Dosimetry for lung tumorigenesis induced by urethane, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[a]pyrene (B[a]P) in A/JJmsSlc mice.

Some chemicals are known to be lung carcinogens in rodents. While many studies using two-stage models have administered medium or high doses to mice, few have tested lower doses. The dose dependence of urethane, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[a]pyrene (B[a]P), three well-known lung carcinogens at high doses, has not been sufficiently reported in lower dose ranges. Our study evaluated the tumorigenicity of urethane, NNK, and B[a]P at 26 weeks after a single intraperitoneal administration of each compound within medium to low dose in male and/or female A/JJmsSlc (A/J) mice. Dose-dependent tumorigenesis was demonstrated histopathologically for the three compounds. These results suggested that the tumorigenicity of these chemicals is dose dependent in A/J mice, even at lower doses than previously reported.

Characterization of biochemical, functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke.

Female C57BL/6 mice were exposed to mainstream cigarette smoke at 600 µg WTPM/L, 4 h/day and 5 days/week for up to 52 weeks. At 26, 52 and 65 weeks (52 weeks of exposure plus 13 weeks of no exposure), lungs were assessed for inflammation, function, histopathology and morphometry. Structural changes were observed and accompanied by altered lung function at 26 and 52 weeks (e.g. increase of static compliance and hysteresis, and decrease of elastance). Lung morphometry quantified significant increase in airspace enlargement at 52 weeks. Chronic smoke exposure induced inflammation in respiratory organs, e.g. mixed inflammatory cell infiltrates, perivascular lymphocyte infiltrates and pigmented alveolar macrophages in the lungs. Minimal or mild alveolar emphysema was diagnosed in 70% by 26 weeks or 80% by 52 weeks. After 13 weeks of recovery, most biochemical, histopathological and morphometrical alterations were restored, while emphysema was observed to persist at 18% incidence by 65 weeks. In conclusion, the employed exposure conditions induced emphysematous changes in the lungs, accompanied by altered lung function and morphological/histopathological changes. Following the 13 weeks of no exposure, morphological changes persisted, although some functional/biochemical alterations regressed.

Biological responses in rats exposed to mainstream smoke from a heated cigarette compared to a conventional reference cigarette.

The heated cigarette (HC) generates mainstream smoke by vaporizing the components of the tobacco rod using a carbon heat source at the cigarette tip. Mainstream smoke of HC contains markedly less chemical constituents compared to combusted cigarettes. Mainstream smoke from HC was generated under Health Canada Intense regimen and its biological effects were compared to those of Reference (3R4F) cigarettes, using nose-only 5-week and 13-week inhalation studies. In the 13-week study, SD rats were necropsied following exposure to mainstream smoke from each cigarette at 200, 600 or 1000 µg wet total particulate matter/L for 1 h/day, 7 days/week or following a 13-week recovery period. Histopathological changes in the respiratory tract were significantly lesser in HC groups; e.g. respiratory epithelial hyperplasia in the nasal cavity and accumulation of pigmented macrophages in alveoli. After a 13-week recovery, the lesions were completely or partially regressed, except for accumulation of pigmented macrophages in alveoli, in both HC and 3R4F groups. In the 5-week study, SD rats were necropsied following exposure to mainstream smoke of either cigarette at 600 or 1000 µg/L for 1 h, two times/day (with 30 min interval), 7 days/week or following a 4-week recovery period. Bronchoalveolar lavage fluid (BALF) analysis of neutrophil percentages and enzyme levels like γ-GT, ALP and LDH indicated that pulmonary inflammation was significantly less in HC groups compared to 3R4F groups. In conclusion, HC demonstrated significantly lower biological effects compared to 3R4F, based on the BALF parameters and histopathology.

Comparison of dermal tumor promotion activity of cigarette smoke condensate from prototype (heated) cigarette and reference (combusted) cigarette in SENCAR mice.

Test cigarette (prototype "heated" cigarette) was evaluated on its dermal tumor promotion activity in SENCAR mice relative to conventional 3R4F cigarette. Mainstream cigarette smoke was generated under the modified Health Canada Intensive Regimen, and smoke condensate (CSCs) were collected using cold traps and extracted with acetone. Female mice received a topical application of 7,12-dimehtylbenz(a)anthracene (DMBA) as the tumor initiator on the back skin during Week 1. Subsequently, CSC was repeatedly applied as the tumor promoter at 5 doses, up to 30 mg tar/application, three times per week for 30 weeks. Test groups showed a clearly longer latency at lower doses (⩽15 mg), but the difference was less clear at higher doses (⩾22.5 mg), while mortalities were not affected throughout the study. Test groups also had consistently lower incidence and multiplicity of neoplasms, as well as lower incidences of non-neoplastic changes (e.g., inflammations and squamous epithelial hyperplasia on the site of application). The group without DMBA initiation did not induce any neoplasm but the respective Reference group showed an increase in tumorigenicity. In conclusion, the study demonstrated significant reduction in dermal irritancy and tumorigenicity of Test CSC compared to Reference CSC.

Frequent aberrant methylation of the promoter region of sterile alpha motif domain 14 in pulmonary adenocarcinoma.

Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor-suppressor and tumor-related genes. In order to identify novel hypermethylated genes in early stage lung adenocarcinoma, we carried out methylated CpG island amplification, modified suppression subtractive hybridization, and methylation-specific polymerase chain reaction to identify aberrant methylation of CpG islands in the A/J mouse lung adenoma model, which histologically mimics the early stage of human pulmonary adenocarcinoma. Through methylated CpG island amplification, suppression subtractive hybridization, and differential screening, we detected five genes, three of which have human homologs. Two of them showed downregulation of their expression in human lung adenocarcinoma. Of these two genes, we selected sterile alpha motif domain 14 (SAMD14) and further analyzed its methylation status and expression level by methylation-specific polymerase chain reaction and quantitative real-time polymerase chain reaction. Most of the lung adenocarcinoma cell lines showed suppressed expression of SAMD14 together with hypermethylation at the promoter region, although an immortalized bronchial epithelium cell line (PL16B) did not show hypermethylation and did express SAMD14. The expression of SAMD14 in A549 was rescued by treatment with the demethylation agent 5-aza-2'-deoxycytidine. These data indicate that hypermethylation of the SAMD14 gene promoter region is associated with silencing of its expression. Hypermethylation at the CpG site of the SAMD14 promoter region was detected frequently in early invasive adenocarcinoma (8/24, 33.3%) but not in in situ adenocarcinoma (0/7, 0%) or normal lung tissue (0/31, 0%). Hypermethylation of the SAMD14 gene is a specific event in pulmonary adenocarcinogenesis and malignant progression.

Neuronatin expression and its clinicopathological significance in pulmonary non-small cell carcinoma.

INTRODUCTION: Neuronatin is a protein that is specifically expressed in the nervous system in the course of embryonal brain development, and its expression is limited to the pituitary gland in normal human adults. Neuronatin expression has been reported in some types of tumor. The purpose of this study was to clarify the significance of neuronatin expression in pulmonary non-small cell carcinoma. METHODS: We determined the frequency of neuronatin expression in surgically resected samples from non-small cell lung carcinoma (51 adenocarcinoma and 41 squamous cell carcinoma) by immunohistochemical staining, and investigated the correlations between expression level and various clinicopathological features. RESULTS: Expression of neuronatin was observed more frequently in squamous cell carcinoma (63%) than in adenocarcinoma (25%). In most cases, nontumorous lung tissue did not react with the antibody against neuronatin. In both adenocarcinoma and squamous cell carcinoma, less differentiated tumors expressed neuronatin more frequently than did differentiated tumors. In adenocarcinoma, but not squamous cell carcinoma, the prognosis of neuronatin-positive cases was significantly worse than that of neuronatin-negative cases. CONCLUSION: Neuronatin expression is specific for tumor tissue and was detected in both pulmonary adenocarcinoma and squamous cell carcinoma at high frequency, particularly in less differentiated tumors. Neuronatin expression is associated with poor prognosis in patients with adenocarcinoma, and may be useful as a prognostic marker for lung adenocarcinoma.

Phenotypic characterization of endometrial stromal sarcoma of the uterus.

Endometrial stromal sarcoma (ESS) of the uterus is a rare uterine malignancy that has not been characterized in detail. To characterize the phenotype of ESS of the uterus, we extracted RNA from ESS and the stroma of normal endometrium using a tissue microdissection system and compared the expression profiles in the two tissues. After suppression subtractive hybridization and differential screening, we detected the metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) gene as one of the major genes upregulated in ESS, and a full-length placental cDNA clone (CS0DI066YJ10) as one of the major genes downregulated. The results were confirmed by in situ hybridization in four resected specimens of ESS and 36 biopsy specimens of normal endometrial tissue. All ESS (4/4) and all cases of endometrial stromal cells in the proliferative phase (13/13) were positive for MALAT-1, but samples of normal stroma in the secretory phase and menopausal state included some that were negative or weakly positive for MALAT-1 (5/13 and 3/10, respectively). In contrast, all ESS and 12 of 13 cases of stromal cells in the proliferative phase were negative for the full-length placental cDNA clone but 10 of 13 cases of endometrial stromal cells in the secretory phase were positive for transcripts of the gene (P < 0.05). These results indicated that endometrial stromal cells have different phenotypic characteristics between proliferative and secretory phases and the tumor cells of ESS have the phenotypic character of endometrial stromal cells in the proliferative phase.

Analysis of differentially expressed genes in neuroendocrine carcinomas of the lung.

INTRODUCTION: Large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC) show considerable differences in their histology but share neuroendocrine (NE) characteristics and also genetic and/or expression patterns. METHODS: We used the subtractive expression method to identify differences in gene expression that would allow discrimination between these two types of NE lung carcinoma. RESULTS: Eight cDNA fragments were transcribed at a higher level in LCNEC compared with SCLC, and these corresponded to five mitochondrial genes, two ribosomal genes, and one fetal regulation factor, neuronatin (NNAT). Immunohistochemically, NNAT protein was detected in 43% (6/14) of LCNECs but in only 8% (1/13) of SCLCs (p < 0.05). Positive staining for NNAT was observed in areas that did not show the NE morphology, such as palisading and rosettes. CONCLUSIONS: The present results suggest that NNAT has the potential to be used as a differential maker between LCNEC and SCLC.

The ACIN1 gene is hypermethylated in early stage lung adenocarcinoma.

INTRODUCTION AND HYPOTHESIS: In recent years, many studies have performed genome-wide searching for differentially methylated genes in cancer. We hypothesized that characteristic aberrant hypermethylation of CpG islands of certain genes may exist in the early stages of lung adenocarcinoma and that such alterations may be useful in the detection and treatment of early lung adenocarcinoma. METHODS: A pair of immortalized cell lines originating from atypical adenomatous hyperplasia (PL16T) and from the resected end of the bronchus of the same patient (PL16B) was searched for aberrantly and differentially hypermethylated DNA fragments by a combination of the methylated CpG island amplification and suppression subtractive hybridization methods. RESULTS: From 229 clones, we selected 15 fragments that had a genomic region meeting the criteria for a CpG island. We identified a gene, apoptotic chromatin condensation inducer 1 (ACIN1), that was hypermethylated in PL16T. A higher frequency of hypermethylation at a locus at the 5': end of the DNA fragment isolated from the ACIN1 gene was found in small-sized adenocarcinoma (2 cm or less) (30/37, 81%) compared with normal lung tissue (9/37, 24%, p < 0.05). Interestingly, hypermethylation of ACIN1 was detected relatively frequently in the normal counterpart of adenocarcinoma without bronchioloalveolar carcinoma (BAC) component (7/16, 44%), but was rare in the normal counterpart of adenocarcinoma with BAC component (2/21, 10%, P < 0.05). CONCLUSIONS: We found hypermethylation of the ACIN1 gene in early stage lung adenocarcinoma. The role of methylation status in the development and malignant transformation of lung adenocarcinoma requires clarification.

Establishment of an immortalized cell line from a precancerous lesion of lung adenocarcinoma, and genes highly expressed in the early stages of lung adenocarcinoma development.

Atypical adenomatous hyperplasia (AAH) is classified as a precancerous lesion of lung adenocarcinoma. We established an immortalized AAH cell line (PL16T) and a human non-neoplastic bronchial epithelial cell line (PL16B) from the same patient by transfection with the gene for SV40 large T antigen. The expression profile of PL16T was compared with that of PL16B by the suppression subtractive hybridization method. From 704 selectively hybridized clones, we finally selected 25 fragments of mRNA that showed transcription levels more than three times higher in PL16T than in PL16B. Thirteen (52%) and eight (32%) of them encoded tumor-associated calcium signal transducer 2 (TACSTD2) and S100 calcium binding protein A2 (S100A2), respectively. The high transcription of TACSTD2 and S100A2 in PL16T was confirmed by in situ hybridization. In normal lung tissue, both TACSTD2 and S100A2 were expressed at very low levels, but seven and five of 14 AAH were positive for TACSTD2 and S100A2, respectively. The frequency of TACSTD2 positivity was increased in 16 of 22 bronchioloalveolar carcinomas (BAC) and adenocarcinoma with mixed subtype with BAC component (mixed BAC). Positivity for S100A2 occurred in four of 22 BAC and mixed BAC. The abnormal transcription of TACSTD2 and S100A2 are thought to be unique molecular markers of the preinvasive stage of lung adenocarcinoma.

Phenotypic characteristics of mouse lung adenoma induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The expression profile of adenoma induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice was compared with that of normal lung tissue by suppression subtractive hybridization (SSH). The mRNAs of surfactant-associated protein A (SP-A) and lysozyme showed characteristically higher transcription in the adenoma tissue than in normal lung. High expression of both SP-A and lysozyme in tumor cells was confirmed by in situ hybridization (ISH). In normal lung, alveolar type II pneumocytes were positive for both SP-A and lysozyme, indicating that tumor cells retained the phenotypic characteristics of the murine alveolar type II pneumocytes. Previous studies of human adenocarcinomas have shown that the two proteins are expressed reciprocally; SP-A and lysozyme are differential markers of atypical adenomatous hyperplasia (AAH) and non-goblet cell type adenocarcinoma, and of goblet cell type adenocarcinoma, respectively. Thus, the present results indicate that the phenotype of NNK-induced A/J mouse adenoma differs from that of AAH, which is thought to be a preinvasive lesion of human adenocarcinoma.