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The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. Hamp;E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

Lung cancer chemoprevention, especially in high-risk former smokers, has great potential to reduce lung cancer incidence and mortality. Thiazolidinediones prevent lung cancer in preclinical studies, and diabetics receiving thiazolidinediones have lower lung cancer rates which led to our double-blind, randomized, phase II placebo-controlled trial of oral pioglitazone in high-risk current or former smokers with sputum cytologic atypia or known endobronchial dysplasia. Bronchoscopy was performed at study entry and after completing 6 months of treatment. Biopsies were histologically scored, and primary endpoint analysis tested worst biopsy scores (Max) between groups; Dysplasia index (DI) and average score (Avg) changes were secondary endpoints. Biopsies also received an inflammation score. The trial accrued 92 subjects (47 pioglitazone, 45 placebo), and 76 completed both bronchoscopies (39 pioglitazone, 37 placebo). Baseline dysplasia was significantly worse for current smokers, and 64% of subjects had mild or greater dysplasia at study entry. Subjects receiving pioglitazone did not exhibit improvement in bronchial dysplasia. Former smokers treated with pioglitazone exhibited a slight improvement in Max, while current smokers exhibited slight worsening. While statistically significant changes in Avg and DI were not observed in the treatment group, former smokers exhibited a slight decrease in both Avg and DI. Negligible Avg and DI changes occurred in current smokers. A trend toward decreased Ki-67 labeling index occurred in former smokers with baseline dysplasia receiving pioglitazone. While pioglitazone did not improve endobronchial histology in this high-risk cohort, specific lesions showed histologic improvement, and further study is needed to better characterize responsive dysplasia.

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Primary Biopsy Analysis

Supplementary Figure Legend from Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-κB and Signal Transducers and Activators of Transcription 3

Supplementary Figure 1 from Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-κB and Signal Transducers and Activators of Transcription 3

Legends for Supplementary Tables 1 and 2

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell–cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2–M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention.Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell–cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971–83. ©2018 AACR.

Supplemental table S1 documents comparability between final groupings of baseline biopsies used for gene expression comparisons between persistent and regressive sites demonstrating comparability in all key histologic and clinical variables with the exception of higher inflammation in the regressive group. Supplemental table S2 describes the number of differentially expressed genes that distinguish the four initial groups of bronchial biopsies including persistent bronchial dysplasia (BD), regressive BD, progressive non-dysplasia (ND) and stable ND. The number of genes differentially expressed between the groups highlight the finding that differences are greater in respect to the outcome of the lesions at a given site versus the degree of dysplasia in the baseline biopsy. Most notably the difference between regressive BD and stable ND, two groups with histologically distinct baseline biopsies show fewer differentially expressed genes than all but one other group comparison including the histologically indistinguishable persistent and regressive BD groups. These findings are consistent with persistent and regressive sites representing biologically distinct lesions. Supplemental table S3 shows airway locations, temporal histologic features of full airways and clinical characteristics associated with each specimen used for gene expression analyses. The table is arranged by persistent versus regressive status and presents specimens in descending order of baseline histologic score within each group. The specimen numbers correspond to those notated in figure 1B of the manuscript. Supplemental figure S1 shows a heat map using differentially expressed genes to demonstrate the rationale for combining groups in the final comparison. The heatmap demonstrates that persistent BD and progressive ND sites, which are combined to represent persistent sites, show similar gene expression profiles, with only cases Group3_051, Group3_086, Group3_113 and Group4_070 showing profiles that demonstrate similarity to the regressive BD/stable ND group. The regressive BD and stable ND, which are combined to represent regressive sites, also show similar gene expression profiles that are opposite to those seen in the persistent site group with only cases Group2_037, Group2_085 and Group2_092 showing profiles that demonstrate similarity to the persistent BD/progressive ND group. Supplemental tables S4 - S6 present full data genelists for genes associated with pathways showing significantly altered activity in comparisons of persistent and regressive BD (supplemental table S3), genes associated with pathways that are altered in relation to increasing histologic score (supplemental table S4) and upstream regulators related to persistence and histologic score (supplemental table S5). Supplemental table S7 shows documents comparability between final groupings of baseline biopsies used for analyses of the composition of inflammatory infiltrates in comparisons between a validational set of persistent and regressive sites. Comparability is demonstrated in all key histologic and clinical variables including mean inflammation score and percent of high inflammation score biopsies between the two groups. A higher mean baseline histologic score was seen in the persistent group. Supplemental figure S2 shows subset analyses of the validational set of persistent and regressive sites with comparison of these groups by tissue compartment (epithelial and stromal) and overall degree of inflammation (supplemental figures S2A and S2B). Supplemental figure S2C shows the changes in expression of genes from the original gene expression array analysis data for those genes associated with polarization of subsets of inflammatory cells. The genes with significant or near significant change provide evidence that the activity of certain subsets of macrophages and T-lymphocytes correlate with persistence or regression of BD. These subsets are further studied in figure 5 of the main text.

The transmembrane receptor Frizzled 9 (FZD9) is important for fetal neurologic and bone development through both canonical and non-canonical WNT/FZD signaling. In the adult lung, however, Fzd9 helps to maintain a normal epithelium by signaling through peroxisome proliferator activated receptor γ (PPARγ). The effect of FZD9 loss on normal lung epithelial cells and regulators of its expression in the lung are unknown. We knocked down FZD9 in human bronchial epithelial cell (HBEC) lines and found that downstream EMT targets and PPARγ activity are altered. We used a FZD9-/- mouse in the urethane lung adenocarcinoma model and found FZD9-/- adenomas had more proliferation, increased EMT signaling, decreased activation of PPARγ, increased expression of lung cancer associated genes, increased transformed growth, and increased potential for invasive behavior. We identified PPARγ as a transcriptional regulator of FZD9. We also demonstrated that extended cigarette smoke exposure in HBEC leads to decreased FZD9 expression, decreased activation of PPARγ, and increased transformed growth, and found that higher exposure to cigarette smoke in human lungs leads to decreased FZD9 expression. These results provide evidence for the role of FZD9 in lung epithelial maintenance and in smoking related malignant transformation. We identified the first transcriptional regulator of FZD9 in the lung and found FZD9 negative lesions are more dangerous. Loss of FZD9 creates a permissive environment for development of premalignant lung lesions, making it a potential target for intervention.

Prevention of premalignant lesion progression is a promising approach to reducing lung cancer burden in high-risk populations. Substantial preclinical and clinical evidence has demonstrated efficacy of the prostacyclin analogue iloprost for lung cancer chemoprevention. Iloprost activates peroxisome proliferator-activated receptor gamma (PPARG) to initiate chemopreventive signaling and

Lung cancer chemoprevention, especially in high-risk former smokers, has great potential to reduce lung cancer incidence and mortality. Thiazolidinediones prevent lung cancer in preclinical studies, and diabetics receiving thiazolidinediones have lower lung cancer rates which led to our double-blind, randomized, phase II placebo-controlled trial of oral pioglitazone in high-risk current or former smokers with sputum cytologic atypia or known endobronchial dysplasia. Bronchoscopy was performed at study entry and after completing 6 months of treatment. Biopsies were histologically scored, and primary endpoint analysis tested worst biopsy scores (Max) between groups; Dysplasia index (DI) and average score (Avg) changes were secondary endpoints. Biopsies also received an inflammation score. The trial accrued 92 subjects (47 pioglitazone, 45 placebo), and 76 completed both bronchoscopies (39 pioglitazone, 37 placebo). Baseline dysplasia was significantly worse for current smokers, and 64% of subjects had mild or greater dysplasia at study entry. Subjects receiving pioglitazone did not exhibit improvement in bronchial dysplasia. Former smokers treated with pioglitazone exhibited a slight improvement in Max, while current smokers exhibited slight worsening. While statistically significant changes in Avg and DI were not observed in the treatment group, former smokers exhibited a slight decrease in both Avg and DI. Negligible Avg and DI changes occurred in current smokers. A trend toward decreased Ki-67 labeling index occurred in former smokers with baseline dysplasia receiving pioglitazone. While pioglitazone did not improve endobronchial histology in this high-risk cohort, specific lesions showed histologic improvement, and further study is needed to better characterize responsive dysplasia.

Lung cancer is one of the deadliest types of cancer and as such requires disease models that are useful for identification of novel pathways for biomarkers as well as to test therapeutic agents. Adenocarcinoma (ADC), the most prevalent type of lung cancer, is a subtype of non-small cell lung carcinoma (NSCLC) and a disease driven mainly by smoking. However, it is also the most common subtype of lung cancer found in non-smokers with environmental exposures. Chemically driven models of lung cancer, also called primary models of lung cancer, are important because they do not overexpress or delete oncogenes or tumor suppressor genes, respectively, to increase oncogenesis. Instead these models test tumor development without forcing a specific pathway (i.e., Kras). The primary focus of this chapter is to discuss a well-established 2-stage mouse model of lung adenocarcinomas. The initiator (3-methylcholanthrene, MCA) does not elicit many, if any, tumors if not followed by exposure to the tumor promoter (butylated hydroxytoluene, BHT). In sensitive strains, such as A/J, FVB, and BALB, significantly greater numbers of tumors develop following the MCA/BHT protocol compared to MCA alone. BHT does not elicit tumors on its own; it is a non-genotoxic carcinogen and promoter. In these sensitive strains, promotion is also associated with inflammation characterized by infiltrating macrophages, lymphocytes, and neutrophils, and other inflammatory cell types in addition to increases in total protein content reflective of lung hyperpermeability. This 2-stage model is a useful tool to identify unique promotion specific events to then test in future intervention studies.

Lung cancer is one of the deadliest types of cancer and as such requires disease models that are useful for identification of novel pathways for biomarkers as well as to test therapeutic agents. Adenocarcinoma (ADC), the most prevalent type of lung cancer, is a subtype of non-small cell lung carcinoma (NSCLC) and a disease driven mainly by smoking. However, it is also the most common subtype of lung cancer found in non-smokers with environmental exposures. Chemically driven models of lung cancer, also called primary models of lung cancer, are important because they do not overexpress or delete oncogenes or tumor suppressor genes, respectively, to increase oncogenesis. Instead these models test tumor development without forcing a specific pathway (i.e., Kras). The primary focus of this chapter is to discuss a well-established 2-stage mouse model of lung adenocarcinomas. The initiator (3-methylcholanthrene, MCA) does not elicit many, if any, tumors if not followed by exposure to the tumor promoter (butylated hydroxytoluene, BHT). In sensitive strains, such as A/J, FVB, and BALB, significantly greater numbers of tumors develop following the MCA/BHT protocol compared to MCA alone. BHT does not elicit tumors on its own; it is a non-genotoxic carcinogen and promoter. In these sensitive strains, promotion is also associated with inflammation characterized by infiltrating macrophages, lymphocytes, and neutrophils, and other inflammatory cell types in addition to increases in total protein content reflective of lung hyperpermeability. This 2-stage model is a useful tool to identify unique promotion specific events to then test in future intervention studies.

Tobacco smoke-induced squamous cell lung cancer (SCC) develops from endobronchial dysplastic lesions that progress to invasive disease. A reproducible murine model recapitulating histologic progression observed in current and former smokers will advance testing of new preventive and therapeutic strategies. Previous studies show that prolonged topical application of N-nitroso-tris-chloroethylurea (NTCU) generates a range of airway lesions in sensitive mice similar to those induced by chronic tobacco smoke exposure in humans. To improve the current NTCU model and better align it with human disease, NTCU was applied to mice twice weekly for 4–5 weeks followed by a recovery period before cigarette smoke (CS) or ambient air (control) exposure for an additional 3–6 weeks. Despite the short time course, the addition of CS led to significantly more premalignant lesions (PML; 2.6 vs. 0.5; P < 0.02) and resulted in fewer alveolar macrophages (52,000 macrophages/mL BALF vs. 68,000; P < 0.05) compared with control mice. This improved NTCU + CS model is the first murine SCC model to incorporate tobacco smoke and is more amenable to preclinical studies because of the increased number of PML, decreased number of mice required, and reduced time needed for PML development.

Polycyclic aromatic hydrocarbons (PAHs), prevalent contaminants in our environment, in many occupations, and in first and second-hand smoke, pose significant adverse health effects. Most research focused on the genotoxic high molecular weight PAHs (e.g., benzo[a]pyrene), however, the nongenotoxic low molecular weight (LMW) PAHs are emerging as potential co-carcinogens and tumor promoters known to dysregulate gap junctional intercellular communication (GJIC), activate mitogen activated protein kinase pathways, and induce the release of inflammatory mediators. We hypothesize that inflammatory mediators resulting from LMW PAH exposure in mouse lung epithelial cell lines are involved in the dysregulation of GJIC. We used mouse lung epithelial cell lines and an alveolar macrophage cell line in the presence of a binary PAH mixture (1:1 ratio of fluoranthene and 1-methylanthracene, PAH mixture). Parthenolide, a pan-inflammation inhibitor, reversed the PAH-induced inhibition of GJIC, the decreased CX43 expression, and the induction of KC and TNF. To further determine the direct role of a cytokine in regulating GJIC, recombinant TNF (rTNF) was used to inhibit GJIC and this response was further enhanced in the presence of the PAH mixture. Collectively, these findings support a role for inflammation in regulating GJIC and the potential to target these early stage cancer pathways for therapeutics.

Prostacyclin (prostaglandin I2, PGI2) overproduction in FVB/N mice prevents the formation of carcinogen and tobacco smoke–induced adenomas, and administration of the oral prostacyclin analogue iloprost to wild-type mice also prevented carcinogen-induced mouse lung adenoma formation. Former smokers taking oral iloprost showed improved bronchial dysplasia histology compared with placebo. Next-generation oral prostacyclin analogues, like treprostinil, were developed for the treatment of pulmonary arterial hypertension (PAH). On the basis of our prior studies with iloprost, we performed preclinical studies examining the ability of treprostinil to chemoprevent urethane-induced murine lung adenocarcinoma. We determined the MTD in chow (prior studies had delivered treprostinil by gavage), and this dose produced serum levels in the experimental animals similar to those found in PAH patients treated with treprostinil. We then examined the chemopreventive efficacy of treprostinil exposure initiated both before (1 week) and after (6 weeks) urethane exposure to better model chemoprevention studies conducted in former smokers. Neither of these dosing strategies prevented murine lung cancer; however, we did detect changes in pulmonary inflammatory cell infiltrate and expression of CXCR4 (a chemokine receptor previously shown to increase in response to treprostinil exposure) in tumor-bearing, treprostinil-treated animals, indicating that the drug was bioavailable. One potential explanation stems from iloprost and treprostinil differentially activating cell surface prostaglandin receptors and intracellular peroxisome proliferator-activated receptors. When murine lung tumor cells were treated with treprostinil, their proliferation rate increased; in contrast, iloprost had no effect on proliferation. Future investigations comparing these two agents will provide insight into iloprost's chemopreventive mechanisms. Cancer Prev Res; 10(11); 671–9. ©2017 AACR.

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. © 2011 Wiley Periodicals, Inc.

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.

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-α (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-1α 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-κB (NF-κB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-κB (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-1α, NF-κB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.

The classical Cheng–Minkowycz study of convection past a vertical plate embedded in a porous medium has been extended to the case of a bidisperse porous medium (BDPM). The boundary layer analysis leads to expressions for the velocity and temperature fields in terms of a geometrical parameter, an inter-phase momentum transfer parameter, a thermal diffusivity ratio, a permeability ratio, a thermal conductivity ratio, and an inter-phase heat transfer parameter. For the leading edge region, and for an inner layer, a similarity solution is obtained numerically. This involves the first four parameters, each of which is a characteristic of the BDPM.

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.

The problem of fully developed forced convection in a parallel-plates channel filled with a saturated porous medium (involving a Brinkman model for the momentum equation), with the effect of viscous dissipation (involving a Brinkman number), is discussed. Some general matters relating to the possibility of fully developed convection are also discussed.

The study of heat transfer in rectangular passages with prescribed wall heat flux is of practical interest. These passages could be open or filled with saturated porous materials. A solution that uses the Green's function can accommodate the inclusion of heat flux over the entire surface area or over isolated sections of the boundary. Also, this solution permits the inclusion of frictional heating. Two different boundary conditions are considered. The computed heat transfer coefficients show that the thermally fully developed condition may not be attainable in practical applications for very narrow passages with prescribed wall heat flux.

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 A2(cPLA2) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA2in this response was examined by determining cPLA2expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA2genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA2null 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 cPLA2null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA2is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.

A two-velocity two-temperature model for bi-dispersed porous media is formulated. Using the model, an analytic solution is obtained for the problem of forced convection in a channel between parallel plane walls that are held either at uniform temperature or uniform heat flux. In each case, Nusselt number values are given as functions of a conductivity ratio, a velocity ratio, a volume fraction, and an internal heat exchange parameter.

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. © 2004 Wiley-Liss, Inc.

Forced convection in a plane channel filled with a saturated bi-disperse porous medium, coupled with conduction in plane slabs bounding the channel, is investigated analytically on the basis of a two-velocity, two-temperature model. It is found that the effect of the finite thermal resistance due to the slabs is to reduce both the heat transfer to the porous medium and the degree of local thermal non-equilibrium. An increase in value of the Peclet number leads to a decrease in the rate of exponential decay in the downstream direction but does not affect the value of a suitably defined Nusselt number. The dependence of Nusselt number on Biot number associated with the boundary slabs, the interphase heat exchange parameter, the interphase thermal conductivity ratio, the interphase effective permeability ratio, and the macroscopic void fraction, is investigated.

Fully developed forced convection in a parallel plate channel filled by a saturated porous medium, with walls held either at uniform temperature or at uniform heat flux, with the effects of viscous dissipation and flow work included, is treated analytically. The Brinkman model is employed. The analysis leads to expressions for the Nusselt number, as a function of the Darcy number and Brinkman number.

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.

Cyclooxygenase (COX) enzyme expression is elevated in human and rodent lung tumors, and non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin reduce lung tumor formation in mice. These observations, along with the well-characterized protection that NSAID treatment engenders for colon cancer, have prompted clinical trials testing whether celecoxib, a COX-2-specific inhibitor, can prevent lung cancer development in populations at high risk. Protection by celecoxib in murine models of pulmonary inflammation and lung tumorigenesis has not yet been evaluated, however, and we now report such studies. Chronic administration of butylated hydroxytoluene (BHT) to mice stimulates pulmonary inflammation characterized by vascular leakage and macrophage infiltration into the air spaces, increased PGE2 production, and translocation of 5-lipoxygenase (5-LO) from the cytosol to the particulate fraction. Dietary celecoxib limited macrophage infiltration, abrogated PGE2 production and reduced particulate 5-LO content. Celecoxib and aspirin were ineffective at preventing lung tumorigenesis in a two-stage carcinogenesis protocol in which 3-methylcholanthrene administration is followed by chronic BHT. Celecoxib also did not reduce the multiplicity of lung tumors after induction by urethane; lung tumors in celecoxib-treated mice were larger than those in mice that did not receive celecoxib. Tumors induced in celecoxib-fed mice contained 60% less PGE2 than tumors in mice fed control diets, so reducing lung PGE2 levels was insufficient to prevent lung tumor formation. As the production of eicosanoids in addition to PGE2 is also inhibited by celecoxib, and as celecoxib has COX-independent interactions, its effects on tumor formation may vary in different organ systems.

Acute pulmonary toxicity and tumor promotion by the food additive 2,6-di-tert-butyl-4-methylphenol (BHT) in mice are well documented. These effects have been attributed to either of two quinone methides, 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) formed through direct oxidation of BHT by pulmonary cytochrome P450 or a quinone methide formed by hydroxylation of a tert-butyl group of BHT (to form BHTOH) followed by oxidation of this metabolite to BHTOH-QM. BHTOH-QM is a more reactive electrophile compared to BHT-QM due to intramolecular interactions of the side-chain hydroxyl with the carbonyl oxygen. To further examine this bioactivation pathway, an analogue of BHTOH was prepared, 2-tert-butyl-6-(1'-hydroxy-1'-methyl)ethyl-4-methylphenol (BPPOH), that is structurally very similar to BHTOH but forms a quinone methide (BPPOH-QM) capable of more efficient intramolecular hydrogen bonding and, therefore, higher electrophilicity than BHTOH-QM. BPPOH-QM was synthesized and its reactivity with water, methanol, and glutathione determined to be >10-fold higher than that of BHTOH-QM. The conversions of BPPOH and BHTOH to quinone methides in lung microsomes from male BALB/cByJ mice were quantitatively similar, but in vivo the former was pneumotoxic at one-half of the dose required for the latter and one-eighth of the dose required for BHT, as determined by increased lung weight:body weight ratios following a single i.p. injection. Similar differences were found in the doses of BHT, BHTOH, or BPPOH required for tumor promotion after a single initiating dose of 3-methylcholanthrene followed by three weekly injections of the phenol. The downregulaton of calpain II, previously shown to accompany lung tumor promotion by BHT and BHTOH, also occurred with BPPOH. The correlation between biologic activities of these phenols and the reactivities of their corresponding quinone methides provides additional support for the role of BHTOH-QM as the principal metabolite responsible for the effects of BHT on mouse lung.

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIα mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Cα catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.

Gap junctions provide direct pathways for the exchange of molecules and ions between neighboring cells, a process known as gap junctional intercellular communication (GJIC). This GJIC is important for homeostasis and regulation of mitosis, differentiation, and apoptosis. Gap junctions are present in lung airway and alveolar epithelial cells and, in addition to the above roles, might coordinate ciliary beating and surfactant secretion. GJIC is decreased in human and mouse lung carcinoma cells because of reduced expression of the gap junction protein, connexin43 (Cx43), and defects in signal transduction pathways that mediate Cx43 function. This reduced GJIC is important in the behavior of lung carcinoma cells because forced expression of Cx43 in lung carcinoma cells inhibits their growth and tumorigenicity. In this report, we summarize our studies on the role of GJIC in lung neoplasia.

Strain A/J mice, which are predisposed to experimentally induced asthma and adenocarcinoma, have the lowest pulmonary protein kinase (PK) C activity and content among 22 inbred mouse strains. PKC in neonatal A/J mice is similar to that in other strains, so this difference reflects strain-dependent postnatal regulation. PKC activity is 60% higher in C57BL/6J (B6) than in A/J lungs, and the protein and mRNA concentrations of PKC-α, the major pulmonary PKC isozyme, are two- to threefold higher in B6 mice. These differences result from more than a single gene as assessed in F1, F2, and backcross progeny of B6 and A/J parents. Quantitative trait locus (QTL) analysis of 23 A×B and B×A recombinant inbred strains derived from B6 and A/J progenitors indicates a major locus regulating lung PKC-α content that maps near the Pkcα structural gene on chromosome 11 ( D11MIT333; likelihood ratio statistic = 12.5) and a major locus controlling PKC activity that maps on chromosome 3 ( D3MIT19; likelihood ratio statistic = 15.4). The chromosome 11 QTL responsible for low PKC-α content falls within QTLs for susceptibilities to lung tumorigenesis and ozone-induced toxicity.

The modelling of viscous dissipation in a porous medium saturated by an incompressible fluid is discussed, for the case of Darcy, Forchheimer and Brinkman models. An apparent paradox relating to the effect of inertial effects on viscous dissipation is resolved, and some wider aspects of resistance to flow (concerning quadratic drag and cubic drag) in a porous medium are discussed. Criteria are given for the importance or otherwise of viscous dissipation in various situations.

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G1-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G1-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27kip-1 protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-β, epidermal growth factor receptor, protein kinase C-α, protein kinase A regulatory subunit-Iα, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G1 growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27kip-1, but not with changes in other growth-related functions. J. Cell. Biochem. 79:347–354, 2000. © 2000 Wiley-Liss, Inc.

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, AIN76A-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 K 3 tumors/ mouse (mean 6 SEM, control diet) to 12.3 K 3 (250), 5.3 K 1 (500) and 2.1 K 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.

Comments are made about limitations of the model proposed by Hennenberg et al. for convection in a porous medium layer driven by the Marangoni effect, and an alternative model for this problem is presented. The predictions of the two models are significantly different.

Several dozen lung epithelial cell lines have been established in culture over the past 20 years from normal lung explants and their spontaneous transformants, and from lung tumors that arose spontaneously or were induced with chemicals, viruses, or oncogenic transgenes. To provide information from which to choose appropriate lines for investigating problems in lung cell biology and pulmonary neoplasia, this review describes the origins of these lines and some of their characteristics. These include growth, morphology, tumorigenicity, ability to metastasize, xenobiotic metabolism, mutational status, signal transducing activities, cytogenetics, ability to form domes, and electric conductance. In addition to collecting this information in a single place for the first time, we describe previously unpublished apoptosis features of some of these lines. An increasing number of investigations are beginning to use these lines and this review contains references into 1997.

Butylated hydroxytoluene (BHT)-induced lung damage in mice is an excellent model system for studying mechanisms of chemically-induced, reversible alveolar injury. Changes in the pulmonary contents of protein kinase C (PKC) and the calcium-dependent protease, calpain, were previously noted during the repair phase following BHT-induced pneumotoxicity. Calpain is believed to initiate PKC down-regulation. PKC-alpha is the major PKC isozyme and calpain II the major calpain isozyme in mouse lung. We have now studied the time course of these enzymatic changes in detail. Pulmonary PKC-alpha concentrations decreased as early as 45 min after an i.p. injection of 200 mg/kg BHT. Calpain II levels rose within the first 40 min after BHT injection, and then declined below control levels. The rapidity of these changes implies a role of these enzymes in mediating the onset of injury. Lung damage and repair, as estimated by measuring the lung weight/body weight ratio, is maximal 6 days after administration of this dose of BHT. The extent of the decreased PKC-alpha and calpain II concentrations at this time was linearly related to the estimated degree of injury based on increased lung weight. This correlation suggests the value of monitoring these enzymes as putative early biomarkers of alveolar injury.