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The sulfone derivative of the non-steroidal anti-inflammatory drug (NSAID), sulindac, has been reported to inhibit mammary and colon tumor formation in rodent models of chemically-induced carcinogenesis. Unlike its parent compound, this metabolite lacks cyclo-oxygenase inhibitory activity. A tumor induction protocol, consisting of NNK administration in the drinking water over several weeks to model chronic human exposure, was used to test whether the sulfone (called FGN-1) could inhibit the formation of primary lung tumors in mice. A total of 150 female, AIN-76A-fed, A/J mice received 9 mg of NNK each. Concentrations of FGN-1 that had been previously determined not to affect body weight gain were added to the food at levels of 0, 250, 500 and 750 mg/kg of diet (30 mice/group) starting 2 weeks before NNK administration and continuing for 22 weeks. At that time pleural surface tumors were counted. Tumor incidence decreased significantly from 96% in the control diet and 93% in the 250 FGN-1 mg/kg diet to 63 and 67% in the 500 and 750 mg FGN-1/kg diet groups, respectively (P < 0.001 by chi-square analysis). Lung tumor multiplicity decreased from 18.1 ± 3 tumors/mouse (mean ± SEM, control diet) to 12.3 ± 3 (250), 5.3 ± 1 (500) and 2.1 ± 1 (750) (P < 0.0005 by post hoc ANOVA). In previous studies using this carcinogenesis protocol, the maximum tolerated dose of sulindac inhibited lung tumor multiplicity by no more than 50% with no effect on incidence. This dose-dependent reduction in tumorigenesis by a non-toxic dose of FGN-1 indicates a strong chemopreventive activity against experimental induction of lung carcinogenesis. The greater potency of the sulfone over sulindac and its lack of toxic side effects because of its inability to affect cyclo-oxygenase activity suggests that clinical testing in individuals at high risk for lung cancer should be considered. [ABSTRACT FROM PUBLISHER]

The reduced gap junctional intercellular communication (GJIC) and gap junction protein (connexin) expression that have been noted in many neoplastic cell types may contribute to the neoplastic phenotype. We assessed GJIC (by fluorescent dye micro-injection) and connexin expression (by Northern blotting, Western blotting and immunohistochemistry) in five mouse and 17 human lung carcinoma cell lines; both measures were lower in neoplastic cells compared to non-transformed lung epithelial cells. Other connexins were not detected in these cells. Co-culture experiments indicated that carcinoma cell lines able to transfer dye among themselves (homologous GJIC) had little capacity for dye-coupling with non-transformed cells (heterologous GJIC). Southern blot analyses indicated that reduction in GJIC and connexin43 expression were not due to deletions or rearrangements of this gene, but were more likely accounted for by transcriptional down-regulation and/or post-transcriptional factors. No correlations between GJIC and known oncogene and tumor suppressor gene alterations in the human lung carcinoma cells were apparent, suggesting that other mechanisms down-regulate GJIC in these cells. Since the neoplastic cell lines exhibited low GJIC (either homologous or heterologous, this characteristic may be involved in expression of the neoplastic phenotype. [ABSTRACT FROM PUBLISHER]

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4-6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

Silibinin inhibits mouse lung tumorigenesis in part by targeting tumor microenvironment. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) can be pro- or anti-tumorigenic, but in lung cancer cell lines they induce pro-inflammatory enzymes cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS). Accordingly, here we examined mechanism of silibinin action on TNF-α + IFN-γ (hereafter referred as cytokine mixture) elicited signaling in tumor-derived mouse lung epithelial LM2 cells. Both signal transducers and activators of the transcription (STAT)3 (tyr705 and ser727) and STAT1 (tyr701) were activated within 15 min of cytokine mixture exposure, while STAT1 (ser727) activated after 3 h. Cytokine mixture also activated Erk1/2 and caused an increase in both COX2 and iNOS levels. Pretreatment of cells with a MEK, NF-κB, and/or epidermal growth factor receptor (EGFR) inhibitor inhibited cytokine mixture-induced activation of Erk1/2, NF-κB, or EGFR, respectively, and strongly decreased phosphorylation of STAT3 and STAT1 and expression of COX2 and iNOS. Also, janus family kinases (JAK)1 and JAK2 inhibitors specifically decreased cytokine-induced iNOS expression, suggesting possible roles of JAK1, JAK2, Erk1/2, NF-κB, and EGFR in cytokine mixture-caused induction of COX2 and iNOS expression via STAT3/STAT1 activation in LM2 cells. Importantly, silibinin pretreatment inhibited cytokine mixture-induced phosphorylation of STAT3, STAT1, and Erk1/2, NF-κB-DNA binding, and expression of COX2, iNOS, matrix metalloproteinases (MMP)2, and MMP9, which was mediated through impairment of STAT3 and STAT1 nuclear localization. Silibinin also inhibited cytokine mixture-induced migration of LM2 cells. Together, we showed that STAT3 and STAT1 could be valuable chemopreventive and therapeutic targets within the lung tumor microenvironment in addition to being targets within tumor itself, and that silibinin inhibits their activation as a plausible mechanism of its efficacy against lung cancer., (Copyright © 2011 Wiley Periodicals, Inc.)

BRCA1 mRNA overexpression is correlated with poor survival in NSCLC. However, BRCA1 functions depend on the interaction with BARD1 for its stability, nuclear localization and ubiquitin ligase activity. Expression of alternatively spliced BARD1 isoforms that lack the BRCA1-interaction domain was found upregulated and correlated with poor prognosis in breast and ovarian cancer. These BARD1 isoforms are essential for proliferation of cancer cells in vitro. We investigated whether BARD1 isoforms are expressed in NSCLC. While in lung tissues from healthy controls BARD1 expression was undetectable on the mRNA level and protein level, we found two novel isoforms in addition to previously identified mRNAs expressed in all NSCLC samples tested. Furthermore, the pattern of BARD1 isoform expression was similar in tumor and morphologically normal peri-tumor tissues, and only one novel isoform π was specifically upregulated in tumors. Immunohistochemistry revealed that all 100 NSCLC cases tested expressed isoform-specific BARD1 epitopes, while BARD1 expression was undetectable in biopsies from healthy controls. Statistical analysis showed that the expression of epitopes PVC and WFS, present on isoform π, or epitope WFS alone, expressed on isoforms π, κ and β, were significantly correlated with decreased patient survival. These findings were corroborated in a mouse model of chemically induced lung cancer. Immunostaining of mouse tumors showed that BARD1 epitopes PVC and WFS were specifically upregulated in invasive, but not in confined lung tumors. Thus, BARD1 isoforms might be involved in tumor initiation and invasive progression and might represent a novel prognostic marker for NSCLC., (Copyright © 2011 UICC.)

Background: Worldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear., Methods: After culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection., Results: Primary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth., Conclusions: Macrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.

Purpose: Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS(-/-) mice., Experimental Design: Male B6/129-Nos2(tm1Lau) (iNOS(-/-)) and B6/129PF2 WT mice were injected i.p. with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742 mg/kg body weight) for 5 d/wk for 18 weeks., Results: Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did not show any such considerable effect in iNOS(-/-) mice. Tumors from WT mice expressed more iNOS (P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors., Conclusions: The lack of effect of silibinin in iNOS(-/-) mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents., (©2010 AACR.)

The inflammatory cytokines tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) stimulate production of the inflammatory mediators prostaglandin E₂ (PGEγ), prostacyclin (PGIγ), and nitric oxide (NO) in cultured lung epithelial cells. Pretreatment of these cells with the selective MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase 1/2) inhibitor U0126 blocked ERK1/2 activation and inhibited cytokine-induced production of these inflammatory mediators. Primary bronchiolar epithelial Clara cells treated with TNFα and IFNγ also produced increased PGE₂, PGI₂, and NO, and PG and NO production was decreased by MEK inhibition. U0126 differentially affected cyclooxygenase (COX)-1, COX-2, and inducible NO synthase (iNOS) expression in cell lines, however, suggesting that MEK1/2 regulates prostanoid and NO production by means other than inducing their biosynthetic enzymes. Functionally, inhibition of MEK1/2 caused G1 cell cycle arrest and decreased cyclin D1 expression, but these effects were not related to decreased prostanoid production. These results indicate separate proinflammatory and proliferative roles for ERK1/2 in lung epithelial cells. During lung tumor formation in vivo, ERK1/2 phosphorylation increased as lung tumors progressed. Since tumor-derived cells were more sensitive than nontumorigenic cells to the antiproliferative effects of U0126, MEK1/2 inhibition may serve as an attractive chemotherapeutic target.

Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models-repeated exposure to BHT and infection with Mycobacterium tuberculosis-to establish whether similar macrophage phenotype changes occur in non-neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN-gamma and IL-4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN-gamma content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN-gamma content and BAL macrophage iNOS expression decreased, and BALF IL-4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis-induced granulomas remained M1-polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma-associated, are exposed to different activating cytokines. BDMCs from BHT-treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis-infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT-induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.

Tumor-associated macrophages (TAMs) encourage and coordinate neoplastic growth. In late stage human lung adenocarcinoma, TAMs exhibited mixed M1 (classical; argI(low)iNOS(high)) and M2 (alternative; argI(high)iNOS(low)) polarization based on arginine metabolism. In several murine cancer models including chemically and genetically-induced primary lung tumors, prostate tumors, colon xenografts, and lung metastases, TAMs expressed argI(high)iNOS(low) early during tumor formation; argI(low)iNOS(high) polarization also occurred during malignancy in some models. In a chemically-induced lung tumor model, macrophages expressed argI(high)iNOS(low) within one week after carcinogen treatment, followed by similar polarization of bone marrow-derived monocytes (BDMCs) a few days later. TAMs surrounding murine prostate tumors also expressed argI(high)iNOS(low) early during tumorigenesis, indicating that this polarization is not unique to neoplastic lungs. In a human colon cancer xenograft model, the primary tumor was surrounded by argI(high)iNOS(low)-expressing TAMs, and BDMCs also expressed argI(high)iNOS(low), but pulmonary macrophages adopted argI(high)iNOS(low) polarization only after tumors metastasized to the lungs. Persistence of tumors is required to maintain TAM polarization. Indeed, in both conditional mutant Kras- and FGF10-driven models of lung cancer, mice expressing the transgene develop lung tumors that regress rapidly when the transgene is silenced. Furthermore, pulmonary macrophages expressed argI(high)iNOS(low) on tumor induction, but then returned to argI(low) iNOS(low) (no polarization) after tumors regressed. Manipulating TAM function or depleting TAMs may provide novel therapeutic strategies for preventing and treating many types of cancer.

Background: Human lung cancer patients exhibit different KRAS mutations depending on smoking status. In a mouse model of human cancer, A/J and BALB/cBy mice treated with the tobacco carcinogen, 3-methylcholanthrene (MCA), followed by butylated hydroxytoluene (BHT)-elicited chronic inflammation develop a high multiplicity of lung tumors., Methods: DNA was isolated from MCA-induced lung tumors in A/J and BALB/cByJ mice. Kras codon 12 sequences from these tumors were compared to those in human lung tumors from smokers and never-smokers., Results: The distribution of Kras codon 12 mutations in MCA-induced A/J lung tumors is strikingly similar to those found in adenocarcinomas from human smokers. In contrast, codon 12 mutations in BALB/cBy mice contain predominantly G --> D mutations, which is the most common mutation in never smokers., Conclusions: A single lung carcinogen induces different tumor initiating mutations in different strains of mice. This may be useful for investigating the role of specific KRAS mutations in adenocarcinoma pathogenesis in smokers versus never smokers, identifying mechanisms that select for certain KRAS mutations and developing new drugs that specifically target cells with different KRAS mutations.

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.

Clara cells were first described as a morphologically distinct cell type by Kolliker in 1881, but they take their name from the seminal study of human and rabbit bronchioles by Max Clara in 1937. Since their discovery, Clara cells have been identified as central players in protecting the airway from environmental exposures. The diverse functions of Clara cells in lung homeostasis include roles in xenobiotic metabolism, immune system regulation, and progenitor cell activity. Recent identification of a sub-population of Clara cells as a bronchiolar tissue-specific stem cell and a potential tumor initiating cell has focused the attention of cell and molecular biologists on the Clara cell and its behavior under normal and disease conditions.

Background: Alternative (M2) macrophage activation is associated with tumor development in many tumor types, including those in the lung. Herein the biological consequences of forcing classical (M1) or alternative (M2) macrophage activation on lung tumor development are examined., Materials and Methods: Urethane-induced lung tumor multiplicity and size were compared in IFN-gamma(-/-) mice which lack M1 macrophage activation, IL-4Ralpha(-/-) mice which lack M2 macrophage activation, and wild-type BALB/cJ (background strain of the IFN-gamma(-/-) and IL-4Ralpha(-/-) mice) mice. Tumor-associated macrophage (TAM) and bone marrow-derived monocyte (BDMC) activation were each examined., Results: The TAMs and BDMCs in the IFN-gamma(-/-) mice exhibited M2 activation, and their lung tumors were significantly larger than those in the wild-type mice. In contrast, urethane-treated IL-4Ralpha(-/-) mice, whose TAMs and BDMCs were M1 activated, developed smaller tumors than the wild-type mice., Conclusion: Altered innate immunity can diminish or accelerate lung tumor progression in response to defective cytokine signaling.

The latency period for lung tumor progression offers a window of opportunity for therapeutic intervention. Herein, we studied the effect of oral silibinin (742 mg/kg body weight, 5 d/wk for 10 weeks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibinin strongly decreased both tumor number and tumor size, an antitumor effect that correlates with reduced antiangiogenic activity. Silibinin reduced microvessel size (50%, P < 0.01) with no change in the number of tumor microvessels and reduced (by 30%, P < 0.05) the formation of nestin-positive microvessels in tumors. Analysis of several proteins involved in new blood vessel formation showed that silibinin decreased the tumor expression of interleukin-13 (47%) and tumor necrosis factor-alpha (47%), and increased tissue inhibitor of metalloproteinase-1 (2-fold) and tissue inhibitor of metalloproteinase-2 (7-fold) expression, without significant changes in vascular endothelial growth factor levels. Hypoxia- inducible factor-1 alpha expression and nuclear localization were also decreased by silibinin treatment. Cytokines secreted by tumor cells and tumor-associated macrophages regulate angiogenesis by activating nuclear factor-kappaB (NF-kappaB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-kappaB (ser276, 38%; P < 0.01) and STAT-3 (ser727, 16%; P < 0.01) in tumor cells and decreased the lung macrophage population. Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased by silibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression by antiangiogenic mechanisms seem to be mediated by decreased tumor-associated macrophages and cytokines, inhibition of hypoxia-inducible factor-1 alpha, NF-kappaB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.

Purpose: Erlotinib, a small molecule inhibitor of the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR), increases survival of advanced non-small cell lung cancer patients who failed standard chemotherapy (Phase III study). We evaluated whether erlotinib is also effective at an early stage of primary lung tumorigenesis in a carcinogen-induced lung tumor model in mice., Methods: Sixteen weeks after carcinogen (urethane) injection, when small self-contained adenomas are evident, male and female A/J mice were treated IP with 10 mg/kg erlotinib or Captisol vehicle daily over 3.5 weeks (15 mice per group). The efficacy, metabolism and mechanism of action of erlotinib were evaluated., Results: Erlotinib reduced tumor burden in males by twofold compared to vehicle (12.7 +/- 1.2 vs 26.2 +/- 2.5 mg, respectively; p < 0.0001), while tumor burden in erlotinib-treated females slightly increased compared to vehicle by 21% (15.1 +/- 1.2 vs 11.9 +/- 0.9 mg, respectively; p < 0.05). Tumor multiplicity, in contrast, was unaffected by erlotinib. The levels of erlotinib that accumulated in plasma, lung tumor tissue and adjacent uninvolved (UI) lung were comparable in males and females. Males, however, accumulated more OSI-420, an active and pharmacologically equipotent metabolite of erlotinib, than females in plasma, lung tumors, and UI lung. In both genders, 80% of tumors contained Kras mutations at codon 61, but no EGFR mutations were detected. The cellular distribution and concentration of EGFR were also similar between genders. In control mice, however, phosphorylated EGFR (pEGFR) levels were nearly 2.5-fold higher in males compared to females in UI lungs and sevenfold higher in lung tumors. Further, erlotinib decreased the contents of pEGFR in UI lungs and lung tumors, particularly in males., Conclusions: Adenomas from male mice in this early lung cancer model are responsive to erlotinib treatment, possibly because of a greater dependence of male tumor growth on the EGFR pathway compared to females. Importantly, these results indicate that small lung adenomas from male mice that utilize EGFR signaling but also harbor Kras mutations shrink in response to erlotinib, suggesting that erlotinib may be beneficial for some patients very early during lung cancer progression.

Microarray experiments designed to identify genes differentially expressed in the E11.5 lung and trachea showed that melanoma inhibitory activity (Mia1) was expressed only in the lung. Mia1 was abundantly expressed during early lung development, but was virtually absent by the end of gestation. Distal embryonic lung epithelium showed high levels of Mia1 expression, which was suppressed by treatment with either retinoic acid or the FGF signaling antagonist SU5402. Late-gestation fetuses in which lung epithelial hyperplasia was induced by misexpression of FGF7 or FGF10 showed continued expression of Mia1 in areas of aberrant morphogenesis. Mia1 expression was also significantly increased in urethane-induced lung adenomas. Treatment of E18.5 lung explants with exogenous MIA caused significant reductions in the expression of the lung differentiation markers Sftpa, Sftpb, Sftpc, and Abca3. Bitransgenic mice expressing MIA under the control of the SFTPC promoter after E16.5, the age when Mia1 is normally silenced, died from respiratory failure at birth with morphologically immature lungs associated with reduced levels of saturated phosphatidylcholine and mature SP-B. Microarray analysis showed significant reductions in the expression of Sftpa, Sftpb, Abca3, Aqp5, Lzp-s, Scd2, and Aytl2 in lungs misexpressing MIA. These results suggest that the silencing of Mia1 that occurs in late gestation may be required for maturation of the surfactant system.

One area of intensive investigation is to understand complex cellular and signaling interactions in the tumor microenvironment. Using a novel, although straightforward, microarray approach, we defined a gene expression signature from the lung tumor microenvironment in the murine A/J-urethane model of human lung adenocarcinoma. The tumor microenvironment is reflected by the composition of the cell types present and alterations in mRNA levels, resulting in a "Field Effect" around the tumor. The genes composing the Field Effect expression signature include proteases and their inhibitors, inflammation markers, and immune signaling molecules. By several criteria, the Field Effect expression signature can be attributed to the macrophage lineage, suggesting a qualitative change in the expression pattern of tumor-associated macrophages (TAM) observed in lung tumors. The protein expression levels for a number of Field Effect genes were verified by Western blot analysis of lung homogenates, and for their expression in macrophages and parenchymal cells outside of the tumors by immunohistochemistry. In addition, the Field Effect expression signature was used to classify bronchoalveolar lavage (BAL) cells from tumor-bearing or age-matched control mice. Using a variety of statistical measures, the Field Effect expression signature correctly classified the BAL cells >94% of the time. Finally, the protein levels for several Field Effect genes were higher in cell-free BAL fluid, indicating they may be secreted by the TAMs. This work suggests that TAMs generate a unique gene expression signature within the tumor microenvironment, and this signature could potentially be used for identifying lung cancer from BAL cells and/or fluid.

Our previous proteomic investigation of lung neoplasia in vitro demonstrated a high concentration of the lung cancer biomarker and splicing factor, hnRNP A2/B1, in the transformed mouse lung epithelial cell line, E9. Since changes in pre-mRNA splicing profoundly affect neoplastic progression, we examined hnRNP A2/B1 expression in chemically induced primary mouse lung tumors, an in vivo model of pulmonary adencocarcinoma. Tumor hnRNP A2/B1 content and spatial distribution assessed by immunohistochemistry varied with stage of progression, genetic background, and whether tumors were induced by a single agent (urethane) or by 2-stage initiation/promotion (3-methylcholanthrene/butylated hydroxytoluene) carcinogenesis. To address mechanisms governing hnRNP A2/B1 expression changes, we utilized in vitro models. hnRNP A2/B1 protein was overexpressed in E9, the spontaneous tranformant of immortalized but non-neoplastic E10 cells, but expression was not strictly a function of enhanced proliferative rate in neoplastic cells. Elevated mRNA content was positively associated with cell division in both E10 and E9, but hnRNP A2/B1 protein levels decreased in proliferating E10 cells. The increased mRNA reflected enhanced mRNA stability, as shown by measuring time-dependent mRNA decay after inhibiting transcription. Dysregulation of hnRNP A2/B1 expression during lung neoplasia in vivo thus depends on complex gene-environmental interactions that affect cell type-specific changes in mRNA processing and, most probably, the rates of translation and/or protein degradation., (Copyright (c) 2007 Wiley-Liss, Inc.)

Little is known about how the composition of stromal cells within the lung cancer microenvironment varies during tumor progression. We examined by immunohistochemistry each of six different stromal cell populations during the development of chemically induced primary lung cancer in mice. Blood vessels were seen even in microscopic lesions, and their numbers increased with tumor size. Neutrophils infiltrated the alveoli of tumor-bearing lungs and within the periphery of macroscopic adenomas and adenocarcinomas. The numbers of peritumoral lymphocytes and macrophages increased during oncogeny, but quantitative changes in mast cells and fibroblasts were not evident. Because macrophage depletion reduces tumor growth and these cells are thus important to tumorigenesis, we also investigated their phenotype. Pulmonary macrophages expressed arginase I (subtype M2) but not inducible nitric-oxide synthase in lungs with premalignant lesions, whereas macrophages in carcinoma-bearing lungs expressed inducible nitric-oxide synthase (subtype M1) but not arginase I. Local pulmonary stimuli did not seem responsible for this shift in macrophage activation state because monocytes still residing within the bone marrow adopted these expression patterns before entering the circulation, presumably in response to tumor-derived signals. These biochemical markers of macrophage activation states would have diagnostic and/or therapeutic value if analogous systemic shifts occur in humans.

Introduction: Human bronchioloalveolar carcinoma (BAC) is a disease with an evolving definition. "Pure" BAC, characterized by a bronchioloalveolar growth pattern and no evidence of stromal, vascular, or pleural invasion, represents only 2 to 6% of non-small cell lung cancer (NSCLC) cases, but up to 20% of NSCLC cases may contain elements of BAC. This imprecise definition makes it difficult to perform epidemiologic analyses or to generate accurate animal models. However, because BAC appears to behave clinically differently from adenocarcinoma, a better understanding of this disease entity is imperative., Methods/results: At the BAC Consensus Conference in 2004, our committee discussed issues relevant to BAC epidemiology, pathogenesis, and preclinical models., Conclusions: Elucidation of molecular events involved in BAC tumorigenesis will allow for more precise epidemiologic studies and improved animal models, which will enable development of more effective treatments against the disease.

Inbred mice vary in their susceptibility to develop macroscopic, chemically-induced, pulmonary neoplasias. It is not known, however, whether microscopic lesions appear in resistant strains but do not grow or if no early lesions arise at all. We show herein that resistant C57BL/6J (B6) and intermediately resistant BALB/cByJ (BALB) mice form very few urethane-induced early microadenomas (i.e. adenomas larger than hyperplasic foci, but detectable only by light microscopy). Additionally, while all urethane-induced microadenomas in sensitive A/J mice gave rise to adenomas, most microscopic tumors induced in BALB mice by 2-stage, 3-methylcholanthrene/butylated hydroxytoluene carcinogenesis spontaneously regressed. The formation of microscopic lesions is thus genetically dependent, but whether they continue to grow or regress depends on how they were induced.

Background: Manipulating prostaglandin (PG) production modulates tumor development. Elevated PGI2 production prevents murine lung cancer, while decreasing PGE2 content protects against colon cancer. PGE2 receptor subtype 2 (EP2) -deficient mice were hypothesized to be resistant to lung tumorigenesis., Materials and Methods: EP2 null BALB/c mice and their wild-type littermates were exposed to an initiation-promotion carcinogenesis protocol and lung tumorigenesis was examined. Chronic lung inflammation was induced to determine whether EP2 ablation influenced inflammatory cell infiltration., Results: Tumor multiplicity in EP2 null mice was 34% lower than in their wild-type littermates (21.9+/-3.0 vs. 14.5+/-2.9 tumors/mouse, p<0.001). The lung tumor burden, an indicator of growth rate, also declined (57%, p<0.05). All the mice exhibited similar inflammatory cell infiltration., Conclusion: PGE2, acting through EP2, enhanced lung tumorigenesis through a mechanism that may be distinct from its proinflammatory activity. Thus, EP2 is a potential target for novel chemoprevention strategies.

Background: Silibinin, a flavanone from milk thistle, inhibits the growth of tumors in several rodent models. We examined the effects of dietary silibinin on the growth, progression, and angiogenesis of urethane-induced lung tumors in mice., Methods: A/J mice (15 per group) were injected with urethane (1 mg/g body weight) or saline alone and fed normal diets for 2 weeks, after which they were fed diets containing different doses of silibinin (0%-1% [wt/wt] silibinin) for 18 or 27 weeks. Immunohistochemistry and Western blot analysis were used to examine angiogenesis and enzymatic markers of inflammation, proliferation, and apoptosis. All statistical tests were two-sided., Results: Urethane-injected mice exposed to silibinin had statistically significantly lower lung tumor multiplicities than urethane-injected mice fed the control diet lacking silibinin (i.e., control mice). Mice that received urethane and 1% (wt/wt) dietary silibinin for 18 weeks had 93% fewer large (i.e., 1.5-2.5-mm-diameter) lung tumors than control mice (mean number of tumors/mouse: 27 in the urethane group versus 2 in the urethane + 1% silibinin group, difference = 25 tumors/mouse, 95% confidence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibinin-fed mice had 41%-74% fewer cells positive for the cell proliferation markers proliferating cell nuclear antigen and cyclin D1 than lung tumors of control mice. Tumor microvessel density was reduced by up to 89% with silibinin treatment (e.g., 56 microvessels/400x field in tumors from control mice versus 6 microvessels/400x field in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400x field, 95% CI = 46 to 54 microvessels/400x field; P<.001]). Silibinin decreased lung tumor expression of vascular endothelial growth factor (VEGF) and of inducible nitric oxide synthase and cyclooxygenase-2, two enzymes that promote lung tumor growth and progression by inducing VEGF expression., Conclusions: Silibinin inhibits lung tumor angiogenesis in an animal model and merits investigation as a chemopreventive agent for suppressing lung cancer progression.

Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA(2) in this response was examined by determining cPLA(2) expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA(2) genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA(2) null mice. Monocyte chemotactic protein-1 (MCP-1) content in bronchoalveolar lavage fluid increases after BHT treatment but before monocyte influx, suggesting a causative role. Bronchiolar Clara cells isolated from cPLA(2) null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA(2) is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.

Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.

Studies using transgenic and knockout mice have demonstrated that particular cytokines influence lung tumor growth and identified prostaglandin E2 (PGE2), prostacyclin (PGI2) and nitric oxide (NO) as critical mediators of this process. PGE2 and NO were pro-tumorigenic while PGI2 was antitumorigenic. We describe herein an in vitro experimental approach to examine interactions among cytokines, prostaglandins (PGs) and NO. PGE2, PGI2, and NO levels were assayed in culture media from non-tumorigenic mouse lung epithelial cell lines, their spontaneous transformants and mouse lung tumor-derived cell lines, before or after exposure to the cytokines TNFalpha, IFNgamma and IL1beta, alone and in combination. More PGE2 than PGI2 was produced by neoplastic cells, while the opposite was observed in non-tumorigenic lines. Cytokine exposure magnified the extent of these differential concentrations. The PGE2 to PGI2 ratio was also greater in chemically-induced mouse lung tumors than in adjacent tissue or control lungs, supporting the physiological relevance of this in vitro model. Expression of PG biosynthetic enzymes in these cell lines correlated with production of the corresponding PGs. Cytokine treatment enhanced NO production by inducing the inflammation-associated biosynthetic enzyme, inducible NO synthase (iNOS), but this did not correlate with the neoplastic status of cells. Inhibition of iNOS or cyclooxygenase 2 activity using aminoguanidine or NS-398 respectively, demonstrated that NO did not affect PG production nor did PGs influence NO production. Since lack of iNOS inhibits mouse lung tumor formation, we propose that this is independent of any modulation of PG synthesis in epithelial cells. The similar normal/neoplastic trends in PGE2 to PGI2 ratios both in vitro and in vivo, together with an amplification of this difference upon cytokine exposure, are consistent with the hypothesis that cytokines released during inflammation exacerbate differences in the behavior of neoplastic and normal lung cells.

Peritumoral and intratumoral macrophages are associated with human and mouse lung cancer The mouse model allows manipulation of the macrophage population to experimentally evaluate its contribution to tumor growth. Genetic and pharmacologic strategies also permit testing the invol vement of specific inflammatory mediators in tumor progression. Among those endogenous mediators thus identified are interleukin (IL)-10, glucocorticoids, prostacyclin, nitric oxide, and surfactant apoprotein D (SP-D); serum SP-D levels are a useful biomarker to monitor tumor growth rate. The importance of understanding the mutually antagonistic roles of individual prostaglandins downstream from cycloxygenase (COX) and how this affects the efficacy of COX-inhibitory drugs is discussed. Promising drug candidates include synthetic glucocorticoids such as budesonide and the sulfone derivative of sulindac, apotosyn.

Pre-mRNA processing is an important mechanism for globally modifying cellular protein composition during tumorigenesis. To understand this process during lung cancer, expression of two key pre-mRNA alternative splicing factors was compared in a mouse model of early lung carcinogenesis and during regenerative growth following reversible lung injury. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and alternative splicing factor/splicing factor 2 (ASF/SF2) act antagonistically to modulate splice site selection. Both hnRNP A1 and ASF/SF2 contents rose in adenomas and during injury-induced hyperplasia compared to control lungs, as measured by immunoblotting. While both proteins increased similarly during compensatory hyperplasia, hnRNP A1 increased to a much greater extent than ASF/SF2 in tumors, resulting in a 6-fold increase of the hnRNP A1 to ASF/SF2 ratio. Immunohistochemical analysis showed that hnRNP A1 localized exclusively within tumor nuclei, while ASF/SF2 appeared in cytoplasm and/or nuclei, depending on the growth pattern of the tumor cells. We also demonstrated cancer-associated changes in the pre-mRNA alternative splicing of CD44, a membrane glycoprotein involved in cell-cell and cell-extracellular matrix interactions. hnRNP A1 and ASF/SF2 expression is thus differentially altered in neoplastic lung cells by mechanisms that do not strictly arise from increased cell division. These changes are influenced by tumor histology and may be associated with production of variant CD44 mRNA isoforms., ((c) 2004 Wiley-Liss, Inc.)

Chronic inflammation predisposes toward many types of cancer. Chronic bronchitis and asthma, for example, heighten the risk of lung cancer. Exactly which inflammatory mediators (e.g., oxidant species and growth factors) and lung wound repair processes (e.g., proangiogenic factors) enhance pulmonary neoplastic development is not clear. One approach to uncover the most relevant biochemical and physiological pathways is to identify genes underlying susceptibilities to inflammation and to cancer development at the same anatomic site. Mice develop lung adenocarcinomas similar in histology, molecular characteristics, and histogenesis to this most common human lung cancer subtype. Over two dozen loci, called Pas or pulmonary adenoma susceptibility, Par or pulmonary adenoma resistance, and Sluc or susceptibility to lung cancer genes, regulate differential lung tumor susceptibility among inbred mouse strains as assigned by QTL (quantitative trait locus) mapping. Chromosomal sites that determine responsiveness to proinflammatory pneumotoxicants such as ozone (O3), particulates, and hyperoxia have also been mapped in mice. For example, susceptibility QTLs have been identified on chromosomes 17 and 11 for O3-induced inflammation (Inf1, Inf2), O3-induced acute lung injury (Aliq3, Aliq1), and sulfate-associated particulates. Sites within the human and mouse genomes for asthma and COPD phenotypes have also been delineated. It is of great interest that several susceptibility loci for mouse lung neoplasia also contain susceptibility genes for toxicant-induced lung injury and inflammation and are homologous to several human asthma loci. These QTLs are described herein, candidate genes are suggested within these sites, and experimental evidence that inflammation enhances lung tumor development is provided.

Activating mutations in Ras oncoproteins represent attractive targets for cancer immunotherapy, but few vectors capable of generating immune responses required for tumor killing without vector neutralization have been described. Whole recombinant yeast heterologously expressing mammalian mutant Ras proteins were used to immunize mice in a carcinogen-induced lung tumor model. Therapeutic immunization with the whole recombinant yeast caused complete regression of established Ras mutation-bearing lung tumors in a dose-dependent, antigen-specific manner. In combination with the genomic sequencing of tumors in patients, the yeast-based immunotherapeutic approach could be applied to treat Ras mutation-bearing human cancers.

Epidemiological investigations suggest that chronic lung inflammation increases lung cancer risk. Pharmacologic and genetic evidence in mouse models indicates that lipid mediators released during pulmonary inflammation enhance lung tumor formation. Cytosolic phospholipase A2 (cPLA2) catalyzes arachidonic acid (AA) release from membrane phospholipids. AA can then lead to the synthesis of several classes of lipid mediators, including prostaglandin (PG) biosynthesis through the cyclooxygenase (COX) pathway. We investigated a role for cPLA2 in mouse lung tumorigenesis by using mice genetically deficient in cPLA2. After multiple urethane injections into cPLA2 null mice and wild-type littermates, the number of lung tumors was determined. cPLA2 null mice developed 43% fewer tumors (from 16 +/- 2 to 9 +/- 2 tumors/mouse; P < 0.05) than wild-type littermates. cPLA2, COX-1, COX-2 and microsomal prostaglandin E2 synthase (mPGES), examined by immunohistochemistry, are present in alveolar and bronchiolar epithelia and in alveolar macrophages in lungs from naive mice and tumor-bearing mice. Tumors express higher levels of each of these four enzymes than control lungs, as determined by immunoblotting. No differences were detected in the contents of COX-1, COX-2 and mPGES between wild-type and cPLA2 null mice. Although the steady-state levels of prostaglandin E2 and prostaglandin I2 in lung tissue extracts prepared from wild-type or cPLA2 (-/-) mice were not significantly different, both prostaglandins markedly increased in tumors from wild-type mice, an increase that was significantly blunted in tumors from cPLA2 (-/-) mice. These results demonstrate a role for cPLA2 in mouse lung tumorigenesis that may be mediated by decreased prostaglandin synthesis.

Epidemiologic evidence suggests that pulmonary diseases with a prominent chronic inflammatory component elevate lung cancer risk. Genetic manipulations of mouse models of lung inflammation and tumorigenesis can be used to investigate this association. The genes encoding pro-inflammatory tumor necrosis factor-alpha (TNFalpha) and antiinflammatory IL-10 cytokines map within quantitative trait loci that regulate susceptibility to lung tumor development in mice; sensitive A/J and resistant C57BL/6J (B6) mice have different Tnfa and Il-10 alleles. Genetic ablation studies were performed to examine whether these genes would qualify as candidate tumor modifiers. Tnfa null (-/-) mice on a B6 background and B6.129 Il-10(-/-) mice were intercrossed with A/J mice and subjected to urethane carcinogenesis; lung tumor multiplicity was determined 20 weeks later. In the absence of one copy of Tnfa, tumor number. Male Il-10(+/+) mice developed more tumors than did female mice (P < 0.001), absence of one copy of Il-10 raised tumor number in female mice to that observed in +/+ males, but no change in multiplicity occurred in Il-10 hemizygous males. Thus, a deficit of pro-inflammatory TNFalpha decreased the number of tumors, whereas diminished gene copy number of anti-inflammatory IL-10 increased tumorigenesis; manifestation of an effect of Il-10 haploinsufficiency is gender dependent. These studies support a role for inflammation in lung cancer susceptibility., (Copyright 2003 Wiley-Liss, Inc.)

Most murine lung tumors are composed of differentiated epithelial cells. We have reported previously that surfactant protein (SP)-D is expressed in urethane-induced tumors. Serum levels of SP-D are increased in patients with interstitial lung disease and acute respiratory distress syndrome and in rats with acute lung injury but have not been measured in mice. In this study, we sought to determine whether SP-D could be detected in murine serum and discovered that it was increased in mice bearing lung tumors. Serum SP-D concentration was 5.0 +/- 0.2 ng/ml in normal C57BL/6 mice, essentially absent in SP-D nulls, and 63.6 +/- 9.0 ng/ml in SP-D-overexpressing mice. SP-D in serum was verified by immunoblotting. Serum SP-D was increased in mice bearing tumors induced by three different protocols, and the SP-D level correlated with tumor volume. However, in mice with a single adenoma or a few adenomas, SP-D levels were usually within the normal range. SP-D was expressed by the tumor cells, and there was also a field effect whereby type II cells near the tumor expressed more SP-D than type II cells in the remainder of the lung. Serum SP-D was also increased by lung inflammation. In airway inflammation induced by aerosolized ovalbumin in sensitized BALB/c mice, the serum levels were elevated after challenge. In conclusion, serum SP-D concentration is increased in mice bearing lung tumors and generally reflects the tumor burden but is also elevated during lung inflammation.

Murine pulmonary adenomas progress to malignancy with many similarities to human pulmonary adenocarcinoma, the most common form of lung cancer. Inbred mice vary in their susceptibility to lung tumor development, and induced genetic modifications are a powerful tool for understanding this susceptibility. Many transgenic and null mutations relevant to lung cancer pathogenesis were derived on the highly resistant C57BL/B6 (B6) background. Since the inability to reliably induce lung tumors in B6 mice limits these studies, we systematically examined several carcinogenesis protocols in B6 mice. Ten weekly ethyl carbamate (EC) doses caused a nearly 100% lung tumor incidence with a tumor multiplicity >2; multiple EC dosing is thus an alternative to the time-consuming transfer of transgenes and null mutations to susceptible backgrounds.

In the present study, we used newly developed F(11) generation mouse advanced intercross lines (AIL) to fine map Pas1-3 quantitative trait loci (QTL). The (A/J x C57BL/6) F(11) AIL mouse population was created by crossing lung tumor-resistant C57BL/6 mice with lung tumor-susceptible A/J mice. By selectively genotyping 30% of the population, we have confirmed the Pas1 QTL and narrowed it to an interval of approximately 1.0 cM or 1.3 Mb in the vicinity of the Kras2 gene. The Pas2 QTL was detected by both ANOVA and regression analysis but not by MapMaker EXP/QTL software. In addition, an interaction between the Pas1 and Pas2 QTLs was revealed. However, the Pas3 QTL was not confirmed in this study. It was either lost during the development of the AIL or too weak to be detected using AIL. The Pas1 locus is now sufficiently fine-mapped that candidate gene(s) for the Pas1 locus can be characterized by positional cloning. In this study, all 27 of the known or predicted genes located in the Pas1 candidate region were characterized as possible candidate Pas genes. Six genes were selected for additional analyses because of their relevant function in tumorigenesis or allelic changes between A/J and C57BL/6 mice. The Lrmp gene bears amino acid polymorphisms among various mouse strains that are highly correlated with the Pas1 allele status. The Pas1c1 gene (RIKEN Ak016641), encoding an intermediate filament tail domain-containing protein, produces alternatively spliced transcripts across inbred strains of mice, and its splicing pattern cosegregates with the Pas1 allele. The genetic and expression data support these two genes as strong candidates for the Pas1 locus. Of the other four genes (Eca39, RIKEN Ak015530, mHoj-1, and Krag), no functional polymorphisms or differential gene expression were found in Eca39, mHoj-1, and Krag between lung tumor-susceptible and -resistant strains. The Ak015530 carries an amino acid polymorphism, but this polymorphism does not cosegregate with mouse lung tumor susceptibility. Thus, these 4 genes are less likely candidates for the Pas1 locus.

In order to examine how tumorigenicity is abrogated by gap junctional intercellular communication (GJIC), protein expression was analyzed in four related mouse lung epithelial cell lines that vary in their GJIC status and neoplastic potential. Since alterations in protein expression underlie neoplastic behavior, this proteomic analysis provides insights into the molecular pathogenesis of lung cancer. E10, an immortalized but non-tumorigenic cell line derived from alveolar type II pneumocytes, has functional GJIC. E9, a spontaneous transformant of E10, is GJIC-deficient and is tumorigenic upon injection into a syngeneic mouse. Stable transfection of E9 with Gja1, the gene for the gap junctional protein, connexin 43, re-established GJIC and rendered this line (designated E9-2) non-tumorigenic; the vector transfection control line, E9-41, remains tumorigenic. Proteins extracted from these cell lines were separated by two-dimensional electrophoresis (2DE) and visualized by Coomassie blue staining. We consistently observed differential expression of 27 proteins between E10 and E9 and identified 11 of these by peptide mass mapping. The functions of these proteins include stress response, cytoskeletal structure, signal transduction, apoptosis, immune response, pre-mRNA processing, and carbohydrate metabolism. Gja1 transfection affected the concentrations of four of these proteins, viz. PDI, alpha-enolase, aldolase A, and gelsolin-like protein. PDI concentration was most profoundly affected; E10 cells contain twice as much PDI as E9, and PDI was restored to E10-like levels in the E9-2 transfectant line while remaining at E9-like levels in the vector control E9-41 cells. An association between connexin 43 and PDI expression was also observed in a second set of independently derived type II cell lines. The PDI superfamily has multiple cellular roles including chaperoning assembled glycoproteins, regulating the activities of transcription factors, and regulating disulfide bond formation.

A model system to investigate the promotion phase of pulmonary carcinogenesis involves chronic exposure of carcinogen-initiated mice to the food additive, butylated hydroxytoluene (BHT). Previous studies strongly suggested that this activity is due to the cytochrome p450-catalyzed formation of quinone methides 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and 6-tert-butyl-2-(1',1'-dimethyl-2'-hydroxy)ethyl-4-methylenecyclohexa-2,5-dienone (BHTOH-QM). The effects of these electrophiles on non-tumorigenic C10 and E10 epithelial cell lines derived from a normal mouse lung explant were compared with effects on their corresponding neoplastic siblings, the A5 and E9 spontaneous transformants, respectively. The tumorigenic cells were more resistant to cell killing, with LC(50) values of 165-180 microM for BHT-QM and 12-22 microM for BHTOH-QM, versus LC(50) values in the non-tumorigenic cells of 105-118 microM and 5.0-6.0 microM, respectively. Constitutive glutathione (GSH) concentrations were 12-20 nmol/10(6) cells, and BHT-QM toxicity was enhanced >2-fold by depleting GSH with buthionine sulfoximine (BSO). Formation of the GSH conjugate of BHT-QM accounted for a substantial fraction of the cellular GSH lost by quinone methide exposure. Enhanced lipid peroxidation and superoxide formation occurred in all cell lines treated with BHT-QM, but both tumorigenic lines contained higher levels of GSH S-transferase and superoxide dismutase (SOD) activities. These data suggest the possibility that BHT-derived quinone methides may exert their promoting effects by inducing oxidative stress; such stress is better tolerated by tumorigenic cells, which have higher levels of antioxidant enzymes. Normal cells are destroyed more readily which allows neoplastic cells to expand their proliferation.

Studies using normal or neoplastically transformed established mouse lung epithelial cell lines revealed a reduction in gap junctional, intercellular communication (GJIC) with transformation. To determine the stage in tumor development at which GJIC is interrupted, we used the well-established model of lung tumors induced in strain A/J mice by urethane. In this system, tumor development follows a well-characterized pattern; hyperplasias, adenomas, and carcinomas are manifested at approximately 8, 16, and 40 weeks after urethane treatment, respectively. GJIC levels were examined using a novel technique where cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse that opens transient pores on the plasma membrane is applied in the presence of the fluorescent dye, Lucifer yellow, causing dye penetration into cells growing on the conductive part of the slide. Migration of the dye through gap junctions to nonelectroporated cells growing on the nonconductive area is then microscopically observed under fluorescence illumination. Unexpectedly, primary cells cultured from urethane-induced tumors, even late stage carcinomas, possessed extensive GJIC immediately upon isolation. Upon passage for several months however, these cells lost GJIC. These results suggest that the molecular changes that lead to the formation of the tumor in vivo are not sufficient to interrupt gap junctions. Propagation of tumor cells in culture induces additional alterations that can lead to gap junction closure.

Inducible nitric oxide synthase (iNOS) content is elevated in human lung adenocarcinomas, and lung cancer patients exhale more nitric oxide (NO) than healthy individuals. The mechanism of this association of chronically elevated NO with tumorigenesis has not been defined. We investigated the role of iNOS in murine lung tumorigenesis, a model of human lung adenocarcinoma, using wild-type (+/+) and iNOS (-/-) mice. Genetic disruption of iNOS decreased urethane-induced lung tumor multiplicity by 80% (P < 0.0001). iNOS protein was expressed in lung tumors growing in wild-type mice and bronchiolar Clara cells isolated from normal mouse lungs, but was undetectable in whole lung extracts by immunoblotting. Because NO regulates vascular endothelial growth factor (VEGF) expression in other systems, we examined the effect of iNOS deficiency on VEGF protein concentration in mouse lung tumors. VEGF concentration was 54% lower in lung tumors isolated from iNOS (-/-) mice versus controls, implying that NO modulates angiogenesis in these tumors. Lung tumors also have elevated levels of cyclooxygenase (COX) -1 and COX-2 contents relative to normal lungs, but iNOS deficiency did not change COX expression in the tumors. Chronic inflammation predisposes mice to lung tumorigenesis; accordingly, we examined whether butylated hydroxytoluene-induced chronic lung inflammation was influenced by iNOS deficiency. Butylated hydroxytoluene-induced alveolar macrophage infiltration was unaffected by iNOS (-/-) status, suggesting that although NO is a critical mediator of mouse lung tumorigenesis, it is not essential in this model of lung inflammation. The substantial (80%) reduction in lung tumor multiplicity in iNOS (-/-) mice strongly supports examining iNOS-specific inhibitors as potential lung cancer chemopreventive agents.