among the invertebrate and vertebrate species
The BEP2D cell line was found to be homozygous for the rs10009228 polymorphism and express an a9 nicotinic acetylcholine receptor (nAChR) protein with the N442S aa substitution
We demonstrated for the first time that different variants of human a9 nAChR subunit protein differentially influence regulation of bronchial cell proliferation and susceptibility to the tumorigenic transformation by NNK
Summary
The functional components of cholinergic system are highly conserved among the invertebrate and vertebrate species. Previous genome wide association studies revealed single nucleotide polymorphisms (SNPs) in the CHRNA5-CHRNA3-CHRNB4 gene cluster on chromosome 15 that encodes the a5, a3 and b4 subunits of the nicotinic acetylcholine receptors (nAChRs) associated with nicotine dependence and lung cancer [1,2,3]. The genetic variations in other nAChR subunits expressed in lung epithelia [4,5,6,7] remain largely unknown. We have established an important role for nAChRs expressed on respiratory cells in the malignant transformation caused by pharmacological doses of tobacco-derived carcinogenic nitrosamines, and demonstrated that nicotinic antagonists can abolish these pathobiologic effects [8]. Nicotine and its carcinogenic derivatives can contribute to the development of lung cancer by acting as tumor promoters that facilitate the outgrowth of cells with genetic damage [9]. Pulmonary nAChRs are widely considered as novel drug targets for prevention and treatment of lung cancer [13,14,15]
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