Lori D. Dwyer-Nield

Mouse lung epithelial cell lines--tools for the study of differentiation and the neoplastic phenotype.

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 In addition to collecting this information in a single place for the first time, we describe previously unpublished apoptosis features of some of these lines.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.

Differential Expression and Localization of the mRNA Binding Proteins, AU‐Rich Element mRNA Binding Protein (AUF1) and Hu Antigen R (HuR), in Neoplastic Lung Tissue

Modulation of gene expression at the level of mRNA stability has emerged as an important regulatory paradigm. In this context, differential expression of numerous mRNAs in normal versus neoplastic tissues has been described. Altered expression of these genes, at least in part, has been demonstrated to be at the level of mRNA stability. Two ubiquitously expressed mRNA binding proteins have recently been implicated in the stabilization (Hu antigen R/HuR) or destabilization (AU‐rich element mRNA binding protein [AUF1]/heterogeneous nuclear ribonucleoprotein D) of target mRNAs. Further, their functional activity appears to require cytoplasmic localization. In the present study, we demonstrate a strong correlation between increased cytoplasmic expression of both AUF1 and HuR with urethane‐induced neoplasia and with butylated hydroxytoluene–induced compensatory hyperplasia in mouse lung tissue. In addition, when compared with slower growing cells, rapidly growing neoplastic lung epithelial cell lines expressed a consistently higher abundance of both AUF1 and HuR proteins. Moreover, in nontumorigenic cell lines, both AUF1 and HuR protein abundance decreased with confluence and growth arrest. In contrast, in spontaneous transformants, AUF1 and HuR abundance was unaffected by changes in cell density. We suggest that growth‐regulated alterations in AUF1 and HuR abundance may have pleiotropic effects on the expression of a number of highly regulated mRNAs and that this significantly impacts the onset, maintenance, and progression of the neoplastic phenotype. Mol. Carcinog. 28:76–83, 2000.

Growth inhibition in G1 and altered expression of cyclin D1 and p27kip-1after forced connexin expression in lung and liver carcinoma cells

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.

Differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogen-induced chronic lung inflammation

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‐γ and IL‐4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN‐γ 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‐γ 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.

Growth inhibition and regression of lung tumors by silibinin: modulation of angiogenesis by macrophage-associated cytokines and nuclear factor-kappaB and signal transducers and activators of transcription 3.

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.

Mutation-Selective Tumor Remission with Ras-Targeted, Whole Yeast-Based Immunotherapy

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.

The lung tumor promoter, butylated hydroxytoluene (BHT), causes chronic inflammation in promotion-sensitive BALB/cByJ mice but not in promotion-resistant CXB4 mice.

An inflammatory response accompanies the reversible pneumotoxicity caused by butylated hydroxytoluene (BHT) administration to mice. Lung tumor formation is promoted by BHT administration following an initiating agent in BALB/cByJ mice, but not in CXB4 mice. To assess the contribution of inflammation to this differential susceptibility, we quantitatively characterized inflammation after one 150 mg/kg body weight, followed by three weekly 200 mg/kg ip injections of BHT into male mice of both strains. This examination included inflammatory cell infiltrate and protein contents in bronchoalveolar lavage (BAL) fluid, cyclooxygenase (COX)-1 and COX-2 expression in lung extracts, and PGE(2) and PGI(2) production by isolated bronchiolar Clara cells. BAL macrophage and lymphocyte numbers increased in BALB mice (P<0.0007 and 0.02, respectively), as did BAL protein content (P<0.05), COX-1 and COX-2 expression (P<0.05 for each), and PGI(2) production (P<0.05); conversely, these indices were not perturbed by BHT in CXB4 mice. BALB mice fed aspirin (400 mg/kg of chow) for two weeks prior to BHT treatment had reduced inflammatory cell infiltration. Our results support a hypothesis that resistance to BHT-induced inflammation in CXB4 mice accounts, at least in part, for the lack of effect of BHT on lung tumor multiplicity in this strain.

Relative amounts of antagonistic splicing factors, hnRNP A1 and ASF/SF2, change during neoplastic lung growth: implications for pre-mRNA processing.

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.

Butylated hydroxytoluene (BHT) induction of pulmonary inflammation: a role in tumor promotion.

Chronic pulmonary inflammatory diseases predispose towards lung cancer by unknown mechanisms. Butylated hydroxytoluene (BHT) administration to mice causes lung injury and a subsequent inflammatory response, and when administered chronically to certain inbred strains following carcinogen treatment, increases lung tumor multiplicity. We hypothesize that inflammation promotes lung tumor growth in this model system and have begun to examine this hypothesis by assessing inflammatory parameters in inbred strains that vary in their susceptibility to promotion. Positive correlations were found between susceptibilities to tumor promotion and BHT induction of alveolar macrophage and lymphocyte infiltration into alveolar airspaces, and increased vascular permeability (P < .03, P < .04, and P < .005, respectively). The amounts of pulmonary cyclooxygenase (COX)-1 and COX-2 did not strongly correlate with promotion. Because persistent elevation of macrophage content is the hallmark of a chronic inflammatory response, the alveolar macrophage population was depleted by adding chlorine to the drinking water prior to carcinogenesis. This treatment reduced lung tumor multiplicity following 2-stage carcinogenesis (P < .05). These correlations between inflammatory and tumorigenic responses to BHT, along with decreased tumorigenesis after macrophage depletion, are consistent with a role of inflammation in promotion. Inflammatory mediators may provide targets for early diagnosis and chemoprevention.Chronic pulmonary inflammatory diseases predispose towards lung cancer by unknown mechanisms. Butylated hydroxytoluene (BHT) administration to mice causes lung injury and a subsequent inflammatory response, and, when administered chronically to certain inbred strains following carcinogen treatment, increases lung tumor multiplicity. We hypothesize that inflammation promotes lung tumor growth in this model system and have begun to examine this hypothesis by assessing inflammatory parameters in inbred strains that vary in their susceptibility to promotion. Positive correlations were found between susceptibilities to tumor promotion and BHT induction of alveolar macrophage and lymphocyte infiltration into alveolar airspaces, and increased vascular permeability (P <. 03, P <. 04, and P <. 005, respectively).The amounts of pulmonary cyclooxygenase (COX)-1 and COX-2 did not strongly correlate with promotion. Because persistent elevation of macrophage content is the hallmark of a chronic inflammatory response, the alveolar macrophage population was depleted by adding chlorine to the drinking water prior to carcinogenesis. This treatment reduced lung tumor multiplicity following 2-stage carcinogenesis (P <. 05).These correlations between inflammatory and tumorigenic responses to BHT, along with decreased tumorigenesis after macrophage depletion, are consistent with a role of inflammation in promotion. Inflammatory mediators may provide targets for early diagnosis and chemoprevention.

Tumor Progression Stage and Anatomical Site Regulate Tumor-Associated Macrophage and Bone Marrow-Derived Monocyte Polarization

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.