Showing papers in "Experimental Lung Research in 1998"

Expression of TNF and the necessity of TNF receptors in bleomycin-induced lung injury in mice.

Abstract: Bleomycin (BLM) induction of lung fibrosis in mice is an established model to study the mechanism of pulmonary fibrosis. Cytokine secretion has been implicated as a fundamental component of the lung fibrotic process observed in response to BLM. Among the cytokines implicated in lung fibrosis, Tumor necrosis factor (TNF) alpha has been considered to play a fundamental role. In the present study, we characterized the cellular sources of TNF during BLM-induced lung injury and examined the importance of TNF receptors in this process. To characterize the expression of TNF, we utilized two strains of mice, one sensitive (C57BL/6) and one resistant (BALB/c) to BLM-induced lung injury. Mice received BLM (120 mg/kg total) or saline, as control, by multiple subcutaneous injections. BLM induced the development of inflammation in subpleural areas only in the lungs of BLM-sensitive mice. These subpleural areas were characterized by infiltration of CD68-positive macrophages and increased collagen deposition. BLM enhanced the expression of TNF mRNA in BLM-sensitive, but not in BLM-resistant, mice. In situ hybridization studies localized the expression of TNF in the areas of BLM-induced inflammation in 6% and 27% of macrophages at 14 and 21 days post BLM treatment. In addition to TNF, BLM exposure resulted in the upregulated expression of transforming growth factor (TGF)-beta 1, but not interleukin (IL)-1, mRNA in the lungs of both murine strains at 14 and 21 days. This upregulated expression of TGF-beta 1 mRNA was greater in the lungs of BLM-sensitive mice. In separate experiments, double TNF receptor knockout mice were exposed to BLM. These animals demonstrated an increased expression of TNF, but not TGF-beta 1, mRNA in response to BLM and did not exhibit histologic evidence of lung injury following BLM exposure. In summary, the upregulation of TNF mRNA in macrophages correlated with the appearance of inflammation following BLM exposure and was limited to the BLM-sensitive strain. Furthermore, in addition to the release of the TNF ligand, it appears that the presence of TNF receptors is necessary for the development of BLM-induced lung injury, and signaling through these receptors may contribute to the regulation of the TGF-beta 1 mRNA expression observed in response to bleomycin. These results provide further support for a role of macrophages and TNF in the induction of lung inflammation.

Molecular comparison of human and mouse pulmonary adenocarcinomas.

Abstract: Mice develop lung tumors similar in their histogenesis and molecular features to peripheral adenocarcinomas in humans. The advantage of this model system is that events early in tumorigenesis can be delineated and their biological consequences tested by transgenic and knockout strategies. Both human and murine adenocarcinomas contain Kras mutations; in mice these occur within weeks following carcinogen administration. Decreased expression of similar tumor suppressor genes occur in both species due to mutation, deletion, altered DNA methylation, or unknown mechanisms. These genes include p15, p16, Rb, cyclin D1, p53, Apc, Mcc, and Gjal. Some genes have only been examined in one of these species, such as the deletions in chromosome 3p and the overexpression of bcl 2 in human adenocarcinoma. Not all molecular changes are identical to the two species, however. Quinone oxidoreductase (DT-diaphorase) levels rise in the human tumors but fall in the mouse; the extent of both changes is very dramatic. Similarly, EGF-receptor content often increases in human lung adenocarcinomas but decreases in the mouse tumors. In general, however, the nature of the molecular changes is quite similar.

Diesel Exhaust Particles Induced Release of Interleukin 6 and 8 by (Primed) Human Bronchial Epithelial Cells (Beas 2b) In Vitro

Abstract: Several epidemiological studies have recently shown associations of increased premature mortality rates with ambient particulate air pollution. Diesel exhaust particles (DEP) may constitute an important part of (ultra)fine particulate air pollution in urban areas and may therefore contribute to its toxicity. Epithelial lining of the respiratory tract may be the first target of the toxic effects of DEP, that upon exposure may release pro-inflammatory mediators such as interleukin 6 and 8 (IL-6, IL-8), ultimately causing airway tissue damage and immune alterations. In this study the effects of in vitro DEP exposure (0.04-0.33 mg/mL) on IL-6, IL-8 production by a human bronchial epithelial cell line (BEAS-2B) were investigated. For comparison, the production of interleukins during exposure to silica and titanium oxide (TiO2) were also studied, representing relatively toxic and non-toxic particles, respectively. Scanning and transmission electron microscopy showed that the size of the DEP particles ranged between 25 to 35 nm and that DEP was phagocytized by BEAS-2B cells. An increase in IL-6 and IL-8 production (11- and 4-fold, respectively) was found after 24 or 48 h of exposure to DEP compared to the non-exposed cells. This increase was lower compared to silica (17- and 3.3-fold) and higher as compared to TiO2 which showed no increase for IL-6 and IL-8. To study the DEP effect on inflammation-primed cells, BEAS-2B cells were exposed to both tumor necrosis factor-alpha (TNF-alpha) and subsequently to DEP. Exposure to TNF-alpha caused a strong increase in IL-6 and IL-8 production. Additive effects on the IL-6 and IL-8 production by BEAS-2B cells were found after TNF-alpha priming and subsequently exposure to DEP, only at a low dose of DEP and TNF-alpha (0.05-0.2 ng/mL). In conclusion, BEAS-2B phagocytized DEP and produced an increased amount of IL-6 and IL-8. In TNF-alpha primed BEAS-2B cells, DEP increased interleukin production only at low concentrations of DEP and TNF-alpha. Whether this increased production of pro-inflammatory interleukins affects vulnerable balances in the immune system, such as T help-1 and T help-2 subsets ratios, resulting in an altered resistance to respiratory tract infections or altering the expression of respiratory allergy, is the subject of further studies.

Lipoxygenase inhibitors prevent lung carcinogenesis and inhibit non-small cell lung cancer growth

Abstract: The effects of lipoxygenase inhibitors were investigated using human lung cancer cell lines and A/J mice. By RT-PCR, 5-, 12-, and 15-lipoxygenase mRNA was detected in NSCLC cells. NDGA inhibited 5-LO activity in adenocarcinoma cell line NCI-H1264. Using an MTT assay, NDGA, MK591 and AA861 inhibited the growth of NSCLC cell lines tested with IC50 values of 3, 2, and 7 microM, respectively. Using a clonogenic assay, 10 microM NDGA significantly reduced NSCLC colony number. NDGA significantly slowed NSCLC xenograft growth in nude mice. When the tumors were excised and analyzed, nude mice treated with NDGA had significantly more apoptotic figures than did untreated tumors. A/J mice treated with urethane developed adenomas after 4 months and NDGA administration significantly reduced lung adenoma number. These data indicate that lipoxygenase inhibitors inhibit lung cancer growth and prevent lung carcinogenesis.

Lung Fibrosis is Ameliorated by Pirfenidone Fed in Diet After the Second Dose In A Three-Dose Bleomycin-Hamster Model

Abstract: Interstitial lung fibrosis (ILF) is a life-threatening disease which has no known drug for prevention and cure. In the present study, we evaluated the antifibrotic potential of pirfenidone (PD) (5-methyl-1-phenyl-2-(1H)-pyridone) in a three-dose bleomycin (BL)-hamster model of lung fibrosis. Hamsters were intratracheally (IT) instilled with three consecutive doses of bleomycin sulfate (2.5 U/kg/5mL, 2.0 U/kg/5mL, 1.5 U/kg/3.75 mL) or an equivalent volume of saline at weekly intervals. Hamsters were fed a diet after the second dose of BL containing 0.5% PD and hamsters in the control groups were fed the same diet without the drug. The four groups were saline-instilled fed control diet (SCD); saline-instilled fed the same diet containing PD (SPD); BL-instilled fed control diet (BCD); and BL-instilled fed the diet containing PD (BPD). Hamsters were sacrificed at 28 days after IT instillation of last dose of saline or BL and their lungs processed for various assays. Lung hydroxyproline, an index of fibrosis, in SCD, SPD, BCD and BPD were 830, 804, 1609, 1235 micrograms/lung, respectively. Lung prolyl hydroxylase activities in the SPD, BCD and BPD groups were 103%, 313%, 157% of the control SCD group (5.99 x 10(4) dpm/lung/30 min) respectively. Malondialdehyde equivalent levels and superoxide dismutase activity in the corresponding groups were 99, 79, 240 and 145 nmoles/lung and 412, 433, 538 and 410 units/lung respectively. Lung myeloperoxidase activities in the corresponding groups were 56%, 179%, and 116% of the control group (0.44 units/lung). It is concluded that PD is a novel antifibrotic drug that has therapeutic potential in arresting the progression of an ongoing fibrotic process in the lung.

Tea and Tea Polyphenols Inhibit Cell Hyperproliferation, Lung Tumorigenesis, and Tumor Progression

Abstract: Both green and black tea have been shown to inhibit lung tumorigenesis in laboratory animal experiments. Green tea inhibited N-nitrosodiethylamine-induced lung tumor incidence and multiplicity in female A/J mice when tea was given either during the carcinogen treatment period or during the post-carcinogen treatment period. In a separate tumorigenesis model, both decaffeinated black tea and decaffeinated green tea inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor formation. Studies in which tea was administered during different time periods in relation to the NNK suggest that tea can inhibit lung tumorigenesis at both the initiation and promotion stages. The antiproliferative effects of tea may be responsible for these anti-carcinogenic actions. Black tea polyphenol preparations decreased NNK-induced hyperproliferation. Black tea also inhibited the progression of pulmonary adenomas to adenocarcinomas and the formation of spontaneous lung tumors in A/J mice. Growth inhibition...

Alterations in the expression of chemokine mRNA levels in fibrosis-resistant and -sensitive mice after thoracic irradiation.

Abstract: Fibrosis, characterized by the accumulation of collagen, is a consequence of a chronic inflammatory response. The purpose of this study was to determine if the mRNA expression of the chemokines, lymphotactin (Ltn), RANTES, eotaxin, macrophage inflammatory protein (MIP)-1 alpha, -1 beta, and -2, interferon-inducible protein 10 (IP-10), and monocyte chemotactic protein-1 (MCP-1), are altered during the development of radiation-induced pneumonitis and fibrosis. Further, we wished to determine if these changes differ between two strains of mice that vary in their sensitivity to radiation fibrosis. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 12.5 Gy to the thorax. Total lung RNA was prepared and hybridized utilizing RNase protection assays. Data were quantified by phosphorimaging and results normalized to a constituitively expressed mRNA L32. 8 weeks post-irradiation most chemokines measured were elevated to varying degrees. The degree of elevation of each chemokine was identical in both strains. This suggested that chemotactic activity for neutrophils, macrophages, and lymphocytes were occurring during pneumonitis. By 26 weeks post-irradiation, messages encoding Ltn, RANTES, IP-10, and MCP-1 were elevated only in fibrosis sensitive (C57BL/6) mice. In situ hybridization demonstrated that MCP-1 and RANTES transcripts were produced predominantly from macrophages and lymphocytes. These studies suggest that lymphocytic recruitment and activation are key components of radiation-induced fibrosis.

Inflammatory and epithelial responses in mouse strains that differ in sensitivity to hyperoxic injury.

Abstract: The pulmonary response to various toxicants including bleomycin, ozone, ionizing radiation, and hyperoxia is highly variable among mouse strains. The current study tests the hypothesis that at a similar stage of injury, regardless of strain, expression of inflammatory cytokine and epithelial marker genes would be similar, indicating a common pathway of injury progression. Three strains of mice, C57B1/6J, 129/J, and C3H/HeJ, ranging from sensitive to resistant, were exposed to > 95% O2 for varying times. Ribonuclease protection was used to quantify changes in cytokine mRNA. Despite differences in the kinetics, each strain demonstrated similar hyperoxia-induced changes in the abundance of interleukin (IL)-6, IL-1 beta, IL-3, and tumor neucrosis factor (TNF)-alpha. For each strain, death was accompanied by similar increases in cytokine mRNAs above steady-state control levels. Other inflammatory cytokines, including IL-1 alpha, IL-4, and interferon (IFN)-gamma, were unaltered in all strains at all times. In situ hybridization analysis of the epithelial markers, surfactant protein B (SPB), and clara cell secretory protein (CCSP) at the time of proinflammatory induction showed a similar pattern of expression in all strains. Increased SPB was detected in bronchiolar epithelium, while the number of type II cells expressing this message declined. Both the number of cells expressing CCSP as well as abundance per cell declined. These results suggest that although differences in acute sensitivity to hyperoxia exist between mouse strains, once initiated, acute epithelial cell injury and associated inflammatory changes follow the same pattern in all strains.

Inhibition of Lung Tumorigenesis by Nsaids: A Working Hypothesis

Abstract: A 7-week treatment with the tobacco carcinogen NNK induced 8-10 lung adenomas per A/J mouse. NNK suppressed humoral and cellular immune responses and increased plasma PGE2 and LTB4 levels. This protocol is particularly suitable for testing NSAIDs and lipoxygenase inhibitors as cancer preventive agents. Sulindac and ASA inhibited lung tumorigenesis by 52 and 60%, respectively, attenuated the suppressive effect of NNK, and lowered the plasma PGE2 to basal levels. In contrast, naproxen neither inhibited lung tumorigenesis nor increased NNK-suppressed NK cell cytoxicity. NSAIDs and lipoxygenase inhibitors had additive preventive efficacies against NNK-induced lung tumorigenesis. However, sulindac was not effective in preventing lung tumorigenesis induced by B[a]P, which lacks immunosuppressive activity. These results and those published by other investigators lead to the following hypothesis: Reactive intermediates derived from NNK interfere with the stimulation of the complex NF-KB/IKB. NF-KB is involved in ...

Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines

Abstract: Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunosuppressed patients, especially transplant recipients. In this population, infection is frequently due to reactivation of latent virus. The major immediate early promoter of HCMV controls production of immediate early gene products, which are both trans-and cis-active and are responsible for reactivation. Activation of this promoter is therefore a crucial step in regulation of reactivation infection. It is known that there are cAMP-response elements in the HCMV major immediate early promoter. We hypothesized that prostaglandins (PG), like PGE2, which are known to increase cAMP, as well as cytokines known to be released during acute inflammation, may be important in the regulation of this promoter and thus in reactivation of HCMV. To examine this, we transected pCAT760, a plastid containing the major immediate early promoter of HCMV upstream of a chloramphenicol acetyltransferase (CAT) gene, into THP-1 cells. These cells wer...

Differential expression of arginase and iNOS in the lung in sepsis

Abstract: The primary metabolic fates of L-arginine are conversion to L-citrulline by nitric oxide synthase (NOS) and to L-ornithine by arginase. In the lung, arginine utilization is increased after the inducible form of NOS (iNOS) is expressed during inflammation. The expression of arginase in normal lung and after sepsis, and its potential relationships with iNOS, however, are not known. Since arginase and iNOS share the substrate L-arginine, we tested the hypothesis that lung arginase would be co-induced with iNOS in sepsis and its cellular distribution would be related to that of iNOS in the lung. Lungs from cecal ligation and puncture (CLP) and sham-operated (S) rats were harvested 6 or 16 hours after the procedures. Lung wet-to-dry weight ratio, myeloperoxidase content, and lipid peroxidation products were measured as indices of lung injury. Western blot analyses were performed with polyclonal antibodies against two isoforms of rat arginase (I and II) and iNOS. Additional lungs from CLP and S animals were inflation-fixed for immunohistochemistry using the same antibodies. We found by Western blot that arginase II at 39 kDa was the main isoform present in normal rat lung. The enzyme was distributed diffusely in alveolar and bronchial epithelial cells, endothelial cells, and alveolar macrophages. After CLP, arginase II was almost undetectable in rat lungs at 16 hours. In contrast, in normal lung, the iNOS was not detectable by Western blot or immunohistochemistry. After CLP, strong expression of iNOS was found in similar cell types to arginase II. These data demonstrate loss of constitutive expression of arginase II in rat lung as iNOS is upregulated by the response to sepsis.

Additional evidence that the K-ras protooncogene is a candidate for the major mouse pulmonary adenoma susceptibility (Pas-1) gene.

Abstract: A locus for mouse pulmonary adenoma susceptibility, Pas-1, has been mapped on distal chromosome 6, where the K-ras gene is located. Allele-specific activation and expression of the K-ras allele from the susceptible parent has been observed in lung tumors from F1 hybrid mice. We report here genetic mapping of lung tumor susceptibility genes in urethane-treated A x B and B x A recombinant inbred (RI) mice using microsatellite markers to seek further evidence for the K-ras gene as candidate for Pas-1. The K-ras genotype differs between the A/J and C57BL/6J progenitors of the RI strains, and distal chromosome 6 contained a major lung tumor susceptibility determinant in the RI mice. Additional evidence that Pas-1 is K-ras involved linkage analysis of (A/JOLaHsd x BALB/ cOLaHsd) F2 intercross mice whose parents shared the same K-ras genotype. In contrast to the results with the A x B and B x A RI strains, no distal chromosome 6 site was significantly associated with tumor development in these F2 mice. In addition to this major locus, linkage analysis of the RI mice revealed additional quantitative trait loci for susceptibility on chromosomes 10, 17, and 19. These loci may serve as modifiers of Pas-1. The relationship between the K-ras genotype and the frequency of K-ras mutations in urethane-induced lung tumors from the RI mice was also explored. All 18 tumor DNAs from RI mice with high susceptibility contained an AT-->TA transversion at the second base of K-ras codon 61. This was also true for DNAs from 27 of 27 (100%) tumors in mice with high intermediate susceptibility. In RI strains with a low intermediate susceptibility, the DNA from 39 of 47 (83%) tumors contained an AT-->TA transversion at codon 61, and only 13 of 21 (62%) tumors had this mutation in the most resistant group. This reflects a positive correlation between the frequency of K-ras mutations in lung tumors of A x B or B x A RI strains and their susceptibility to lung carcinogenesis. Since K-ras appears to be Pas-1, these results suggest that some RI mice that have the resistant K-ras or Pas-1 allele undergo tumor development by a K-ras-independent route.

Mechanistic relationships between DNA adducts, oncogene mutations, and lung tumorigenesis in strain A mice.

Abstract: This paper describes a series of studies on the lung tumorigenic activities of polycyclic aromatic hydrocarbons (PAHs) in strain A/J mice, their ability to form PAH-DNA adducts in lung tissues, and their ability to mutate the Ki-ras oncogene in PAH-induced tumors. Seven PAHs were studied: cyclopenta[cd]pyrene (CPP), benzo[a]pyrene (B [a]P), benzo[b]fluoranthene (B[b]F), dibenz[a,h] anthracene (DBA), 5-methylchrysene (5MC), benz[j]aceanthrylene (B[j]A), and dibenzo[a,1]pyrene (DB[a,1]P). The dose-response data for the PAHs revealed 100-fold differences in tumor potency based on dose, with the order of activity DB[a,1]P, DBA > B[j]A > 5MC > CPP > B[a]P > B[b]F. Large differences in tumor multiplicity were also observed between the PAHs. DNA adducts were measured by 32P-postlabeling techniques on DNA from lungs of mice treated with these PAH's. DB[a,l]P gave syn- and anti-fjord-region diol-epoxide adducts of dAdo and dGuo; DBA gave both bay-region diol-epoxide-dGuo and bisdihydrodiol-epoxide adducts; CPP gav...

Lung injury and degradation of extracellular matrix components by Aspergillus fumigatus serine proteinase.

Abstract: Aspergillus fumigatus produces a variety of extracellular proteinases that are believed to be virulence factors towards Aspergillus-related lung disease Among Aspergillus proteinases, the serine proteinase is thought to play a major virulent role because of its widespread production Nevertheless, evidence of direct pulmonary injury caused by the A fumigatus serine proteinase is still lacking The purpose of our work was: (1) to provide evidence for a pivotal role of A fumigatus serine proteinase in producing lung injury in an animal model, and (2) to investigate the broadness of the substrate specificity of the proteinase towards extracellular matrix components To achieve this aim, the proteinase from an A fumigatus strain isolated from human airways was purified by a four-step procedure, including cation exchange and hydrophobic interaction High-performance capillary electrophoresis, SDS-PAGE, determination of Km towards synthetic substrates, and inhibitory studies were used to further characteriz

Chemokine mRNA alterations in newborn and adult mouse lung during acute hyperoxia.

Abstract: Chemokines play a major role in the recruitment of inflammatory cells during acute lung injury. Adult and newborn C57BL/6 mice were exposed to >95% oxygen for up to 72 hours and 7 days, respectively. Chemokine mRNA abundance was evaluated in whole lung RNA by ribonuclease protection assay and in tissue sections by in situ hybridization. Monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and interferon γ-induced protein (IP)-10 mRNAs were present in whole newborn lung by 4 days of hyperoxia and were markedly elevated by 7 days. Levels of mRNA for MCP-1, MIP-1α, and MIP-2 were elevated to a lesser extent by 72 hours of hyperoxia in adults. MCP-1 mRNA abundance was moderately elevated in scattered areas of perivascular tissue, peribronchiolar tissue, and the alveolar interstitium in 4-day hyperoxic newborns and markedly upregulated diffusely throughout the peripheral airspaces in 7-day hyperoxic newborns. MCP-1 mRNA abundance was limited to scattered perivascular areas and air...

Role of Gap Junctions in Lung Neoplasia

Abstract: Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas ...

Tobacco Smoke as a Mouse Lung Carcinogen

Abstract: Male and female strain A/J mice were exposed to environmental tobacco smoke that was generated by burning Kentucky 1R4F reference cigarettes. Exposures lasted 6 hours per day, 5 days per week for a total of 5 months, followed by a 4-month recovery period in air. Chamber concentrations of total suspended particulate matter (TSP) ranged from 50 to 90 mg/m3. Under these conditions, the average lung tumor multiplicity was 1.2 to 1.4 tumors per lung, significantly higher (p < 0.05) than in concomitant controls. ETS exposure led to a comparatively modest increase in cell proliferation in the alveolar zone during the first 2 weeks and in the terminal airways during the first 6 weeks. In the nasal passages cell proliferation was increased throughout, but reverted down to normal when the animals were placed in air. Smoke exposure increased immunostaining for cytochrome P4501A1 in airways and parenchyma. Exposure to the smoke gas phase only produced a similar increase in lung tumor multiplicity as did exposure to f...

Radiological and functional assessment of radiation-induced lung injury in the rat

Abstract: The purpose of this study is to develop an experimental model to measure localized radiation-induced lung injury using multiple end-points including breathing frequency, high-resolution computed tomography (CT), and radionuclide perfusion. The rats were anesthetized and the right lung irradiated with a single dose of 18 Gy using 200-kVp x-rays. The lung function of the animals was measured every 2 weeks after irradiation with the breathing rate assay. CT scanning and radionuclide lung perfusion assay were performed prior to and 2, 4, 10, 16, and 34 weeks after irradiation. Significant elevation in breathing rate occurred after 16 weeks, with a maximal increase between 22 and 28 weeks. An increase in the right lung density started 4 weeks after irradiation. Regional measurements indicated a relatively uniform increase in density at 4 and 10 weeks, while foci of high-density areas were observed at the later time points. Changes in rat lung volume indicated shrinkage of the irradiated right lung and accompanying compensatory hypertrophy of the shielded left lung. Radionuclide perfusion assay showed significant decrease in relative blood flow in the irradiated right lung 4 weeks after hemithoracic irradiation. Changes in breathing rate provide an index of overall lung function while changes in lung density, volume, and perfusion are of particular importance for evaluating loco-regional differences in lung sensitivity. This study is the first demonstration that CT can be used to measure volume changes after thoracic irradiation in rats.

Role of the cytosine DNA-methyltransferase and p16INK4a genes in the development of mouse lung tumors.

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, t...

Differential effects in vivo of thyroid hormone on the expression of surfactant phospholipid, surfactant protein mRNA and antioxidant enzyme mRNA in fetal rat lung.

Abstract: Antenatal administration of triiodo-L-thyronine (T3) to late gestation rats resulted in decreased lung antioxidant enzyme (AOE) activity but increased surfactant phospholipids. In fetal rat lung explant cultures, T3 decreased the expression of surfactant proteins (SP) A and B. There have been no reported studies of the simultaneous in vivo developmental influence of T3 on both pulmonary AOE and SP gene expression. We hypothesized that antenatal T3 treatment would cause differential regulation of surfactant phospholipid, SP, and AOE genes in the late gestation fetal rat. Timed pregnant rats received intramuscular injections of either T3 (7 mg/kg) or placebo on days 19 and 20 of gestation and fetuses were delivered on day 21. Fetal lung SP-A, SP-B, SP-C, and AOE mRNA levels were studied by Northern analysis. AOE mRNA levels were further quantitated by solution hybridization. Total lung phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) content were quantitated by a phosphorus assay. T3 significa...

Taurine Protects rat Bronchioles from Acute Ozone Exposure: A Freeze Fracture and Electron Microscopic Study

Abstract: Dietary taurine has been shown to protect rat and hamster lung epithelia from acute oxidant injury. One of the earliest morphologic criteria of oxidant injury is the alteration of tight junctions of the peripheral lung airways. In the present study, we have used this criteria to evaluate whether taurine was capable of protecting rat lungs from ozone exposure. Rats were treated for 10 days with 50% taurine in their drinking water, prior to exposure to 2 ppm of ozone for 3 hours. The lungs from rats pretreated with taurine and exposed to ozone were compared to untreated rats exposed to ozone and air-exposed controls. At 2, 6, 12, 24, 48, and 72 hours after exposure to air or ozone, rats were anesthetized and the lungs perfusion-fixed through the right side of the heart with a solution of glutaraldehyde and paraformaldehyde. Light microscopy revealed the typical, mild inflammatory cell infiltrate beginning at 6 hours after ozone exposure in bronchioles, alveolar ducts, and surrounding alveoli which was absen...

Immunophenotyping of lymphocytes in the lung interstitium and expression of osteopontin and interleukin-2 mRNAs in two different murine models of pulmonary fibrosis.

Abstract: In the surfactant protein C/tumor necrosis factor (SP-C/TNF) transgenic mouse, the TNF-α transgene is overexpressed in type II pneumocytes. Pulmonary lymphocytic infiltration develops which is followed by fibrotic changes including accumulation of fibroblasts and deposition of extracellular matrix. We hypothesized that lymphocytes played a role in the development of pulmonary fibrosis in this model. Lymphocytes were recovered from the interstitium of the lung and analyzed by flow cytometry. The absolute number of lymphocytes recovered from transgenic mice were approximately four times of that in littermates. Flow cytometric analysis showed the presence of γ$dL T cells and B1 cells in the former group but these cells were almost absent in the lung of non-transgenic littermates. We also studied lymphocytes accumulating in the lung during bleomycin (BLM)-induced pneumopathy. Serial analyses showed a progressive increase of CD4/CD8 ratio after injection of BLM, reaching a peak at day 14, then decreased to the...

Expression of cell cycle proteins in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung tumors.

Abstract: Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined by immunohistochemistry in 4-(methylnitrosamino)-1-(3 -pyridyl)-1-butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mut...

Genetics of quantitative and qualitative aspects of lung tumorigenesis in the mouse: multiple interacting Susceptibility to lung cancer (Sluc) genes with large effects.

Abstract: Inbred strains of mice exhibit large differences in their susceptibility to various complex quantitative genetic traits, among which is the susceptibility to lung cancer. These differences are caused by the combined effects of multiple quantitative trait loci (QTLs). Due to their multiplicity, it is relatively difficult and laborious to study the effects of individual QTLs. To dissect complex genetic traits the authors make use of recombinant congenic strains (RCS), a system of mouse inbred strains in which the genetic complexity is reduced. The susceptibility to lung cancer is studied by using the series of O20-congenic-B10.O20 (OcB) RC strains. They are derived from the parental background strain O20 and the parental donor strain B10.O20, two mouse inbred strains that differ from each other in both quantitative and qualitative aspects of lung tumorigenesis. This study describes the segregation of lung tumor number, size, and histology among the OcB RC strains, and indicates that these traits are influen...

Introduction to mouse lung tumorigenesis.

Abstract: This article provides a brief overview of some of the characteristics of lung tumors in mice and their application for studies in both chemical and molecular carcinogenesis and in cancer chemoprevention. The reader is referred to the above-mentioned review articles, and the articles to follow in this issue, for more extensive discussions of mouse lung tumorigenesis. It has been very exciting and rewarding to observe the progress made by many dedicated scientists in the field of mouse lung tumorigenesis during the past several years, and I hope that the next few years will be even more exciting.

Silica Deposition in the Lung During Epithelial Injury Potentiates Fibrosis and Increases Particle Translocation to Lymph Nodes

Abstract: Increased respiratory disease and daily mortality rates are associated with higher levels of fine particulate air pollutants. We examined the possibility that deposition of particles to previously injured lungs might accentuate pulmonary damage, by investigating how the lung handled silica deposited during a phase of epithelial injury. A low dose of intratracheal (IT) bleomycin (BL) was used to induce epithelial damage in mice; 3 days later, 0.2 mg silica was instilled. Lung injury, measured by cell numbers and protein levels in bronchoalveolar lavage, was increased at 1 week and many silica particles translocated to the interstitium. At 12 weeks, the silica plus BL group showed increased pulmonary fibrosis biochemically and morphologically, and had significantly higher retained-silica content in the lung. In addition, these mice showed enlarged hilar lymph nodes with many granulomas-containing macrophages and silica. The results indicate that instillation of fine particulates to the alveoli at a time of ...

Molecular pathogenesis of transplacentally induced mouse lung tumors

Abstract: Previous studies from this and other laboratories have shown that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring, the incidence of which correlated with fetal inducibility of Cyplal. Analysis of paraffin-embedded lung tissue for Ki-ras-2 mutations indicated that 79% of the lesions examined contained point mutations in codons 12 and 13 of the Ki-ras-2 gene locus, the majority of which (84%) were G → T transversions. The mutational spectrum was dependent on the tumor stage, as both the incidence of mutation and type of mutation produced correlated with malignant progression of the tumor. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas, and 100% of the adenocarcinomas. In the tumors with mutations, GLY12 → CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas, and 14% of the adenocarcinomas. GLY12 → VAL12 transversions were not observed in hyperplasias and occurred in 42% of the adenomas and 57% of the adenocarcinomas. ...

H441 Pulmonary Epithelial Cell Mitogenic Effects and Signaling Pathways in Response to Hgf and Tgf-α

Abstract: Pulmonary epithelial cells are important in lung growth, development, and injury. H441 pulmonary adenocarcinoma cells may be a useful model for studying pulmonary epithelial cell growth factor responses in vitro. Isolated pulmonary epithelial type II cells proliferate in response to transforming growth factor (TGF)-alpha via the epidermal growth factor (EGF) receptor. Type II cells also proliferate in response to hepatocyte growth factor (HGF). In the present study, H441 cell responses to these growth factors were examined, and compared to type II cells. Both the EGF-R and the c-met proto-oncogene receptor, to which HGF binds, were immunoprecipitated from H441 cells. In H441 cells, addition of TGF-alpha resulted in phosphorylation of the EGF receptor and increased cell number and tritiated thymidine incorporation. Incubation with HGF resulted in phosphorylation of its c-met proto-oncogene receptor in type II and H441 cells, and also increased cell number and tritiated thymidine incorporation. Both HGF and TGF-alpha stimulated phosphorylation of the intracellular signaling molecules p42 and p44 mitogen activated protein kinases in H441 cells. H441 cells exhibited responses to mitogenic growth factors similar to type II cells and may be useful as a model for type II cell growth factor responses and signal transduction.

Roles of K+ and Cl- Channels in cAMP-induced pulmonary vasodilation

Abstract: Increase in intracellular adenosine 3', 5'-cyclic monophosphate (cAMP) is a common pathway for many clinically used drugs to cause pulmonary artery (PA) relaxation. Activity of sarcolemmal K+ and Cl(-)-channels is an important determinant of membrane potential (Em), which, in turn, plays a critical role in regulating pulmonary vascular tone. Whether K+ and Cl- channels were involved in cAMP-induced PA relaxation was tested using isolated rat PA rings. Raising extracellular K+ concentration from 20 to 142.7 mM increased the K(+)-evoked contraction, but significantly decreased the relaxation induced by the adenylate cyclase activator, forskolin (FSK, 2.5 microM), suggesting that FSK-induced PA relaxation depended on transmembrane K+ gradient. Indeed, the FSK-induced relaxation was inhibited by 4-aminopyridine (4-AP, 10 mM), a voltage-gated K+ (Kv) channel blocker. Neither the Ca(2+)-activated K+ channel blocker, charybdotoxin, nor the ATP-sensitive K+ channel blocker, glibenclamide, had this effect. Furthermore, reducing extracellular Cl- concentration from 142.7 to 50 mM significantly decreased the FSK-induced relaxation in PA rings precontracted with 142.7 mM K+ (Ek approximately 0 mV), but negligibly affected the evoked contraction. This indicates that transmembrane Cl- gradient also regulates FSK-induced PA relaxation. Indeed, the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10 microM), significantly inhibited the FSK-induced relaxation in PA rings preconstricted by 142.7 mM K+. In summary, the data suggest that the cAMP-induced PA relaxation is attributable, at least partly, to both activation of the 4-AP-sensitive Kv channels and stimulation of the NPPB-sensitive Cl- channels.

Transforming growth factor-beta expression in mouse lung carcinogenesis

Abstract: Transforming growth factor-beta (TGF-β) is a multifunctional growth modulator that inhibits the proliferation of many epithelial cells while stimulating the proliferation of most fibroblasts. To examine the role of TGF-β in mouse lung chemically induced tumorigenesis, expression of the TGF-β1, -β2, and -β3 proteins was examined in A/J mice treated with the carcinogen urethane to induce lung adenomas using immunohistochemical staining analysis. Immunostaining for the TGF-β ligands was detected in the epithelium of the bronchioles of untreated A/J mice with immunostaining being more intense for TGF-β1 than for TGF-β2 and TGF-β3; immunostaining for each TGF-β ligand was also detected in the bronchiolar epithelium of urethane-treated A/J mice at levels similar to untreated mice. Immunostaining for the TGF-β ligands was also detected in adenomas by 2 months; staining for TGF-β1, -β2, and -β3 in adenomas was detected at levels comparable with bronchioles. Following treatment with urethane for 8 months, immunost...