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The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non–small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of α5 and β3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC. [Cancer Res 2008;68(12):4693–700]

The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non–small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of α5 and β3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC. [Cancer Res 2008;68(12):4693–700]

The expression of nicotinic acetylcholine receptors (nAChR) in fetal lung suggests maternal smoking during pregnancy effects newborn lung structure and function by the direct interaction of nicotine with nAChR in the developing lung. The recent identification of the lynx1 nAChR modulator protein in nicotinic neurons in the brain suggests that lynx1 may be similarly expressed in the lung. To study this, cDNAs encoding lynx1 were cloned from rhesus monkey lung. The temporal expression of lynx1 was studied in pre- and postnatal monkey lungs by in situ hybridization, immunohistochemistry, and realtime polymerase chain reaction (PCR). Lynx1 mRNA signal and lynx1 immunohistochemical staining were localized predominantly in airway epithelial cells, submucous glands, and smooth muscle cells, in endothelial and smooth muscle cells in vessel walls, and in alveolar type II cells. The distribution of lynx1 was similar to that of alpha4, beta2, and beta4 nAChR expression as determined by immunohistochemistry. Immunohistochemical staining also co-localized choline acetyltransferase, the enzyme that synthesizes acetylcholine, with lynx1 expression. Lynx1 expression was first observed in 71-day fetal lungs and increased with age. Immunohistochemistry, Western analysis, and realtime PCR analysis showed increased lynx1 expression in lungs following prenatal nicotine exposure. Thus, lynx1 is co-expressed with nAChR in the lung. Alteration of lynx1 levels is a potential new mechanism by which nicotine affects lung development.

It is well established that maternal smoking during pregnancy is a leading preventable cause of low birth weight and prematurity. Less appreciated is that maternal smoking during pregnancy is also associated with alterations in pulmonary function at birth and greater incidence of respiratory illnesses after birth. To determine if this is the direct result of nicotine interacting with nicotinic cholinergic receptors (nAChRs) during lung development, rhesus monkeys were treated with 1 mg/kg/day of nicotine from days 26 to 134 of pregnancy. Nicotine administration caused lung hypoplasia and reduced surface complexity of developing alveoli. Immunohistochemistry and in situ α-bungarotoxin (αBGT) binding showed that α7 nAChRs are present in the developing lung in airway epithelial cells, cells surrounding large airways and blood vessels, alveolar type II cells, free alveolar macrophages, and pulmonary neuroendocrine cells (PNEC). As detected both by immunohistochemistry and by αBGT binding, nicotine administration markedly increased α7 receptor subunit expression and binding in the fetal lung. Correlating with areas of increased α7 expression, collagen expression surrounding large airways and vessels was significantly increased. Nicotine also significantly increased numbers of type II cells and neuroendocrine cells in neuroepithelial bodies. These findings demonstrate that nicotine can alter fetal monkey lung development by crossing the placenta to interact directly with nicotinic receptors on non-neuronal cells in the developing lung, and that similar effects likely occur in human infants whose mothers smoke during pregnancy. J. Clin. Invest. 103:637–647 (1999)

Realtime PCR (or qPCR) is a widely used gold of standard for quantitative analysis of gene expression. It is also a very sensitive method for viral copy number detection, and genotyping. This Protocol outlines qPCR theory, data analysis and detailed procedures.

Background Rhesus macaques serve a critical role in the study of human biomedical research. While both Indian and Chinese rhesus macaques are commonly used, genetic differences between these two subspecies affect aspects of their behavior and physiology, including response to simian immunodeficiency virus (SIV) infection. Single nucleotide polymorphisms (SNPs) can play an important role in both establishing ancestry and in identifying genes involved in complex diseases. We sequenced the 3' end of rhesus macaque genes in an effort to identify gene-based SNPs that could distinguish between Indian and Chinese rhesus macaques and aid in association analysis. Results We surveyed the 3' end of 94 genes in 20 rhesus macaque animals. The study included 10 animals each of Indian and Chinese ancestry. We identified a total of 661 SNPs, 457 of which appeared exclusively in one or the other population. Seventy-nine additional animals were genotyped at 44 of the population-exclusive SNPs. Of those, 38 SNPs were confirmed as being population-specific. Conclusion This study demonstrates that the 3' end of genes is rich in sequence polymorphisms and is suitable for the efficient discovery of gene-linked SNPs. In addition, the results show that the genomic sequences of Indian and Chinese rhesus macaque are remarkably divergent, and include numerous population-specific SNPs. These ancestral SNPs could be used for the rapid scanning of rhesus macaques, both to establish animal ancestry and to identify gene alleles that may contribute to the phenotypic differences observed in these populations.

Background Nonhuman primates (NHPs) are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays. Results We have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey) orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey), Papio hamadryas (Baboon), and Chlorocebus aethiops (African green monkey, vervet). The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species. Conclusion This study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays.

Smoking during pregnancy leads to decreased pulmonary function and increased respiratory illness in offspring. Our laboratory has previously demonstrated that many effects of smoking during pregnancy are mediated by nicotine. We now report that vitamin C supplementation can prevent some of the effects of maternal nicotine exposure on pulmonary function of offspring. Timed-pregnant rhesus monkeys were treated with 2 mg/kg/day nicotine bitartrate from Gestation Days 26 to 160. On Gestation Day 160 (term, 165 days) fetuses were delivered by C-section and subjected to pulmonary function testing the following day. Nicotine exposure significantly reduced forced expiratory flows, but supplementation of mothers with 250 mg vitamin C per day prevented the effects of nicotine on expiratory flows. Vitamin C supplementation also prevented the nicotine-induced increases in surfactant apoprotein-B protein. Neither nicotine nor nicotine plus vitamin C significantly affected levels of cortisol or cytokines, which have been shown to affect lung development and surfactant expression. Prenatal nicotine exposure significantly decreased levels of elastin content in the lungs of offspring, and these effects were slightly attenuated by vitamin C. These findings suggest that vitamin C supplementation may potentially be clinically useful to limit the deleterious effects of maternal smoking during pregnancy on offspring's lung function.

The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.

It is well established that small cell lung carcinomas (SCLCs) express receptors for acetylcholine (ACh) and that stimulation of these receptors by nicotine or other cholinergic agonists stimulates cell growth via activation of nicotinic cholinergic receptors (nAChRs) and/or muscarinic cholinergic receptors (mAChRs). The aim of this study was to determine whether SCLC cells synthesize and secrete ACh and respond to endogenous ACh to create a functioning cholinergic autocrine loop. Reverse transcription-PCR was used to screen a panel of SCLC cell lines for components of cholinergic signaling. Choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT), as well as alpha3, alpha5, alpha7, beta2, and beta4, nAChR subunits and M3 and M5 mAChRs, were found to be present in most of the SCLC cell lines tested. Real-time PCR showed that mRNA levels for ChAT, VAChT, and alpha7 and beta2 nAChR subunits varied significantly among different SCLC cell lines tested. The H82 cell line was found to express the highest levels of ChAT, and that cell line was chosen for additional studies of ACh release and cell proliferation. ACh was easily detectable in H82 cell culture media, and levels of ACh were increased by the acetylcholinesterase inhibitor neostigmine. Vesamicol, an inhibitor of VAChT, and hemicholinium-3, an inhibitor of choline transport, both reduced H82 cell ACh basal release in a dose-dependent manner. In parallel with the reductions of ACh release, vesamicol and hemicholinium-3 also decreased H82 cell proliferation. H82 cell proliferation was also inhibited by the muscarinic and nicotinic antagonists atropine and mecamylamine, respectively, in dose- and time-dependent manners. Finally, archival cases of SCLC were screened by immunohistochemistry for expression of ChAT. Thirteen of 26 tumors screened were positive for ChAT. These findings demonstrate that SCLC can synthesize, secrete, and degrade ACh and that released ACh stimulates SCLC cell growth. Identification of this new autocrine loop provides a potential new target for therapeutic intervention.

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.