Nicotinic receptor-mediated activation by the tobacco-specific nitrosamine NNK of a Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc in human small cell lung carcinoma cells and pulmonary neuroendocrine cells.
OBJECTIVE
Small cell lung carcinoma (SCLC) expresses phenotypic features of pulmonary neuroendocrine cells and demonstrates a strong etiologic association with smoking. SCLC cell lines express a Raf-1-dependent mitogenic signal transduction pathway, which is thought to transduce the mitogenic signals initiated by neuropeptide autocrine growth factors. Recent studies have identified the tobacco-specific carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a site-selective high-affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), which regulates the growth of a significant subset of SCLC in vitro by stimulating the release of the autocrine growth factor serotonin. The purpose of this study was to identify signaling events initiated by binding of NNK to the alpha7 nAChR.


METHODS
We have used a human SCLC cell line and fetal hamster pulmonary neuroendocrine cells with in vitro kinase activation assays and western blots to assess the levels of expression and activation of Raf-1, MAPK and c-myc to address this issue.


RESULTS
Our data show that NNK activates the Raf-1, MAP kinase pathway, resulting in phosphorylation of c-myc. The activation of this signal transduction pathway by NNK was inhibited by the site-selective antagonist for the alpha7 nAChR alpha-bungarotoxin (alpha-BTX) or by the serotonin reuptake inhibitor imipramine, suggesting that the responses to NNK were mediated by nicotinic receptor-initiated release of serotonin. Accordingly, NNK-induced 5-HT release was blocked by alpha-BTX while NNK-induced DNA synthesis was inhibited by alpha-BTX, imipramine, the PKC inhibitor sphingosine or the MEK inhibitor PD98059. SCLC cells demonstrated high basal levels of 5-HT release, DNA synthesis, and over-expressed Raf-1 and MAPK protein suggesting the constitutive activation of an upstream regulator such as the alpha7 nAChR.


CONCLUSIONS
Our findings link, for the first time, the stimulation of a nicotinic acetylcholine receptor by a cancer-causing agent with the activation of a Raf-1/MAPK/c-myc signaling pathway. Furthermore, our data suggest that serotonin uptake inhibitors may protect against the development or be useful in the clinical management of SCLC.
Jull
B A
BA
Experimental Oncology Laboratory, College of Veterinary Medicine, University of Tennessee, Knoxville 37901-1071, USA.
Plummer
H K
HK
Schuller
H M
HM
D000818:Animals; D002038:Bungarotoxins / Q000494:pharmacology; D002273:Carcinogens / Q000633:toxicity*; D018288:Carcinoma, Small Cell / Q000139:chemically induced* / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D008175:Lung Neoplasms / Q000139:chemically induced* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009490:Neurosecretory Systems / Q000187:drug effects* / Q000378:metabolism; D009602:Nitrosamines / Q000633:toxicity*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology*; D011978:Receptors, Nicotinic / Q000502:physiology*; D012701:Serotonin / Q000502:physiology; D014407:Tumor Cells, Cultured
D002038:Bungarotoxins; D002273:Carcinogens; D009602:Nitrosamines; D016271:Proto-Oncogene Proteins c-myc; D011978:Receptors, Nicotinic; D012701:Serotonin; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D007099:Imipramine