Title: Increased Susceptibility of Mice Lacking Clara Cell 10-kDa Protein to Lung Tumorigenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a Potent Carcinogen in Cigarette Smoke
Abstract: Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events. Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events. Lung cancer is the leading cause of cancer deaths in both men and women in the United States, and 90% of all human lung cancers are related to cigarette smoking (1Minna J.D. Roth J.A. Gazdar A.F. Cancer Cell. 2002; 1: 49-52Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 2Shopland D.R. Environ. Health Perspect. 1995; 103: 131-142Crossref PubMed Scopus (174) Google Scholar). Both tobacco smoke (3Shijubo N. Itoh Y. Yamaguchi T. Shibuya Y. Morita Y. Hirasawa M. Okutani R. Kawai T. Abe S. Eur. Respir. J. 1997; 10: 1108-1114Crossref PubMed Scopus (138) Google Scholar) and lung tumorigenesis (4Linnoila R.I. Jensen S.M. Steinberg S.M. Mulshine J.L. Eggleston J.C. Gazdar A.F. Am. J. Clin. Pathol. 1992; 97: 233-243Crossref PubMed Scopus (90) Google Scholar, 5Linnoila R.I. Szabo E. DeMayo F. Witschi H. Sabourin C. Malkinson A. Ann. N. Y. Acad. Sci. 2000; 923: 249-267Crossref PubMed Scopus (57) Google Scholar) are associated with reduced levels of Clara cell 10-kDa (CC10) 1The abbreviations used are: CC10, Clara cell-specific 10-kDa protein; WT, wild type; KO, knock-out; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; SSCP, single-strand conformational polymorphism; MAPK, mitogen-activated protein kinase; Erk (and ERK), extracellular signal-regulated kinase; PBS, phosphate-buffered saline.1The abbreviations used are: CC10, Clara cell-specific 10-kDa protein; WT, wild type; KO, knock-out; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; SSCP, single-strand conformational polymorphism; MAPK, mitogen-activated protein kinase; Erk (and ERK), extracellular signal-regulated kinase; PBS, phosphate-buffered saline. protein, a steroid-inducible, multifunctional, secreted polypeptide that accounts for ∼7% of the total protein in bronchioalveolar lavage fluid (6Mukherjee A.B. Kundu G.C. Mantile-Selvaggi G. Yuan C.J. Mandal A.K. Chattopadhyay S. Zheng F. Pattabiraman N. Zhang Z. Cell Mol. Life Sci. 1999; 55: 771-787Crossref PubMed Scopus (120) Google Scholar, 7Singh G. Katyal S.L. Ann. N. Y. Acad. Sci. 2000; 923: 43-58Crossref PubMed Scopus (87) Google Scholar). CC10, first identified as blastokinin (8Krishnan R.S. Daniel J.C. Science. 1967; 158: 490-492Crossref PubMed Scopus (230) Google Scholar) or uteroglobin (9Beier H.M. Biochim. Biophys. Acta. 1968; 160: 289-291Crossref PubMed Scopus (240) Google Scholar), is the founding member of a newly formed superfamily of proteins called Secretoglobins (10Mukherjee A.B. Chilton B.S. Ann. N. Y. Acad. Sci. 2000; 923: 348-354PubMed Google Scholar). Most of the proteins of this superfamily are tissue-specifically expressed in the secretory epithelia of virtually all organs. The human CC10 gene is mapped to chromosome 11q12.2-13.1 (11Zhang Z. Zimonjic D.B. Popescu N.C. Wang N. Gerhard D. Stone E. Arbour N.C. de Vries M.A. Scheffer H. Garritsen J. Collie' J. ten Kate L.P. Mukherjee A.B. DNA Cell Biol. 1997; 16: 73-83Crossref PubMed Scopus (40) Google Scholar) and encodes a 16-kDa homodimeric protein in which the two identical 70-amino acid subunits are covalently linked by two disulfide bonds (6Mukherjee A.B. Kundu G.C. Mantile-Selvaggi G. Yuan C.J. Mandal A.K. Chattopadhyay S. Zheng F. Pattabiraman N. Zhang Z. Cell Mol. Life Sci. 1999; 55: 771-787Crossref PubMed Scopus (120) Google Scholar). The altered expression of CC10 (3Shijubo N. Itoh Y. Yamaguchi T. Shibuya Y. Morita Y. Hirasawa M. Okutani R. Kawai T. Abe S. Eur. Respir. J. 1997; 10: 1108-1114Crossref PubMed Scopus (138) Google Scholar) or single nucleotide polymorphism in the CC10 gene (9Beier H.M. Biochim. Biophys. Acta. 1968; 160: 289-291Crossref PubMed Scopus (240) Google Scholar) is associated with a variety of pulmonary diseases in humans (9Beier H.M. Biochim. Biophys. Acta. 1968; 160: 289-291Crossref PubMed Scopus (240) Google Scholar, 11Zhang Z. Zimonjic D.B. Popescu N.C. Wang N. Gerhard D. Stone E. Arbour N.C. de Vries M.A. Scheffer H. Garritsen J. Collie' J. ten Kate L.P. Mukherjee A.B. DNA Cell Biol. 1997; 16: 73-83Crossref PubMed Scopus (40) Google Scholar, 12Hermans C. Bernard A. Am. J. Respir. Crit. Care Med. 1999; 159: 646-678Crossref PubMed Google Scholar, 13Laing I.A. Goldblatt J. Eber E. Hayden C.M. Rye P.J. Gibson N.A. Palmer L.J. Burton P.R. Le Souef P.N. J. Med. Genet. 1998; 35: 463-467Crossref PubMed Scopus (103) Google Scholar). Previous studies have suggested a potential protective role of CC10 in suppressing inflammation or modulating the immune response in the lungs following pulmonary injury or infection (6Mukherjee A.B. Kundu G.C. Mantile-Selvaggi G. Yuan C.J. Mandal A.K. Chattopadhyay S. Zheng F. Pattabiraman N. Zhang Z. Cell Mol. Life Sci. 1999; 55: 771-787Crossref PubMed Scopus (120) Google Scholar, 11Zhang Z. Zimonjic D.B. Popescu N.C. Wang N. Gerhard D. Stone E. Arbour N.C. de Vries M.A. Scheffer H. Garritsen J. Collie' J. ten Kate L.P. Mukherjee A.B. DNA Cell Biol. 1997; 16: 73-83Crossref PubMed Scopus (40) Google Scholar, 12Hermans C. Bernard A. Am. J. Respir. Crit. Care Med. 1999; 159: 646-678Crossref PubMed Google Scholar, 13Laing I.A. Goldblatt J. Eber E. Hayden C.M. Rye P.J. Gibson N.A. Palmer L.J. Burton P.R. Le Souef P.N. J. Med. Genet. 1998; 35: 463-467Crossref PubMed Scopus (103) Google Scholar).It has been reported that CC10 expression is rarely detectable in human non-small cell lung cancers, despite the fact that it is abundantly produced by the progenitor cells in normal airways (4Linnoila R.I. Jensen S.M. Steinberg S.M. Mulshine J.L. Eggleston J.C. Gazdar A.F. Am. J. Clin. Pathol. 1992; 97: 233-243Crossref PubMed Scopus (90) Google Scholar, 14Broers J.L. Jensen S.M. Travis W.D. Pass H. Whitsett J.A. Singh G. Katyal S.L. Gazdar A.F. Minna J.D. Linnoila R.I. Lab. Invest. 1992; 66: 337-346PubMed Google Scholar). Its expression is drastically reduced in SV40-induced carcinogenesis (15Magdaleno S.M. Wang G. Mireles V.L. Ray M.K. Finegold M.J. DeMayo F.J. Cell Growth Differ. 1997; 8: 145-155PubMed Google Scholar, 16Sandmoller A. Halter R. Suske G. Paul D. Beato M.A. Cell Growth Differ. 1995; 6: 97-103PubMed Google Scholar), and it has been reported that CC10 expression induced in certain cancer cells leads to diminished invasiveness and anchorage-independent growth, characteristic of these cells (17Szabo E. Goheer A. Witschi H. Linnoila R.I. Cell Growth Differ. 1998; 9: 475-485PubMed Google Scholar, 18Zhang Z. Kundu G.C. Panda D. Mandal A.K. Mantile-Selvaggi G. Peri A. Yuan C.J. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3963-3968Crossref PubMed Scopus (47) Google Scholar). Moreover, the overexpression of CC10 in immortalized bronchial epithelial cells delayed the induction of anchorage-independent growth in response to a potent carcinogen in cigarette smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; Refs. 5Linnoila R.I. Szabo E. DeMayo F. Witschi H. Sabourin C. Malkinson A. Ann. N. Y. Acad. Sci. 2000; 923: 249-267Crossref PubMed Scopus (57) Google Scholar and 17Szabo E. Goheer A. Witschi H. Linnoila R.I. Cell Growth Differ. 1998; 9: 475-485PubMed Google Scholar, 18Zhang Z. Kundu G.C. Panda D. Mandal A.K. Mantile-Selvaggi G. Peri A. Yuan C.J. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3963-3968Crossref PubMed Scopus (47) Google Scholar, 19Peri A. Bonaccorsi L. Muratori M. Luconi M. Baldi E. Granchi S. Pesciullesi A. Mini E. Cioppi F. Forti G. Serio M. Miele L. Maggi M. Int. J. Cancer. 2000; 88: 525-534Crossref PubMed Scopus (18) Google Scholar, 20Leyton J. Manyak M.J. Mukherjee A.B. Miele L. Mantile G. Patierno S.R. Cancer Res. 1994; 54: 3696-3699PubMed Google Scholar). Thus, it is suggested that CC10 may have a protective role against lung tumorigenesis, induced by NNK. To test this hypothesis, we exposed wild type (WT) and CC10-knockout (KO) mice to NNK and compared the rate of lung adenoma formation in these two groups of animals. In addition, we determined whether compared with WT mice NNK treatment of the CC10-KO mice caused: (a) epithelial hyperproliferation, (b) a higher incidence of mutations in the proto-oncogene, K-ras, (c) a higher level of FasL expression, and (d) increased phosphorylation of MAPK/Erk1, all of which are associated with lung tumorigenesis. Our results show that compared with the lungs of NNK-treated WT mice, those of the NNK-treated CC10-KO mice manifest: (i) a markedly higher incidence of airway epithelial hyperplasia and the formation of adenomas, (ii) a markedly increased frequency of K-ras mutations, (iii) a significantly higher level of FasL expression, and (iv) an increased phosphorylation of MAPK/Erk1. We propose that CC10 plays a critical role in protecting the lungs against NNK-induced hyperplasia and adenoma formation, most likely by suppressing the events that are known to precede tumorigenesis in this organ.EXPERIMENTAL PROCEDURESAnimals, Exposure, and Tissue Collection—CC10-KO mice (21Zhang Z. Kundu G.C. Yuan C.J. Ward J.M. Lee E.J. DeMayo F. Westphal H. Mukherjee A.B. Science. 1997; 276: 1408-1412Crossref PubMed Scopus (117) Google Scholar) were generated by gene targeting in embryonic stem cells. The strain- and age-matched C57BL/6 WT mice (Jackson Laboratory, Bar Harbor, ME) were housed under standard conditions in a National Institutes of Health animal facility. All procedures were approved by the NCI Animal Care and Use Committee. Starting at 8 weeks of age, the mice received NNK intraperitoneally (104 mg/kg of body weight, three times, given every other month) or physiological saline, respectively. The animals were serially sacrificed at 5 and 8 months and at 11 and 12 months. These time periods were chosen because it has been reported that aging B6 and 129 mouse strains spontaneously develop cancers in various organs, including the lung, at a high frequency (22Haines D.C. Chattopadhyay S. Ward J.M. Toxicol. Pathol. 2001; 29: 653-661Crossref PubMed Scopus (120) Google Scholar). Two hours prior to sacrifice, mice received 100 μg/g 5-bromo-2′-deoxyuridine (BrdUrd, Sigma) intraperitoneally. Representative tissue specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, sectioned and stained, and embedded in Tissue-Tek® O.C.T. compound (Miles Laboratories Inc., Elkhart, IN) before freezing or snapfrozen in dry ice-cooled 2-methylbutane and stored at –140 °C for molecular analysis as described below.Immunohistochemistry—Immunohistochemistry was performed essentially as described previously (4Linnoila R.I. Jensen S.M. Steinberg S.M. Mulshine J.L. Eggleston J.C. Gazdar A.F. Am. J. Clin. Pathol. 1992; 97: 233-243Crossref PubMed Scopus (90) Google Scholar) using the Vectastain ABC kit (Vector, Burlingame, CA). Primary antibodies were rabbit polyclonal antibodies anti-CC10 (diluted 1:10,000; a gift from Dr. Francesco De-Mayo, Baylor School of Medicine, Houston, TX), anti-surfactant-associated protein C (SP-C, diluted 1:100, Chemicon International, Inc., Temecula, CA), anti-Fas (diluted 1:3000), anti-FasL (diluted 1:1000, both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Ki67 (diluted 1:1000, Novocastra, Laboratories Ltd., Newcastle, UK), and rat anti-BrdUrd monoclonal antibody (diluted 1:300, Accurate Chemical Science, NY). Expression levels of CC10, Fas, and FasL were analyzed with a Nikon Eclipse 400® microscope and Metamorph® software. Proliferation indexes were generated by counting BrdUrd or Ki67-labeled nucleus per 2000–3000 cells per animal lung with a Nikon Eclipse 400® microscope. Statistical analyses were performed using the Mann Whitney U test.Immunoprecipitation and Western Blot Analysis—Snap-frozen lung tissues were homogenized by ultrasonication in lysis buffer composed of PBS, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, 1% proteinase and phosphatase inhibitor mixtures (Sigma). The levels of CC10 in the lung lysates were analyzed by immunoprecipitation as described previously (17Szabo E. Goheer A. Witschi H. Linnoila R.I. Cell Growth Differ. 1998; 9: 475-485PubMed Google Scholar). The levels of Erk1/2 were directly immunoblotted with a rabbit anti-ERK1 antibody (diluted 1:500; Santa Cruz Biotechnology, Inc.), and the levels of phospho-MAPK and phospho-Elk1 were assessed, respectively, using a rabbit anti-phospho-MAPK polyclonal antibody (diluted 1:1500; New England Biolabs, Inc., Beverly, MA) and a Phospho Plus Elk1 Antibody Kit (New England Biolabs, Inc.) as described in the manufacturer's procedures. The levels of CC10, Erk1, phospho-MAPK and phospho-Elk1 were quantified with a Densitometer (Pharmaceutical Biotech).DNA Isolation and Laser Capture Microdissection—About 500 cells from adenomas and airway epithelia were acquired from deparaffinized sections using PixCell I (Arcturus Engineering, Mountain View, CA) laser capture microdissection. DNA was purified according to manufacturer's procedures and used for amplification of K-Ras DNA by polymerase chain reaction (PCR) as described below.Single-strand Conformational Polymorphism (SSCP)—The SSCP analysis was based on the methods described previously (23Gochuico B.R. Miranda K.M. Hessel E.M. De Bie J.J. Van Oosterhout A.J. Cruikshank W.W. Fine A. Am. J. Physiol. 1998; 274: L444-L449PubMed Google Scholar). Briefly, a 143-base pair K-ras exon 1 DNA fragment was yielded by PCR using primers RasE1F (5′-TTA TTG TAA GGC CTG CTG AA-3′) and RasE1R (5′-GCA GCG TTA CCT CTA TCG TA-3′). In addition, a 192-base pair K-ras exon 2 DNA fragment was generated with primers RasE2F (5′-TTCTCAGGACTCCTACAGGA-3′) and RasE2R (5′-ACC CAC CTA TAA TGG TGA AT-3′). PCR was carried out in a 20-μl PCR reaction mixture containing 3 μl of DNA prepared by laser capture microdissection, 2 μl of 10× PCR buffer, 0.4 μl of 10 mm dNTP, 0.4 μl of a 10 μm concentration of each primer, 0.4 μl of [32P]dCTP (20 μCi/μl) (PerkinElmer Life Sciences) and 1 unit of Taq polymerase (Invitrogen). Each sample was subjected to 35 cycles, and each cycle consisted of denaturing at 94 °C for 45 s, annealing at 58 °C for 45 s, and extending at 72 °C for 90 s, with a final step at 72 °C for 8 min. An equal amount of PCR product was mixed with 2× SSCP loading buffer (98% formamide, 20 mm EDTA, pH 8, 0.1% bromphenol blue, 0.1% xylene cyanol) and denatured at 95 °C for 5 min. Four μl of each sample was loaded onto the SSCP gel (FMC BioProducts, Rockland, ME) and run at 6 watts for 16 h in 0.6× TBE at room temperature. The SSCP gels were dried and exposed to autoradiography film.DNA Sequencing—Mutant DNA derived from a variant SSCP band was amplified by PCR with the primers RasE1F and RasE1R. The amplified DNA was then purified with QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and sequenced with a BigDye Terminator Cycle Sequencing Kit and an ABI PRISM DNA sequencer (PE Applied Biosystems, Foster City, CA).RESULTSMorphology of Epithelial Cells in the Airways of WT and CC10-KO Mice before and after NNK Treatment—To assess the effects of NNK on WT and CC10-KO mouse lungs, we first examined the morphology of airway epithelium and CC10 level (Fig. 1). As expected, while high levels of CC10 expression in the lungs of WT mice are readily detectable (Fig. 1A), none of the airway epithelial cells of CC10-KO mice were positive for CC10 (Fig. 1B). The Clara cells of CC10-KO mice showed a marked reduction in apical cytoplasm, normally the storage site for CC10 protein (Fig. 1, C–F). Most importantly, the NNK treatment of WT mice leads to a precipitous reduction in the levels of lung CC10 as assessed by immunohistochemistry (Fig. 1G) and by immunoblot analysis (Fig. 1H). Morphologically, the apical cytoplasm of bronchial epithelial cells in NNK-treated CC10–/–mice remain flat. These results indicate that CC10 deficiency leads to morphological changes in lung epithelial cells of CC10-KO mice and that NNK treatment of WT mice markedly reduces the production of this protein in the lung.At 5–8 months after NNK treatment, histopathology revealed focal airway epithelial hyperplasia in 33% (4 of 12 mice) and solitary lung adenomas in 25% (3 of 12 mice) of the CC10-KO animals (Table I). At 11–12 months after NNK exposure, epithelial hyperplasia and small solitary lung adenomas were induced in 38% (3 of 8) of the CC10-KO mice (Table I). All NNK-induced lung adenomas in CC10-KO mice were solitary, less than 1 mm in diameter, and of alveolar type II cell lineage (Fig. 2, A–C). Moreover, compared with the cells surrounding the alveoli proliferating cells in adenomas are markedly increased as suggested by an elevated BrdUrd index (Fig. 2, D and E). Only one out of 22 NNK-treated WT mice developed a solitary lung adenoma (p = 0.041). These results indicate that CC10-KO mice are more prone to developing hyperplasia and lung adenomas than their WT counterparts following exposure to NNK.Table IDevelopment of lung epithelial hyperplasia and adenomas in NNK-treated CC10-KO miceTreatmentGenotype (n)Duration of treatment (months)Mice with hyperplasiaMice with adenomasNNKKO(12)5-84 (33%)3 (25%)NNKWT(12)5-801PBSKO(6)5-800PBSWT(12)5-800NNKKO(8)11-123 (38%)3 (38%)NNKWT(10)11-1200PBSKO(6)11-1201PBSWT(10)11-1200 Open table in a new tab Fig. 2Photomicrographs of NNK-induced lung tumors in CC10-KO mice. A, a small solitary adenoma in the peripheral lung of a NNK-treated CC10-KO mouse. All of the NNK-induced adenomas in KO mice were solitary and less than 1 mm in diameter. Note the location in the alveolar compartment without any apparent connection with the airways (hematoxylin-eosin stain, ×15). B, higher magnification (×200) reveals a tubular adenoma with minimal atypia. C, tumor cells are positive for a type II pneumocyte marker SP-C, suggesting alveolar type II cell lineage of the tumor (immunoperoxidase stain, ×700). D, BrdUrd index in the adenomas was increased 5-fold, compared with the surrounding alveoli, while there was no significant difference in tumors or surrounding alveoli between NNK-treated KO and WT mice (immunoperoxidase staining, ×100). E, numerous tumor cells displayed Ki-67 immunoreactivity (immunoperoxidase staining, ×100). Throughout the lung tissue including adenomas, Ki-67-positive cells were observed at higher numbers than BrdUrd-positive cells.View Large Image Figure ViewerDownload (PPT)NNK-treated CC10-KO Manifest a Markedly Elevated Level of Proliferation of Airway Epithelial Cells—We next determined whether lack of CC10 protein had changed the cellular dynamics in the pulmonary epithelia following NNK treatment by studying cell proliferation. Again, at 5–6 months after treatment, about 1.5 and 0.91% of BrdUrd-positive epithelial cells appeared in the airways and alveoli, respectively, of the NNK-treated KO mice (Fig. 3, A and B). This reflected a 3–10-fold increase in the bronchiolar epithelium, as compared with 0.45% of BrdUrd-positive cells in NNK-treated WT mice (p = 0.016), 0.13% in PBS-treated KO mice (p = 0.036), and 0.37% in PBS-treated WT mice (data not shown). These results were confirmed by immunostaining using the cellular proliferation marker Ki-67. There was a 2–6-fold increase in the Ki-67 labeling index in the lungs of NNK-treated CC10-KO, as compared with NNK-treated WT mice, PBS-treated CC10-KO, and WT mice (data not shown). These data suggest that hyperproliferation induced by NNK in KO mice may be responsible for higher incidence of lung adenomas.Fig. 3CC10-KO mice manifest hyperproliferation of airway epithelium and increased FasL expression. Bronchiolar epithelia of mice exposed to 5 months of NNK are shown. A, WT mouse with a single labeled cell (arrow). It should be noted that no statistically significant differences between WT and CC10-KO mice were detected in PBS-treated control groups, although the lung BrdUrd labeling index was 3-fold lower in the airways of CC10-KO mice compared with those in WT mice. B, NNK-exposed KO mouse with numerous labeled bronchiolar cells (arrows), resulting in a significantly higher (up to 10-fold) labeling index than in WT mice. (A and B, immunoperoxidase staining for BrdUrd, ×100.) In contrast, no statistically significant difference of BrdUrd labeling index in the lung was found in WT animals between NNK treatment and PBS treatment. In the alveolar compartment, following NNK exposure there was a 2–3-fold increase in the labeling index of KO mice, as compared with 0.51% of BrdUrd-positive cells in WT mice (p = 0.029). No statistically significant difference in the alveolar BrdUrd labeling index was found between CC10-KO and WT mice in PBS-treated control groups. C, WT control animal reveals minimal immunoreactivity in the bronchiolar epithelial cells for FasL. D, at 5 months of NNK exposure, there is increased staining in the airway epithelium in CC10-KO animals. (C and D, immunoperoxidase staining, ×300).View Large Image Figure ViewerDownload (PPT)NNK Induces Markedly Elevated FasL Expression in CC10-KO Lungs—Since Clara cells are a source of FasL, the expression of which is associated with enhanced tumorigenesis (23Gochuico B.R. Miranda K.M. Hessel E.M. De Bie J.J. Van Oosterhout A.J. Cruikshank W.W. Fine A. Am. J. Physiol. 1998; 274: L444-L449PubMed Google Scholar, 24Hahne M. Rimoldi D. Schroter M. Romero P. Schreier M. French L.E. Schneider P. Bornand T. Fontana A. Lienard D. Cerottini J. Tschopp J. Science. 1996; 274: 1363-1366Crossref PubMed Scopus (1191) Google Scholar), we determined whether CC10-KO mice have an abnormal expression of FasL by immunohistochemistry. While Fas expression appeared similar among the four treatment groups (data not shown), markedly enhanced FasL immunoreactivity occurred in lungs of NNK-treated CC10-KO mice as compared with a minimal to moderate increase in the lungs of NNK-treated WT mice. Using a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay we determined the level of apoptosis in bronchial epithelia of NNK-treated CC10-KO and WT mice and found that NNK treatment markedly increased the level of apoptosis in CC10-KO mice. 2Y. Yang, Z. Zhang, A. B. Mukherjee, and R. I. Linnoila, unpublished results. Moreover, enhanced immunoreactivity for FasL was also detected within alveolar type II cells and lymphocytes in the hyperplasia and adjacent to tumors in NNK-treated KO mice (Fig. 3, C and D). The results indicate that CC10 protein may have a protective role on cellular stability against NNK-induced cellular biochemical alteration such as FasL overexpression.Increased K-ras Mutation in CC10-KO Lungs—Recent reports indicate that most chemically induced lung tumors (∼90%) in A/J mice carry an activating point mutation in the K-ras proto-oncogene (25Matzinger S.A. Crist K.A. Stoner G.D. Anderson M.W. Pereira M.A. Steele V.E. Kelloff G.J. Lubet R.A. You M. Carcinogenesis. 1995; 16: 2487-2492Crossref PubMed Scopus (49) Google Scholar, 26Chen B. Liu L. Castonguay A. Maronpot R.R. Anderson M.W. You M. Carcinogenesis. 1993; 14: 1603-1608Crossref PubMed Scopus (102) Google Scholar). To determine whether there is an increase in K-ras mutations in the lung tumors of NNK-treated KO mice, we microdissected the tissues and carried out PCR-SSCP and sequencing assay to detect mutations in exons 1 and 2 of this gene. This area contains clusters of the known transformation mutations. We found that three out of six lung adenomas (50%) in NNK-treated CC10-KO mice had K-ras mutations in codon 12 of exon 1 (supplemental Fig. 1), in which GGT was transversed to GCT, resulting in an amino acid change from glycine to alanine. A K-ras mutation at codon 32 was found in a lung adenoma of an NNK-treated WT mouse, in which TAC was transitioned to TAT, resulting in a silent mutation. All three K-ras mutations in NNK-exposed KO mice were early phase alveolar adenomas, suggesting that CC10 deficiency confers susceptibility to NNK-mediated genomic instability such as K-ras mutations, which may contribute to carcinogenesis.Elevated Levels of MAPK/Erk1 Phosphorylation in NNK-treated CC10-KO Mouse Lungs—Since the MAPK cascade, situated downstream from activating ras mutations, plays a critical role in regulating cell growth, proliferation, and responses to extracellular signals (27Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1662) Google Scholar, 28Robledo R.F. Buder-Hoffmann S.A. Cummins A.B. Walsh E.S. Taatjes D.J. Mossman B.T. Am. J. Pathol. 2000; 156: 1307-1316Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), we examined the activation stages of MAPK by immunobloting analysis (Fig. 4). We found elevated levels of Erk1 in NNK-treated lungs from both KO and WT mice (Fig. 4A). Moreover, we also found dramatically increased MAPK activity in NNK-treated CC10-KO mouse lungs with adenomas as compared with those in PBS-treated lungs (Fig. 4A). The relative levels of phospho-MAPK in PBS-treated lungs from KO animals were twice as high as from WT mice and enhanced up to 3-fold following the exposure to NNK (Fig. 4B). Western blot analysis of phosphorylated Elk1 also showed similar results. Thus it appears that the lungs of CC10-deficient mice are more sensitive to NNK-induced activation of the MAPK that mediates increased cellular proliferation and cell growth, leading to tumorigenesis.Fig. 4Activation of MAPK kinase pathway associated with NNK exposure in CC10-deficient mice. The levels of Erk1 and phosphorylated MAPK (phospho-MAPK) were analyzed by immunoblotting of lung lysates using state-specific antibodies. A representative gel from three CC10-KO and three WT mice is shown in A. Although the levels were variable, the average levels of Erk1 were unchanged between NNK-and PBS-treated CC10-KO and WT mice. B, the level of phospho-MAPK compared with the total kinase was determined by densitometric analysis and expressed in relative units of phospho-MAPK/ERK1. The level in KO control lung (bar 1) was 2-fold as compared with that of WT control (bar 4), and the exposure to NNK at 8 months was associated with a 3-fold increase in the CC10-KO tumor lung (bar 2) and lung (bar 3) lysates, when compared with the lysates from corresponding WT NNK-treated animals (bars 5 and 6, respectively).View Large Image Figure ViewerDownload (PPT)DISCUSSIONIn this study, we provided evidence that CC10-KO mice are significantly more susceptible to developing hyperproliferation of airway epithelial cells and the formation of pulmonary adenomas in response to NNK-treatment. Smoking remains a significant health hazard throughout the world, and NNK is a potent carcinogen in cigarette smoke. We discovered that mice exposed to this carcinogen express drastically lower levels of CC10 as does cigarette smoking (3Shijubo N. Itoh Y. Yamaguchi T. Shibuya Y. Morita Y. Hirasawa M. Okutani R. Kawai T. Abe S. Eur. Respir. J. 1997; 10: 1108-1114Crossref PubMed Scopus (138) Google Scholar). The results of previous studies (17Szabo E. Goheer A. Witschi H. Linnoila R.I. Cell Growth Differ. 1998; 9: 475-485PubMed Google Scholar, 18Zhang Z. Kundu G.C. Panda D. Mandal A.K. Mantile-Selvaggi G. Peri A. Yuan C.J. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3963-3968Crossref PubMed Scop