Abstract  The pulmonary adenoma susceptibility 1 (Pas1) locus affects inherited predisposition and resistance to chemically induced lung tumorigenesis in mice. The A/J and C57BL/6J mouse strains carry the susceptibility and resistance allele, respectively. We identified and genotyped 65 polymorphisms in the Pas1 locus region in 29 mouse inbred strains, and delimited the Pas1 locus to a minimal region of 468 kb containing six genes. That region defined a core Pas1 haplotype with 42 tightly linked markers, including intragenic polymorphisms in five genes (Bcat1, Lrmp, Las1, Ghiso, and Kras2) and amino-acid changes in three genes (Lrmp, Las1, Lmna-rs1). In (A/J x C57BL/6J)F1 mouse lung tumors, the Lmna-rs1 gene was completely downregulated, whereas allele-specific downregulation of the C57BL/6J-derived allele was observed at the Las1 gene, suggesting the potential role of these genes in tumor suppression. These results indicate a complex multigenic nature of the Pas1 locus, and point to a functional role for both intronic and exonic polymorphisms of the six genes of the Pas1 haplotype in lung tumor susceptibility.

Abstract  Rodent species and strains show wide variations in susceptibility to lung tumorigenesis. In mice, hierarchical clustering of 29 inbred laboratory strains by pulmonary adenoma susceptibility 1 (Pas1) locus polymorphisms separated the strains into either an A/J- or a C57BL/6J-type Pas1 haplotype. A pooled analysis (including >8500 mice) of studies on spontaneous and chemically induced lung tumorigenesis in these strains revealed a significantly higher risk of spontaneous lung tumors [odds ratio (OR) 12.17; 95% confidence interval (CI) 9.00-16.45] as well as of chemically induced lung tumors (OR 15.14; 95% CI 12.51-18.31) in the A/J-type haplotype. Strain differences were observed with six different carcinogens, suggesting that Pas1 locus activity is carcinogen-independent. Thus, the present meta-analysis indicates a link between the genetic control of spontaneous and chemically induced lung tumor susceptibility in mice. The Pas1 susceptibility allele is frequent in the population of inbred mouse strains, whereas a counterpart appears to be absent or rare in rat and hamster strains. These findings might help in the interpretation of results of rodent carcinogenicity bioassays and assessing the risk of lung carcinogenesis from chemicals.

Abstract  Early detection represents one of the most promising approaches to improving lung cancer survival. To date, no screening strategies have been shown to decrease mortality from the disease. Furthermore, no reliable circulating biomarkers of lung cancer have been identified that allow early diagnosis. With the advent of gene targeting technology, new genetically engineered mouse models of lung cancer closely recapitulate the pathobiology of human disease. These mouse models have enabled novel approaches to early detection, including the identification of cancer-associated serum markers using proteomic technologies and the development of new molecular imaging tools. The application of innovative technologies to accurate mouse models promises to accelerate the discovery of new molecular targets and imaging biomarkers for the early detection of lung cancer.

Abstract  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.

Abstract  Pulmonary adenoma susceptibility 1 (Pas1), the major locus affecting inherited predisposition to lung tumor development in mice, maps near the Kras2 gene. We previously reported a significant association between a KRAS2/RsaI polymorphism and the risk and prognosis of lung adenocarcinoma (ADCA) in the Italian population. In the present case-control study, we examined 269 lung ADCA patients, 121 squamous cell lung carcinoma patients, and 632 healthy individuals (general population controls) in the Japanese population with genetic markers spanning approximately 1200 kb in the KRAS2 region. Allele-specific oligonucleotide hybridization revealed the same KRAS2/RsaI polymorphism associated with risk and prognosis as in Italian lung ADCA patients; the polymorphism was significantly associated with clinical stage (P < 0.001) and survival rate (log rank = 0.0014), confirming the mapping of PAS1 and pointing to the role of this locus in human lung cancer.

Abstract  This report shows the tumor rates of control group animals from selected studies.

Abstract  The probability that a mouse develops a pulmonary tumor, as well as the structure of that tumor, are dependent on several genes. Three pulmonary adenoma susceptibility (pas) genes predispose some inbred strains to develop lung tumors, even in the absence of carcinogen exposure, and cause others to be resistant. One pas gene is K-ras, which may also be overexpressed in these tumors in a mutated form capable of transforming cells. Mice with activated Ha-ras transgenes override the resistant pas alleles and are born with lung cancer. Susceptible strains have a higher turnover rate of alveolar type II and bronchiolar Clara cells, those cells from which lung tumors arise, than more resistant strains. A high precursor cell turnover rate correlates with a propensity to neoplasia in other animal models as well, possibly due to low concentrations of endogenous growth regulatory molecules such as corticosterone and protein kinase C (PKC). Neoplastic lung epithelial cells are relatively resistant to glucocorticoids and have low PKC levels. A set of genes other than the pas genes governs the response to tumor modulation by butylated hydroxytoluene (BHT). The genes that determine whether lung tumor multiplicity is enhanced by chronic BHT exposure may regulate the ability to hydroxylate BHT at a tert-butyl position to form BHT-OH, a metabolite with greater tumor-promoting potency than BHT. Inbred and recombinant inbred strain variations in adenoma growth patterns indicate that another set of genes, which we have designated pah for pulmonary adenoma histogenesis, may determine which cell type becomes neoplastic and whether adenomas will undergo malignant conversion.

Abstract  Methylene chloride is a high production chemical used in a variety of applications resulting in estimated occupational and consumer exposures of at least one million people per day. Results of previously reported chronic evaluations of inhaled methylene chloride indicated that it caused mammary tumors in Fischer 344 rats and neoplasia in the lungs and liver of B6C3F1 mice. Mechanism(s) for methylene chloride-induced carcinogenesis have not been adequately elucidated. In this paper we describe the histologic evaluation of animals at a number of intermittent times for the purposes of assessing the progressive development of liver and lung neoplasia. Additionally, a series of stop-exposure treatments was conducted to evaluate the role of different methylene chloride exposure durations on the induction of hepatic and pulmonary neoplasia in female mice. Inhalation exposure to 2000 p.p.m. methylene chloride for 6 h per day, 5 days per week, for 104 weeks resulted in an 8-fold increase in the incidence of exposed animals having a lung adenoma or carcinoma (63 versus 7.5%; P < 0.01) and a 13-fold increase in the total number of pulmonary adenomas and carcinomas per animal at risk (0.97 versus 0.075; P < 0.01). This exposure also caused a 2.5-fold increase in the incidence of mice having liver tumors (69 versus 27%; P < 0.01) and a 3-fold increase in the total number of hepatic adenomas and carcinomas per animal at risk (1.34 versus 0.46; P < 0.01). Methylene chloride exposure hastened the first appearance of lung tumors (by 1 year) compared to that observed in control animals; chemical-induced and spontaneous liver tumors first occurred simultaneously. A shorter exposure duration was sufficient to attain maximal numbers of lung tumors than that needed for a maximal liver tumor burden. Lung tumor multiplicity was substantially increased by having additional time after cessation of the chemical treatment. This contrasts with the findings in liver, where additional post-exposure latency time did not effect tumor multiplicity compared to that of mice evaluated immediately after cessation of exposure. The incidence of lung alveolar hyperplasia in methylene chloride exposed animals was very low, even in tumor-bearing animals and the hyperplasias were not seen until at least 13 weeks after appearance of adenomas and carcinomas. Thus, the genesis of methylene chloride induced lung tumors in B6C3F1 mice is not preceded by overt cytotoxicity, enhanced cell proliferation nor observed hyperplasia.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract  Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). Identified at the bronchioalveolar duct junction, BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties. Furthermore, BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo. These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma. Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma, this work points to BASCs as the putative cells of origin for this subtype of lung cancer.

Abstract  Cumene is produced in a modified Friedel-Crafts reaction process that uses acidic catalysts to alkylate benzene with propylene. Cumene is the principal chemical used in the production of phenol and acetone. Cumene is used to produce acetophenone, α-methylstyrene, diisopropylbenzene, and dicumylperoxide; as a thinner; as a constituent of some petroleum-based solvents; in gasoline blending, diesel fuel, and high-octane aviation fuel; and as a raw material for peroxides and oxidation catalysts. Because cumene is a good solvent for fats and resins, it has been suggested as a replacement for benzene in many industrial applications. Cumene occurs naturally in petroleum and in a variety of foodstuffs. Cumene was nominated for study by the NIEHS because of its high production volume, presence in gasoline and other fuels, potential for human exposure, and lack of existing carcinogenicity test data. Male and female F344/N rats and B6C3F1 mice were exposed to cumene (greater than 99.9% pure) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat bone marrow, and mouse peripheral blood. 2-WEEK STUDY IN RATS Groups of five male and five female rats were exposed to cumene vapor at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats exposed to 4,000 ppm died on day 1, and two male and three female rats exposed to 2,000 ppm died by day 4. Mean body weights of 2,000 ppm rats were significantly less than those of the chamber controls. Rats exposed to 2,000 ppm that died early were severely lethargic following daily exposure. Liver and kidney weights of all exposed groups were increased. Accumulation of minimal to mild hyaline droplets was observed in the renal tubular cortex of males exposed to concentrations of 250 to 2,000 ppm. 2-WEEK STUDY IN MICE Groups of five male and five female mice were exposed to cumene vapor at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days. All mice exposed to 4,000 ppm died on day 1; all mice exposed to 2,000 ppm died on day 2, and four female mice exposed to 1,000 ppm died by day 4. Mean body weights of all exposed groups were similar to those of the chamber controls. Mice exposed to 2,000 ppm were severely lethargic after the first exposure. The four female mice exposed to 1,000 ppm that died early exhibited signs of lethargy and ataxia. Liver weights, both relative and absolute, were increased in all groups of surviving males and in 250 and 500 ppm female groups. 3-MONTH STUDY IN RATS Groups of 10 male and 10 female rats were exposed to cumene vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Kidney and liver weights of 250 ppm or greater males and liver weights of 1,000 ppm females were significantly greater than those of the chamber controls. There were significant differences between exposed and chamber control females in the relative length of time spent in the estrous stages. The amount of α2u-globulin in the right kidneys was significantly increased in male rats exposed to 125 ppm or greater. The incidences of medullary granular casts in males exposed to 250 ppm or greater were significantly increased. The severities of renal tubule cortex hyaline droplet accumulation and regeneration increased with increasing exposure concentration in male rats. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female mice were exposed to cumene vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Eight 1,000 ppm females died during week 1 of the study. Mean body weights of males exposed to 500 or 1,000 ppm were significantly less than those of the chamber controls. The eight 1,000 ppm female mice that died during the first week of the study exhibited clinical signs of acute toxicity, including lethargy or ataxia. Liver weights of mice exposed to 500 or 1,000 ppm were significantly increased. The weight of the cauda epididymis and the spermatid count were significantly decreased in 1,000 ppm males. 2-YEAR STUDY IN RATS Groups of 50 male and 50 female rats were exposed to cumene vapor at concentrations of 0, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Survival of all exposed groups of rats was similar to that of the chamber controls. Mean body weights of 1,000 ppm females were slightly less than those of the chamber controls during the second year of the study but were similar to the chamber controls at the end of the study. Incidences of adenoma of the respiratory epithelium in the nose occurred with a positive trend in males and were significantly increased in all exposed groups of males and in 250 ppm females. Incidences of hyperplasia of basal cells in the olfactory epithelium in the nose of all exposed groups and hyperplasia of the respiratory epithelium in the nose of all exposed groups of males and 1,000 ppm females were significantly increased. The incidences of renal tubule adenoma in all exposed groups of males, renal tubule carcinoma in 500 and 1,000 ppm males, and renal tubule adenoma or carcinoma (combined) in all exposed groups of males were increased; the difference from chamber controls for the combined incidence was significant at 500 ppm. The incidences of hyperplasia of the renal tubule and transitional epithelium of the renal pelvis in 500 and 1,000 ppm males and mineralization of the renal papilla in all exposed groups of males were significantly greater than those of the chamber controls. The incidence of interstitial cell adenoma (including bilateral) of the testis was significantly increased in 1,000 ppm male rats, and there was a positive trend in the incidences across all groups. 2-YEAR STUDY IN MICE Groups of 50 male and 50 female mice were exposed to cumene vapor at concentrations of 0, 125 (female mice only), 250, 500, or 1,000 (male mice only) ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. An exposure concentration-related decrease in survival occurred in male mice, and the survival of 1,000 ppm males was significantly less than that of the chamber controls. Mean body weights of 1,000 ppm males were generally less than those of the chamber controls after week 8 of the study, and those of 500 ppm females were less from week 28 until week 76 of the study. The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) in all exposed groups of mice occurred with positive trends and were significantly greater than those in the chamber controls. The incidences of alveolar epithelial bronchiole metaplasia and bronchiole hyperplasia were significantly increased in all exposed groups of mice. p53 and K-ras mutations were found in 52% and 87% of lung neoplasms in exposed mice compared to 0% and 14% in the chamber controls, respectively. In female mice, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) occurred with positive trends and were significantly increased in the 500 ppm group. In male mice, there were significant increases in the incidences of eosinophilic foci of the liver. The incidences of hemangiosarcoma in the spleen and of follicular cell adenoma in the thyroid gland were significantly increased in 1,000 ppm male mice. In the nose, the incidences of olfactory epithelium atrophy, basal cell hyperplasia of the olfactory epithelium, atypical hyperplasia of the olfactory epithelium, hyperplasia of olfactory epithelium glands, and suppurative inflammation were generally significantly increased in 500 and 1,000 ppm males and 500 ppm females. The incidence of squamous metaplasia of the respiratory epithelium was significantly increased in 500 ppm females. The incidence of basal cell hyperplasia was also significantly increased in 250 ppm females. The incidences of epithelial hyperplasia of the forestomach in the 500 and 1,000 ppm groups of males and the incidences of ulceration and inflammation of the forestomach in 1,000 ppm males were significantly increased. GENETIC TOXICOLOGY Cumene was not mutagenic in S. typhimurium strain TA97, TA98, TA100, or TA1535, when tested with and without liver S9 activation enzymes. Cumene induced small, but significant, increases in micronucleated polychromatic erythrocytes in bone marrow of male rats treated by intraperitoneal injection. In contrast, no increase in micronucleated erythrocytes was observed in peripheral blood of male or female mice exposed to cumene by inhalation for 3 months. CONCLUSIONS Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of cumene in male F344/N rats based on increased incidences of respiratory epithelial adenoma in the nose and renal tubule adenoma or carcinoma (combined). Increased incidences of interstitial cell adenoma of the testis may have been related to exposure to cumene. There was some evidence of carcinogenic activity of cumene in female F344/N rats based on the incidences of respiratory epithelium adenoma in the nose. There was clear evidence of carcinogenic activity of cumene in male B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. The increased incidences of hemangiosarcoma in the spleen and follicular cell adenoma in the thyroid gland in male mice may have been related to cumene exposure. There was clear evidence of carcinogenic activity of cumene in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Increased incidences of hepatocellular adenoma or carcinoma (combined) in female mice were also considered to be related to exposure to cumene. Exposure of male rats to cumene resulted in nonneoplastic lesions of the kidney characteristic of α2u-globulin accumulation. Exposure to cumene resulted in nonneoplastic lesions in the nose of male and female rats; the lung, nose, liver, and forestomach of male mice; and the lung and nose of female mice.

Abstract  Codon 12 mutations are frequent in the Ki-ras oncogene in human lung adenocarcinomas, but the effects of these alterations have not been well characterized in lung epithelial cells. Murine primary lung tumors derived from peripheral epithelial cells also may present Ki-ras mutations and are useful models for study of early phases of tumor development. One hypothesis is that Ki-ras mutation and/or a Ki-ras p21 increase could enhance Ki-ras p21-GTP and cell-cycle stimulation through raf-1 and extracellularly regulated protein kinases (Erks). We examined lung tumors 1-7 mm in largest dimension initiated in male Swiss mice by N-nitrosodimethylamine for pathologic type, Ki-ras mutations and levels of total Ki-ras p21, Ki-ras p21 bound to GTP, raf-1, Erk1 and Erk2 and their phosphorylated (activated) forms, and proliferating cell nuclear antigen. Total Ki-ras p21 and activated ras-GTP were not significantly greater in tumors than in normal lung or in tumors with versus those without Ki-ras mutations. Carcinomas with Ki-ras mutations were significantly smaller than those without mutations. Carcinomas were significantly larger than adenomas only for tumors without mutations. High levels of Erk2 and correlation of Erk2 amount with ras-GTP were specific characteristics of tumors with Ki-ras mutations. Size of all tumors correlated with ras-GTP but not with proliferating cell nuclear antigen. Raf-1 was expressed mainly in alveolar macrophages in normal lung but was focally upregulated in papillary areas of some tumors. The results indicate that Ki-ras influences the characteristics of lung tumors, but a linear ras-raf-Erk-cell-cycle control sequence does not adequately characterize tumorigenic events in this model. Mol. Carcinog. 28:156-167, 2000.

Abstract  Identifying cells of tumor origin is a fundamental question in tumor biology. Answers to this central question will not only advance our understanding of tumor initiation and progression but also have important therapeutic implications. In this study, we aimed to uncover the cells of origin of lung adenocarcinoma, a major subtype of non-small cell lung cancer. To this end, we developed new mouse models of lung adenocarcinoma that enabled selective manipulation of gene activity in surfactant associated protein C (SPC)-expressing cells, including alveolar type II cells and bronchioalveolar stem cells (BASCs) that reside at the bronchioalveolar duct junction (BADJ). Our findings showed that activation of oncogenic Kras alone or in combination with the removal of the tumor suppressor p53 in SPC⁺ cells resulted in development of alveolar tumors. Similarly, sustained EGF signaling in SPC⁺ cells led to alveolar tumors. By contrast, BASCs failed to proliferate or produce tumors under these conditions. Importantly, in a mouse strain in which Kras/p53 activity was selectively altered in type II cells but not BASCs, alveolar tumors developed while BADJs retained normal architecture. These results confirm and extend previous findings and support a model in which lung adenocarcinoma can initiate in alveolar type II cells. Our results establish the foundation for elucidating the molecular mechanisms by which lung cancer initiates and progresses in a specific lung cell type.

Abstract  Lung cancer is a devastating disease that presents a challenge to basic research to provide new steps toward therapeutic advances. The cell-type-specific responses to oncogenic mutations that initiate and regulate lung cancer remain poorly defined. A better understanding of the relevant signaling pathways and mechanisms that control therapeutic outcome could also provide new insight. Improved conditional mouse models are now available as tools to improve the understanding of the cellular and molecular origins of adenocarcinoma. These models have already proven their utility in proof-of-principle experiments with new technologies including genomics and imaging. Integrated thinking to apply technological advances while using the appropriate mouse model is likely to facilitate discoveries that will significantly improve lung cancer detection and intervention.

Abstract  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.

Abstract  Mouse lung tumorigenesis is a convenient model for examining all stages of lung adenocarcinoma (AC) progression. Because enhanced cyclooxygenase 2 (COX-2) expression has been observed in advanced human AC, we investigated the intracellular concentrations of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and COX-2, at different times after carcinogen administration to A/J mice. The concentrations of both proteins were much higher in urethane-induced adenomas and carcinomas compared with control A/J mouse lung tissue (P < 0.03 and P < 0.01 in adenomas and AC, respectively, for COX-1; P < 0.003 and P < 0.004 in adenomas and AC, respectively, for COX-2). Small benign tumors that arose spontaneously in 13-month-old mice also stained for COX-1 and COX-2, showing that this elevated enzyme content does not depend on chemical induction. COX-1 and COX-2 immunostaining was observed in normal bronchiolar and alveolar epithelia, alveolar macrophages and bronchiolar smooth muscle. This is the first report of the cellular distribution of COX-1 and COX-2 in murine lungs and the first in any species to demonstrate their co-localization. COX content in isolated bronchiolar Clara cells, a putative cell of tumor origin, was equal to that found in tumors, suggesting that the high enzyme content in neoplasms is due to their proportionally high concentration of these tumor precursor cells. Different patterns of COX-1 and COX-2 expression were observed in tumors of different growth patterns; only occasional small foci stained in solid adenomas, while most cells in papillary adenomas were immunoreactive. This staining pattern was also seen in adenocarcinomas, but some of the papillary portions also included focally stained and unstained regions. The continued expression during neoplastic progression of these specialized enzymes present in normal cells of tumor origin suggests their function in maintenance of the neoplastic state.

Abstract  CpG island methylation is an epigenetic modification of DNA associated with the silencing of gene transcription. The p16INK4a (p16) tumor suppressor gene is inactivated in human non-small cell lung cancers (NSCLCs) by either homozygous deletion or aberrant methylation. Inactivation of tumor suppressor genes by methylation has been linked in part to altered activity of the cytosine DNA-methyltransferase (DNA-MTase), the enzyme that catalyzes DNA methylation at CpG sites. The purpose of these studies was to define the role of DNA-MTase and p16 in the development of murine lung cancer. DNA-MTase activity was determined in alveolar type II and Clara cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity leading to an increase in overall DNA methylation was found only in alveolar type II cells, the target for murine adenocarcinomas. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. In addition, enzyme activity increased incrementally during lung cancer progression. Expression of p16 was detected in all primary lung tumors from A/J mice; however, levels of expression differed by up to 15-fold between tumors. The apparent low levels of expression seen in approximately half of the tumors was not attributed to methylation of the p16 gene. In contrast to the detection of p16 expression in primary tumors, this gene was deleted in four tumor-derived cell lines induced in the A/J mouse by NNK. The results from these studies indicate that the modulation of DNA-MTase activity was cell specific, segregated with susceptibility, and occurred early in neoplastic evolution. Thus, the marked increase in enzyme activity detected in alveolar type II cells after carcinogen treatment could be a major factor contributing to the high susceptibility for chemical-induced neoplasia associated with the A/J mouse strain. The inactivation of the p16 gene in murine cancers induced by NNK most likely arises as a late event via homozygous deletion.

Abstract  A decrease in the intracellular concentrations of the transcripts for some tumor suppressor genes has been found during murine lung tumorigenesis; for p15INK4b and p16INK4a, this was due to homozygous deletions. We report here a decrease in the mRNA levels of the mutated in colorectal cancer (Mcc) and adenomatous polyposis coli (Apc) genes in mouse lung tumors and some neoplastic cell lines. This was assessed both by northern blotting and reverse transcriptase-polymerase chain reaction of RNA isolated from lung tumors that had been induced by urethane, N-nitrosodiethylamine, or 3-methylcholanthrene in (A/J x C57BL/6) F1 or A/J mice. A reduced amount of both Mcc and Apc messages was also seen when two neoplastic cell lines, a spontaneous transformant (E9) and a line derived from a chemically induced solid tumor (82-132), were compared with two independently derived nontumorigenic cell lines (E10 and C10); E9 was derived from E10, and all of these lines are probably of alveolar type 2 cell origin. A cell line derived from a chemically induced papillary lung tumor probably of bronchiolar Clara cell origin (LM2) had Mcc mRNA levels similar to those of C10 and E10 but reduced Apc mRNA levels. A line (p53-823) derived from a papillary tumor that arose in a mouse with a mutated p53 transgene had a reduced amount of the Mcc gene product only. These differential changes in the relative amounts of Apc and Mcc messages in LM2 and p53-823) cells may serve as useful models for studying the regulation of their expression. Both messages had half-lives of 6-9 h in normal E10 and neoplastic E9 cells, so decreased message stability does not account for these reductions. This is the first report of estimated degradation rates of these mRNAs. Apc and Mcc message content did not vary as a function of growth status of the cell lines. Single-strand conformation polymorphism analysis did not reveal mutations in Apc coding regions known to have a high mutation frequency in human colon tumors. Loss of heterozygosity of Apc and Mcc was not found in tumors that developed in the F1 mice, implying a lack of allelic deletions. These changes in tumor suppressor gene expression may contribute to the development and maintenance of neoplasia in lung epithelium.

Abstract  Lung tumorigenesis in the mouse is controlled by multiple genes, which until now largely escaped detection because of the limitations of the available genetic tools. Therefore, we have developed the Recombinant Congenic Strains (RCS), which can be used to separate and map individual tumor susceptibility genes. The two strains, B10.O20/Dem and O20/A (used to produce the RCS series O20.c.B10.O20/Dem) differ in number, type, and degree of malignancy of lung tumors. Thus the genetic control of the different aspects of lung tumorigenesis can now be analyzed using RCS. The tests on the role of the Major Histocompatibility Complex (MHC; H-2 in mice) in lung tumorigenesis show that the H-2 influence is different for alveolar tumors and papillary tumors. In addition, only the development of papillary tumors is influenced by glucocorticoid administration concomitant with the transplacental carcinogen treatment. The MHC influence on lung tumor development is probably related to H-2 effects on the regulation of lung differentiation. In the H-2 congenic strains used papillary tumors developed early, whereas alveolar tumors appeared later, if at all. This differential impact of genetic and hormonal factors on the development of alveolar and papillary tumors suggests that they arise either from different cell types or from a common cell type at different stages of differentiation.

Abstract  Mouse lung tumors were induced transplacentally in offspring by treating C3H/HeNCrMTV- and Swiss Webster [Tac:(SW)fBR] mice during different periods of gestation with a single i.p. injection of N-nitrosoethylurea (ENU) at 0.5 mmol or 0.74 mmol/kg. Quantitative and qualitative evaluation of the lung tumors in the offspring at ages ranging from 1 week to 52 weeks was carried out by light microscopic study of hematoxylin and eosin-stained (H&E) serial and step sections. By nitroblue tetrazolium enzyme histochemistry, 3-hydroxybutyrate dehydrogenase (seen predominantly in Clara cells) was localized in frozen tissue sections. By avidin-biotin peroxidase complex immunohistochemistry, various specific cellular and nuclear markers were investigated on paraffin sections (antisera against surfactant apoprotein, Clara cell antigen, lysozyme, and 5-bromo-2' deoxyuridine). Normal lung and lung tumors were also studied by electron microscopy. A histological method was developed to assess all lesions present in the entire lung. It was shown that solid and papillary tumor types arose individually and that mixed solid/papillary forms represented a progression of the benign solid adenoma to the malignant papillary carcinoma. Immunocytochemical localization of DNA synthesis with 5-bromo-2' deoxyuridine gave the highest labeling indices at early stages of tumor growth. As the size of the papillary tumors increased, fewer nuclei were labeled/mm2 of tumor section. Lack of both specific Clara cell antigen and 3-hydroxybutyrate dehydrogenase and the absence of typical nonosmiophilic Clara cell granules indicated a cell of origin other than Clara cells. Evidence for alveolar type II cell origin of both solid and papillary neoplasms in spontaneous and induced tumors was found in the expression of surfactant apoprotein, the presence of mature lamellar bodies (solid tumors) or small lamellar bodies, and immature stages of lamellar bodies (papillary tumors). Lysozyme was present in mature alveolar type II cells and solid tumors but absent in fetal lung and papillary neoplasms. Tumors induced on gestation day 14 or day 16 had all developed by 2 weeks of age and generally did not increase in multiplicity with age, whereas those induced on day 18 showed a protracted development with regard to frequency, growth (size), and progression. The multiplicity of mouse lung tumors induced at different stages of fetal development paralleled the number of alveolar type II precursor cells (i.e., followed a bell-shaped pattern peaking on day 16 of gestation).

Abstract  Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in approximately 50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in approximately 40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.

Abstract  Our recent linkage study of urethane-induced pulmonary adenomas in SMXA RI strains of mouse revealed two host resistance genes, Par1 (chromosome 11) and Par3 (chromosome 12). The map positions of Par1 and Par3 correspond to human 17q11-23 and 14q11-24, based on synteny between mouse and human. In this study, we examined the loss of heterozygosity (LOH) in these two homologous human chromosomal regions in 30 primary lung adenocarcinoma samples with matched normal DNA. Using 15 highly polymorphic markers, two commonly deleted regions were identified on human chromosomes 14 and 17, respectively. At 17q21, nine (53%) of 17 informative tumors showed LOH between D17S588 and D17S518. On the other hand, at 14q11-12, seven (32%) of 22 informative tumors showed LOH at loci between D14S261 and D14S80. Subsequently, we examined 25 squamous cell carcinomas (SQ) and 24 small cell carcinomas (SCC). At 14q11-12, six (38%) of 16 informative SQ and five (42%) of 12 informative SCC showed LOH. In contrast, at 17q11-23, one (7%) of 15 informative SQ and two (14%) of 14 SCC showed LOH. Therefore, the gene on 17q seemed to affect selectively adenocarcinomas, whereas the other gene on 14q, all three types of lung carcinomas. These observations indicate that a comparative genetic analysis provides a promising approach to survey genes involved in multifactorial process of human lung carcinogenesis.

Abstract  Recent studies have demonstrated that K-ras mutations in lung epithelial cells elicit inflammation that promotes carcinogenesis in mice (intrinsic inflammation). The finding that patients with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer after controlling for smoking suggests a further link between lung cancer and extrinsic inflammation. Besides exposure to cigarette smoke, it is thought that airway inflammation in COPD is caused by bacterial colonization, particularly with non-typeable Hemophilus influenzae (NTHi). Previously, we have shown that NTHi-induced COPD-like airway inflammation promotes lung cancer in an airway conditional K-ras-induced mouse model. To further test the role of inflammation in cancer promotion, we administered the natural anti-inflammatory agent, curcumin, 1% in diet before and during weekly NTHi exposure. This significantly reduced the number of visible lung tumors in the absence of NTHi exposure by 85% and in the presence of NTHi exposures by 53%. Mechanistically, curcumin markedly suppressed NTHi-induced increased levels of the neutrophil chemoattractant keratinocyte-derived chemokine by 80% and neutrophils by 87% in bronchoalveolar lavage fluid. In vitro studies of murine K-ras-induced lung adenocarcinoma cell lines (LKR-10 and LKR-13) indicated direct anti-tumoral effects of curcumin by reducing cell viability, colony formation and inducing apoptosis. We conclude that curcumin suppresses the progression of K-ras-induced lung cancer in mice by inhibiting intrinsic and extrinsic inflammation and by direct anti-tumoral effects. These findings suggest that curcumin could be used to protract the premalignant phase and inhibit lung cancer progression in high-risk COPD patients.

Abstract  An Eco-RI restriction fragment length polymorphism occurring in a DNA fragment containing the first exon of the murine KRAS2 gene was shown to correlate with the inherited susceptibility of inbred strains of mice to urethan (CAS: 51-79-6)-induced pulmonary adenomas. Eco-RI digestion of murine DNA yielded four KRAS2-specific fragments. Polymorphic variation occurred in the smallest molecular-weight fragment with alleles of either 0.70 or 0.55 kb in size. Genotyping of 14 inbred strains of mice revealed a correlation between KRAS2 Eco-RI polymorphic variation and the differential susceptibility among inbred strains to development of pulmonary adenomas. Strains with a high incidence of pulmonary adenomas, either spontaneously occurring or in response to carcinogen induction, had the 0.55-kb KRAS2 allele whereas adenoma-resistant strains had the 0.70-kb allele. Analysis of a series of recombinant inbred strains (AXB, BXA) that developed from reciprocal crosses between a highly susceptible strain (A/J) and a highly resistant strain (C57BL/6J) revealed a statistically significant threefold difference in lung tumor susceptibility on the basis of KRAS2 genotype. Further analysis of individual F2 mice of a C57BL/6 female X A/J male cross also demonstrated a threefold difference in tumor susceptibility on the basis of KRAS2 allelic variation.