Interaction of tobacco-specific toxicants with the neuronal α7 nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders
Introduction
This manuscript represents a review of our work during the past 3 years and includes new, unpublished data, as well as some data which have been previously published in a different context. Appropriate reference is given in the text and figure legends.
Pulmonary neuroendocrine cells are specialized lung cells which co-express features of neurons and endocrine cells (Becker, 1984). These cells are abundant in the neonate, and decrease rapidly in number by 12 months of age Cutz, 1997, Cutz et al., 1985. Pulmonary neuroendocrine cells are hypoxia-sensitive chemoreceptors and are thought to be important mediators of pulmonary neonatal adaptation, particularly the onset of breathing Cutz, 1997, Cutz et al., 1985. Pulmonary neuroendocrine cells demonstrate hyperplasia accompanied by elevated levels of their secretory products in a variety of pediatric pulmonary diseases, such as bronchopulmonary dysplasia (Johnson and Georgieff, 1989, Johnson et al., 1982, Cutz, 1997, cystic fibrosis Johnson et al., 1988, Cutz, 1997, sudden infant death syndrome Cutz et al., 1988, Cutz et al., 1996, Johnson and Georgieff, 1989, and asthma Stanislawski et al., 1981, Johnson and Georgieff, 1989, Aguayo, 1993. Functional abnormalities of these cells may contribute to the pathophysiology of this family of pediatric lung diseases because their peptide and biogenic amine secretory products modulate the tonus of bronchial and vascular smooth muscle, modulate neurotransmission, and stimulate the growth of epithelial lung cells and fibroblasts Willey et al., 1984, Johnson et al., 1988, Seuwen et al., 1988, Sunday, 1988, Schuller and Hegedus, 1989, Rozengurth and Sinneth-Smith, 1990, Cutz, 1997. The most prominent product of pulmonary neuroendocrine cells for which effects on cell growth, smooth muscle tension and neuromodulation have been documented, is the biogenic amine 5-hydroxytryptamine (Cutz, 1997; 5-HT, serotonin). Research into the role of 5-HT in pediatric pulmonary disease has focused on its well-documented constricting effects on bronchial and vascular smooth muscle Johnson et al., 1988, Lechin et al., 1996, Cutz, 1997. Although it has been known for some time that 5-HT stimulates the growth of fibroblasts, its growth stimulating effects on pulmonary neuroendocrine cells and small cell carcinima has been only recently discovered Schuller, 1989, Schuller and Hegedus, 1989, Schuller and Orloff, 1998.
Epidemiologic studies have established that babies of mothers who smoke during pregnancy are at increased risk to develop sudden infant death syndrome Cutz et al., 1988, Johnson and Georgieff, 1989, Malloy et al., 1992, Schoendorf and Kiely, 1992, Schellscheidt et al., 1997 and asthma Hu et al., 1997, Oliveti et al., 1996. Moreover, both of these diseases as well as bronchopulmonary dysplasia and cystic fibrosis are exacerbated in newborns and infants exposed to second-hand smoke Aguayo, 1993, Cutz et al., 1996, Knight et al., 1998. However, the underlying mechanisms of these effects are far from understood. Nicotine is a well-documented secretagogue for pulmonary neuroendocrine cells and causes exocytosis of their dense-cored cytoplasmic granules which are the storage site of peptides and serotonin Lauweryns et al., 1977, Tabbasian et al., 1989, Cutz, 1997. Similarly, nicotine stimulated the release of 5-HT from human small cell lung carcinoma cells in culture Codignola et al., 1994, Schuller and Orloff, 1998. As it is well established that the biological effects of nicotine are mediated by binding to nicotinic acetylcholine receptors, these findings suggest that the release of 5-HT is under autonomic control via members of the nicotinic acetylcholine receptor family.
Smoking has long been recognized as the single most important risk factor for the development of lung cancer (Weiss, 1983; Cook et al., 1993). While non-small cell lung carcinomas develop also in a significant number of non-smokers, small cell lung carcinoma is extremely rare in non-smokers (Weiss, 1983; Cook et al., 1993). Chronic non-neoplastic pulmonary disease (bronchitis/bronchiolitis, emphysema, chronic obstructive pulmonary disease, asthma) has more recently emerged as a risk factor for lung cancer in both smokers and non-smokers Osann, 1991, Weiss, 1991, Park et al., 1995. In fact, a recent study has shown that elevated CO2 levels comparable to those in the diseased lung stimulate the release of 5-HT, resulting in the activation of the MAP kinase cascade in SCLC (Merryman et al., 1997). Small cell lung carcinoma expresses phenotypic and functional characteristics of pulmonary neuroendocrine cells (Gazdar and Carney, 1984). All pulmonary neuroendocrine cells and small cell lung carcinoma cells have the ability to synthesize and release 5-HT (Becker, 1984; Gazdar, 1984), suggesting an important function of this biogenic amine in this histologic lung cancer type. This interpretation gains strong support from the recent finding that 5-HT is an autocrine growth factor for this cancer type as well as for pulmonary neuroendocrine cells Schuller, 1989, Schuller and Hegedus, 1989, Schuller and Orloff, 1998, Codignola et al., 1994.
The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are formed from nicotine by nitrosation (Fig. 1) during the curing of tobacco and in the mammalian organism Fischer et al., 1990, Hecht and Hoffmann, 1990. NNK is a potent lung carcinogen in all animal species tested, and is thought to contribute significantly to the high lung cancer burden associated with smoking (Hecht and Hoffmann, 1990). On the other hand, NNN, which more closely resembles nicotine in structure than NNK (Fig. 1) is a weak carcinogen in laboratory animals. It is well established that reactive metabolites of NNK or NNN may form DNA-methylating and pyridyloxobutylating products (Fig. 2), resulting in activating point mutations of the Ki-ras gene Belinsky et al., 1988, Hecht et al., 1993a, Hecht et al., 1993b. With respect to the central role of ras as a signal transducer involved in mitogenic pathways of mammalian cells (Lange-Carter and Johnson, 1994), this event is thought to be primarily responsible for the development of lung tumors in response to NNK Belinsky et al., 1988, Hecht et al., 1993a, Hecht et al., 1993b. However, point mutations of the ras gene have only been identified in non-small cell lung carcinomas whereas they are consistently absent in small cell lung carcinoma Mitsudomi et al., 1991, Wagner et al., 1993. The α7 nicotinic acetylcholine receptor and its associated mitogenic signal trasnduction pathway is emerging as an important growth regulator of pulmonary neuroendocrine cells and small cell lung carcinoma Schuller, 1989, Schuller and Hegedus, 1989, Schuller and Orloff, 1998, Cattaneo et al., 1993, Cattaneo et al., 1997, Codignola et al., 1994 and may be critically involved in the development of neoplastic and non-neoplastic pulmonary diseases.
Section snippets
Tissue culture
Cultures of fetal pulmonary neuroendocrine cells were established from fetal hamster lung periphery harvested on day 15 of gestation as previously described Linnoila et al., 1993, Schuller, 1994. Following disaggregation of the tissue (100 mg) with 3 ml trypsin/EDTA for 60 min at 37°C, complete RPMI medium was added to yield 106 cells/ml, and 2 ml each of the resulting cell suspension was used to seed 6 T75 tissue culture flasks. The cultured cells were enriched to >80% of pulmonary
Results
RT-PCR assays with the primer for the human α7 nicotinic acetylcholine receptor revealed mRNA of this receptor in fetal hamster pulmonary neuroendocrine cells and human small cell lung carcinoma cells Fig. 3, Fig. 4. Sequence comparison of the 611 bp between the primers of the RT-PCR fragment from fetal hamster pulmonary neuroendocrine cells with the comparable sequence of human α7 mRNA by a maximum matching program of DNASIS showed a matching percentage of 89%. A Blast search of Genbank using
Discussion
The data presented in this review suggest that pediatric smoking-associated pulmonary diseases and small cell lung carcinoma may be caused by the direct chronic stimulation of an α7 nicotinic acetylcholine receptor-initiated autocrine loop by nicotine or NNK. While it is well established that the Raf-1/MAP kinase cascade is a major mitogenic signal transduction pathway expressed in human small cell lung carcinoma Bunn et al., 1993, Heysley and Johnson, 1998, the expression and function of this
Acknowledgements
This study is supported by Public Health Service grant CA51211 with the National Cancer Institute and a grant with the VERUM Foundation (Munich, Germany).
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