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Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

Supplementary Figures 1-3, Tables 1-5 from Epigenetic Profiles Distinguish Malignant Pleural Mesothelioma from Lung Adenocarcinoma

We assessed the efficacy and safety of cefepime monotherapy (1 g intravenously every 8 h) for febrile neutropenia in patients with lung cancer in a multi-institutional phase II study. Patients treated with chemotherapy with or without radiotherapy for lung cancer were eligible for this study. Other eligibility criteria included fever (temperature of ≥38.0 °C) and an absolute neutrophil count of500/mm(3) or1000/mm(3) with an expected decline to500/mm(3) within the next 48 h. Risk assessment was performed using the Multinational Association of Supportive Care in Cancer risk-index score. Cefepime 1 g was given intravenously every 8 h. The primary endpoint was the response rate at the end of cefepime therapy. Co-administration of granulocyte-colony-stimulating factor was permitted. Of 54 patients enrolled, 39 were classified in the low-risk group and 15 in the high-risk group. Overall response rate was 78% (95% CI: 64.4-88.0%). The response rates were 85% (95% CI: 69.5-94.1%) in the low-risk group and 60% (95% CI: 32.3-83.7%) in the high-risk group, respectively. One patient died from septic shock due to Enterobacter cloacae bacteremia. There was no significant adverse event. Cefepime 1 g intravenously every 8 h appears to be effective for febrile neutropenia in patients with lung cancer, especially in those with low-risk febrile neutropenia, and is well tolerated.UMIN Clinical Trials Registry, UMIN000006157.

Background and objective: Bronchiolitis obliterans (BO) has been reported to develop following ingestion of Sauropus androgynus (SA), a leafy shrub distributed in Southeast Asia. Little is known about direct effects of SA on airway resident cells or haematopoietic cells in vitro. Identification of the SA component responsible for the development of BO would be an important key to elucidate its mechanism. We sought to elucidate the direct effects of SA on airway resident cells or haematopoietic cells and identify the SA element responsible for the pathogenesis of BO. Methods: SA dry powder was partitioned into fractions by solvent extraction. Human and murine monocytic cells, epithelial cells and endothelial cells were cultured with SA solution or fractions eluted from SA. We also investigated the effect of SA in vivo using a murine BO syndrome (BOS) model. Results: The aqueous fraction of SA induced significant increases of inflammatory cytokine and chemokine production from monocytic lineage cells. This fraction also induced significant apoptosis of endothelial cells and enhanced intraluminal obstructive fibrosis in allogeneic trachea allograft in the murine BOS model. We found individual differences in tumour necrosis factor α (TNF-α) production from monocytes of healthy controls stimulated by this aqueous fraction of SA, whereas it induced high-level TNF-α production from monocytes of patients with SA-induced BO. Conclusions: These results suggest that an aqueous fraction of SA may be responsible for the pathogenesis of BO.

Asthma is regarded as a multifactorial inflammatory disorder arising as a result of inappropriate immune responses in genetically susceptible individuals to common environmental antigens. However, the precise molecular basis is unknown. To identify genes for susceptibility to three asthma-related traits, airway hyperresponsiveness (AHR), eosinophil infiltration, and allergen-specific serum IgE levels, we conducted a genetic analysis using SMXA recombinant inbred (RI) strains of mice. Quantitative trait locus analysis detected a significant locus for AHR on chromosome 17. For eosinophil infiltration, significant loci were detected on chromosomes 9 and 16. Although we could not detect any significant loci for allergen-specific serum IgE, analysis of consomic strains showed that chromosomes 17 and 19 carried genes that affected this trait. We detected genetic susceptibility loci that separately regulated the three asthma-related phenotypes. Our results suggested that different genetic mechanisms regulate these asthma-related phenotypes. Genetic analyses using murine RI and consomic strains enhance understanding of the molecular mechanisms of asthma in human.

The rapid improvement in blood oxygenation has been observed in patients with acute respiratory failure placed in a prone position. The gravitational effect relieves upper airway narrowing in the prone position, resulting in an improvement in sleep quality. This study examined the effect of the prone position on autonomic nerve activity and car- diovascular function. Seven healthy adults (mean age 25.2 ± 10.6 years) enrolled in this study. The heart rate variability as a marker of autonomic activity, lactate value as a maker of fatigue, vascular function (brachial-ankle pulse wave velocity; baPWV), and blood pressure in the supine and prone position were evaluated. The heart rate variability was calculated us- ing an electrocardiogram to measure the RR interval. The high-frequency (HF) and low-frequency /high-frequency (LF/HF) were calculated on baseline, after 30-min in the supine and prone position. The ratio of baseline HF to that after 30-min significantly increased in the prone position than the supine position. The ratio of baseline LF/HF to that after 30- min in the prone position significantly decreased compared to the supine position. Meanwhile, the ratio of the baseline LF to that after 30-min did not show any significant difference in the prone position in comparison to the supine position. There were no significant differences on lactate value, baPWV and blood pressure change. The prone position might therefore have a beneficial effect on the autonomic function.

The lung is a primary target for oxygen toxicity because of its constant exposure to high oxygen levels and environmental oxidants. Quercetin is one of the most commonly found dietary flavonoids, and it provides cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways. In the present study, we showed that quercetin increased the levels of heme oxygenase (HO)-1 expression and protected against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in lung epithelial cell lines. Quercetin suppressed H(2)O(2)-induced apoptotic events, including hypodiploid cells, activation of caspase 3 enzyme activity and lactate dehydrogenase release. This cytoprotective effect was attenuated by the addition of the HO inhibitor, tin protoporphyrin IX. In addition, the end products of heme metabolites catalyzed by HO-1, carbon monoxide and bilirubin, protect against H(2)O(2)-induced cytotoxicity in LA-4 cells. Quercetin may well be one of the promising substances to attenuate oxidative epithelial cell injury in lung inflammation.

Quercetin is a flavonoid with a wide variety of cytoprotective and modulatory functions. Heme oxygenase-1 (HO-1) is an inducible enzyme. Its reaction product, carbon monoxide (CO), confers cellular protection in a number of conditions and diseases associated with oxidative or inflammatory lung injury. Furthermore, quercetin was reported to be a potent inducer of HO-1 in several cell types. We hypothesized that quercetin suppresses the production of collagen in fibroblasts via the induction of HO-1. Here, we showed that quercetin suppresses transforming growth factor-β (TGF-β)-induced collagen production in NIH3T3 cells and in normal human lung fibroblasts. This suppressive effect of quercetin was mediated by quercetin-induced HO-1. The suppression of collagen production was conferred by the reaction product of HO-1, CO, but not by bilirubin. Furthermore, the translocation of the nuclear factor E2-related factor-2 (Nrf2), an important transcription factor that regulates the expression of HO-1 from the cytoplasm to the nuclei, was demonstrated in NIH3T3 cells by exposure to quercetin. Assessment of the signal transduction pathway involved in TGF-β signaling showed that quercetin stimulated the Smad and mitogen-activated protein kinase pathway to varying degrees. Our results demonstrate that quercetin exerts suppressive effects on the expression of collagen by the induction of HO-1. Idiopathic pulmonary fibrosis is the most lethal diffuse fibrosing lung disease, and is characterized by the deposition of extracellular matrix. Given that HO-1 is one of the important molecules emerging as a central player in diseases, quercetin or its derivatives, which effectively induced HO-1, will lead to new therapeutic strategies for promoting antifibrotic therapy in respiratory diseases.

The pathological hallmark lesions in idiopathic pulmonary fibrosis are the fibroblastic foci, in which fibroblasts are thought to be involved in the tissue remodeling, matrix deposition, and cross-talk with alveolar epithelium. Recent evidence indicates that some fibroblasts in fibrosis may be derived from bone marrow progenitors as well as from epithelial cells through epithelial-mesenchymal transition. To evaluate whether endothelial cells could represent an additional source for fibroblasts, bleomycin-induced lung fibrosis was established in Tie2-Cre/CAG-CAT-LacZ double-transgenic mice, in which LacZ was stably expressed in pan-endothelial cells. Combined X-gal staining and immunocytochemical staining for type I collagen and alpha-smooth muscle actin revealed the presence of X-gal-positive cells in lung fibroblast cultures from bleomycin-treated mice. To explore the underlying mechanisms, by which loss of endothelial-specific markers and gain of mesenchymal phenotypes could be involved in microvascular endothelial cells, the effects of activated Ras and TGF-beta on the microvascular endothelial cell line MS1 were analyzed. Combined treatment with activated Ras and TGF-beta caused a significant loss of endothelial-specific markers, while inducing de novo mesenchymal phenotypes. The altered expression of these markers in MS1 cells with activated Ras persisted after withdrawal of TGF-beta in vitro and in vivo. These findings are the first to show that lung capillary endothelial cells could give rise to significant numbers of fibroblasts through an endothelial-mesenchymal transition in bleomycin-induced lung fibrosis model.

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

Although stromal-derived factor-1 (SDF-1) via its cognate receptor CXCR4 is assumed to play a critical role in migration of endothelial cells during new vessel formation after tissue injury, CXCR4 expression on endothelial cells is strictly regulated. Erythromycin (EM), a 14-membered ring macrolide, has an anti-inflammatory effect that may account for its clinical benefit in the treatment of chronic inflammatory diseases. However, the effects of EM on endothelial cells and especially their expression of CXCR4 have not been fully evaluated. In this study, we demonstrated that EM markedly induced CXCR4 surface expression on microvascular endothelial cells in vitro and lung capillary endothelial cells in vivo. This ability to induce CXCR4 surface expression on endothelial cells was restricted to 14-membered ring macrolides and was not observed in other antibiotics including a 16-membered ring macrolide, josamycin. Furthermore, this EM-induced expression of CXCR4 on endothelial cells was functionally significant as demonstrated by chemotaxis assays in vitro. These findings suggest that EM-induced CXCR4 surface expression on endothelial cells may promote migration of CXCR4-expressing endothelial cells into sites of tissue injury, which may be associated with the known anti-inflammatory activity of this macrolide.

Although Pneumocystis infection might be one of the causes of secondary pulmonary alveolar proteinosis (PAP), the mechanism of its pathogenesis is uncertain. We analyzed a mouse model of secondary PAP resulting from Pneumocystis infection using mice deficient in CD40 (CD40KO), and evaluated the mechanism of the pathogenesis of secondary PAP from the viewpoint of surfactant-associated protein (SP) homeostasis, the overproduction of SP by type II alveolar epithelial cells, and the phagocytic function of alveolar macrophages (AMs). The effect of CD40 on SP production was also investigated in vitro using the H441 cell line, which has a phenotype similar to type II alveolar epithelial cells and primary alveolar epithelial cells. After long-term exposure to ovalbumin, CD40KO mice showed Pneumocystis infection and accumulation of surfactants in the alveoli (ApCD40KO). The amounts of SP production were up-regulated in ApCD40KO mice compared with wild-type mice treated using the same procedure. On the other hand, AMs from ApCD40KO mice did not show either phagocytic dysfunction or down-regulation of PU.1 expression. Furthermore, the stimulation of CD40-CD40 ligand (CD154) pathway regulated the production of SPs in H441 cells or primary alveolar epithelial cells. These results suggested that CD40KO mice could be one of the models useful for developing secondary PAP resulting from Pneumocystis infection. Surfactant accumulation was due to the overproduction in our model of secondary PAP. The CD40-CD154 interaction plays an important role in the regulation of surfactant-associated protein production.

Summary Background Cyclooxygenase (COX-2) overexpression is seen in many malignancies including lung cancer. Recent pre-clinical studies have shown that selective COX-2 inhibitors have demonstrated promising results when used with chemotherapy. Based on these observations, we assessed the efficacy and tolerability of the combination chemotherapy consisting of carboplatin and paclitaxel with meloxicam, a selective COX-2 inhibitor. Methods Forty-four patients with stage IIIB or IV non-small cell lung cancer (NSCLC), Eastern Cooperative Oncology Group performance status (PS) 0 or 1, who had adequate organ function, were eligible. Patients received paclitaxel 70 mg/m 2 weekly for 3 of 4 weeks with carbopltin (AUC 6) on day 1, as well as daily meloxicam (10 mg/day). The response rate was the primary endpoint. Secondary endpoints were overall survival, toxicity profile and quality of life (using European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and LC13). Results From March 2005 to September 2006, 44 patients were evaluated in this study. Gender M/F, 31/13; median age, 64 years (range, 34–75); stage IIIB/IV, 11/33; PS0/1, 22/22; histology Ad/Sq/Other, 29/6/9. Partial response was observed in 19 patients (43%) with stable disease, and there was no complete response, for an overall response rate of 43% (95% confidence interval, 28.5–57.8%). Ten patients (23%) had grade (G) 3 and three (7%) had G4 neutropenia. Three patients (7%) had G3 thrombocytopenia. As for non-hematological toxicities, one case of G4 toxicity (perforation of jejunum) was observed, but other toxicities were mild (one muscle pain, two liver dysfunction, one fatigue and one nausea G3). Grade 2 peripheral neuropathy was observed in only one patient. Using the EORTC QLQ questionnaire, the global health status did not change significantly during this therapy (before and 4 and 8 weeks later). Median follow-up was 13.6 months (range, 1.8–31.3 months). By the time of the final analysis (October 2007), 26 of the initial 44 patients had died. The 1-year survival rate was 64% and median survival time was 15.9 months. Conclusions Meloxicam in combination with carboplatin and weekly paclitaxel chemotherapy showed promising activity with encouraging survival. This therapy is relatively well tolerated in advanced NSCLC.

We previously reported the results of bacterial artificial chromosome array comprehensive genomic hybridization of malignant pleural mesotheliomas (MPMs), including two cases with high-level amplification in the 11q22 locus. In this study, we found that the YAP1 gene encoding a transcriptional coactivator was localized in this amplified region and overexpressed in both cases, suggesting it as a candidate oncogene in this region. We analyzed the involvement of YAP1 in MPM proliferation, as well as its functional and physical interaction with Merlin encoded by the neurofibromatosis type 2 (NF2) tumor suppressor gene, which is frequently mutated in MPMs. YAP1-RNA interference suppressed growth of a mesothelioma cell line NCI-H290 with NF2 homozygous deletion, probably through cell-cycle arrest and apoptosis induction, whereas YAP1 transfection promoted the growth of MeT-5A, an immortalized mesothelial cell line. We also found that the introduction of NF2 into NCI-H290 induced phosphorylation at serine 127 of YAP1, which was accompanied by reduction of nuclear localization of YAP1, whereas nuclear localization of a YAP1 S 127A mutant was not affected. Furthermore, results of immunoprecipitation and in vitro pull-down assays indicated a physical interaction between Merlin and YAP1. These results suggest that YAP1 is involved in mesothelial cell growth and that the transcriptional coactivator activity of YAP1 is functionally inhibited by Merlin through the induction of phosphorylation and cytoplasmic retention of YAP1. This is the first report of negative regulatory signaling from Merlin to YAP1 in mammalian cells. Future studies of transcriptional targets of YAP1 in MPMs may shed light on the molecular mechanisms of MPM development and lead to new therapeutic strategies.

Background Inflammatory response in pulmonary fibrosis closely resembles a T-helper (Th) 2 immune response. For recruitment to an inflammatory lesion, the majority of Th1 cells express CXC chemokine receptor 3, recognizing monokine induced by interferon-gamma (Mig), interferon γ-inducible protein of 10 kD (IP-10), and interferon-inducible T-cell α chemoattractant (I-TAC). Th2 cells express CC chemokine receptor 4, recognizing thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). We investigated the Th1/Th2 chemokine production patterns by lung fibroblasts and their evaluation in bronchoalveolar lavage (BAL) fluid of interstitial lung disease. Methods The production pattern of Th1/Th2 chemokines by lung fibroblasts was examined in ELISA and quantitative reverse transcriptase polymerase chain reactions. Th1/Th2 chemokine levels in BAL fluid of idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) were examined to evaluate the clinical relevance of Th1/Th2 chemokines. Results The lung fibroblasts were polarized to produce Th1-type chemokines by the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α and the anti-fibrotic cytokine, interferon (IFN)-γ. However, the induction patterns of chemokines by these two cytokines were different, i.e., involving predominant induction of IP-10 and I-TAC by TNF-α and induction of Mig by IFN-γ. Although Mig, IP-10, and I-TAC were produced within the BAL fluid of patients, TARC and MDC were at significantly low levels. Conclusions Our results suggest that lung fibroblasts tend to induce a Th1-type immune response under normal conditions, and that a Th2-type immune response does not play a significant role in smoldering inflammation around the established lesions in IPF and NSIP.

Background and objective: Expression of genes involved in DNA repair and/or DNA synthesis, including ribonucleotide reductase M1 (RRM1) and excision repair cross-complementation 1 (ERCC1) has been reported to be associated with chemosensitivity to platinum agents and gemcitabine. The aim of this study was to test whether similar associations would be seen between mRNA expression for the RRM1, ERCC1 and ERCC2 genes and in vitro chemosensitivity in lung cancer. Methods: Using a panel of 20 lung cancer cell lines, including 15 NSCLC and 5 small cell lung cancers (SCLC), the mRNA expression levels for the RRM1, ERCC1 and ERCC2 genes were examined by quantitative real-time reverse transcription PCR. The in vitro cytotoxicity of cisplatin, carboplatin and gemcitabine was assessed using a tetrazolium-based colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay). Results: Significantly, higher RRM1 mRNA expression was found in SCLC compared with NSCLC. However, there were no correlations between mRNA expression of the ERCC1, ERCC2 and RRM1 genes and chemosensitivity to cisplatin, carboplatin or gemcitabine. Conclusions: These in vitro results suggest that further studies are needed to evaluate the expression of the RRM1, ERCC1 and ERCC2 genes as predictive biomarkers for sensitivity to platinum agents and gemcitabine.

Dermatophagoides farinae are known to be a common environmental allergen causing allergic asthma; however, little is known about their pathophysiological effect via the allergenicities in vivo. Therefore, we first established a mouse model of asthma induced by repeated instillations of D. farinae . Second, to investigate whether the asthmatic responses are Th2-dependent, we examined the effect of the deficiency of interleukin-4 (IL-4) receptor α chain gene. Finally, we examined the effect of fluticasone propionate on this model. Mice were instilled with D. farinae without additional adjuvants into the trachea 8 times. After the final allergen instillation, the airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage and histological examination were carried out. The instillation of the allergen-induced airway hyperresponsiveness, the accumulation of inflammatory cells and increases in the levels of Th2 cytokines and transforming growth factor-β 1 production in the bronchoalveolar lavage fluid dose dependently. The number of goblet cells in the epithelium and the extent of the fibrotic area beneath the basement membrane were also increased in the morphometric study. In contrast, the defect of IL-4/IL-13 signaling through IL-4 receptor α chain completely abrogated all these responses. Furthermore, the simultaneous instillation of fluticasone propionate with the allergen showed significant inhibition or an inhibitory tendency of these changes. These findings demonstrate that the repetitive intratracheal instillations of D. farinae can induce airway remodeling through Th2-type inflammation, and that fluticasone propionate inhibits D. farinae -induced airway remodeling in mice, and this model would be useful for studying mechanisms involved in the development of allergic asthma.

Background and objectives: Macrophage-derived chemokine (MDC/CCL22) is recognized as a T-helper (Th) 2-type chemokine. Both malignant and tuberculous pleural effusions are typically lymphocytic pleural effusions. Tuberculous pleural effusions have a more polarized Th1 reaction than malignant effusions, which are predominantly Th2 in nature. The aim of this study was to compare the levels of MDC in malignant pleural effusions with those in tuberculous pleural effusions to help delineate the role of MDC in Th2 versus Th1 effusions. Methods: Forty-three patients with pleural effusions (32 malignant, 11 tuberculous) were studied. The concentration of MDC in the pleural effusion was measured by ELISA. Results: The median concentration of MDC was lower in malignant pleural effusions than in tuberculous pleural effusions (P

Reactive oxidant species are implicated in the chronic airway inflammation related to asthma and chronic obstructive pulmonary disease. This study was designed to determine mechanisms underlying contraction induced by hydrogen peroxide (H(2)O(2)), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca(2+) concentrations ([Ca(2+)](i)), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H(2)O(2) (0.01-1 mM) caused contraction with an augmentation of [Ca(2+)](i) in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca(2+) concentrations. The contractile force and [Ca(2+)](i) by H(2)O(2) (1 mM) were approximately half of those in response to 1 microM methacholine. However, contraction by H(2)O(2) was not generated under the condition that extracellular Ca(2+) concentrations were less than 0.15 mM. Verapamil (10 microM), an inhibitor of voltage-operated Ca(2+) channels, partially but significantly inhibited the H(2)O(2)-induced contraction. In contrast, SKF-96365 (1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride) (100 microM), a non-selective inhibitor of Ca(2+) channels, completely abolished both the contraction and the increase in [Ca(2+)](i) elicited by H(2)O(2). Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03-10 microM), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H(2)O(2)-induced contraction. In conclusion, both the Ca(2+) influx from the extracellular side and the Ca(2+) sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H(2)O(2). An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress.

要 約 日本肺癌学会および日本呼吸器外科学会では,肺癌登録合同委員会を共同で運営し,2002 年に報告された 1994 年の 肺癌切除症例の全国集計に引き続き,1999 年に切除された肺癌症例についての全国集計を 2006 年に行った.症例数は 13344,全体の 5 年生存率は 61.6% であった.男性(8878 例)の 5 年生存率は 55.4%,女性(4344 例)では 74.2% で あった.c-STAGE 別の 5 年生存率は IA(n=5939):77.0%,IB(n=3242):60.1%,IIA(n=226):53.8%,IIB(n= 1304):43.6%,IIIA(n=1723):38.0%,IIIB(n=567):33.6%,IV(n=211):27.0% であった.p-STAGE 別の 5 年生存率は IA(n=5007):83.3%,IB(n=2803):66.4%,IIA(n=400):60.1%,IIB(n=1388):47.2%,IIIA(n= 1944):32.8%,IIIB(n=1179):30.4%,IV(n=397):23.2% であった.組織型別 5 年生存率は腺癌 67.3%, 平上 皮癌 52.5%,大細胞癌 45.5%,小細胞癌 48.1%,腺 平上皮癌 42.1% であった.術死は 123 例(0.9%),在院死は 146 例(1.1%)に認められた.本登録は個人を特定できる情報を除いて行われており,集計成果は世界に類を見ない大規模 かつ詳細なものと考える.今後予定されている 2004 年切除例登録への参加有資格施設の積極的な取り組みを期待した い.(肺癌.2007;47:299-311)

Cigarette smoking has been clearly pointed out as a risk factor for cardiovascular disease. Endothelial dysfunction contributes to the development of cardiovascular disease. Flow-mediated dilation (FMD) has been known as one of the endothelial function markers. We investigated the acute and chronic effects of smoking on endothelial function in college-aged smokers.Eighteen smokers (mean age: 21 +/- 1 y) and 14 non-smokers (mean age: 20 +/- 1 y) were enrolled in this study. Brachial-ankle pulsed wave velocity (baPWV), systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were measured using a plethysmograph. Endothelium- dependent FMD was induced by reactive hyperaemia. High-resolution ultrasound with a 7.5-MHz linear array transducer was used to measure the diam- eter of the right brachial artery. Measurement of baPWV, SBP, DBP, HR, artery diameter, and %FMD was performed in smokers and non-smokers after 10 min bed rest, and after smoking one cigarette in the smokers. The interval between the two measurements was more than 15 minutes.There were no significant differences on baPWV, SBP, DBP, HR, baseline brachial artery diameter between smokers and non-smokers. Percent FMD after smoking was significantly lower than that at rest values in smokers (8.7 +/- 4.0 vs. 5.3 +/- 2.4, P0.005), although %FMD at rest was not significantly different between smokers and non-smokers. No other markers showed any significant differences between smokers at rest and after smoking.Even the smoking of one cigarette dramatically impaired endothelial function, although habitual cigarette smoking did not decrease FMD in young healthy smokers.

Background: To evaluate the role of FDG-PET in assessing anti-tumor efficacy of molecular targeted drugs, we prospectively performed FDG-PET and CT for response evaluation in patients treated with lapatinib, a dual inhibitor of ErbB1 and ErbB2 tyrosine kinases. Methods: Lapatinib was given orally once a day at doses ranging from 1200 to 1800 mg in a phase I study. CT and FDG-PET were performed before treatment, and at 1, 2 and 3 months after the initiation of the treatment and every 2 months thereafter. Results: A total of 29 FDG-PET examinations were performed in eight patients with various solid tumors and the metabolic activity in the tumor was evaluated as SUVmax. The best responses, as assessed by CT, were as follows; one partial response, four stable disease and three disease progression. The partial response was observed in a patient with trastuzumabresistant breast cancer, whose SUVmax was decreased by 60% from baseline. In all of the four patients whose best response was stable disease, the SUVmax was decreased by 6-42% one month after the start of treatment. Prolonged stable disease (10 months) was observed in a patient with colon cancer, whose SUVmax was decreased by 42%. In the patient group with disease progression, SUVmax was increased in two out of three patients. Conclusions: FDG-PET detected decreases in the metabolic activity of the tumors in patients who experienced clinical benefits on treatment with lapatinib. Thus, FDG-PET may be useful for the evaluation of molecular targeted drugs, such as lapatinib.

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F(340)/F(380). This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca(2+) mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (

This article focuses on pharmacogenetic associations between genetic polymorphism of uridine diphosphate glucuronosyltransferase (UGT) 1A1 gene and irinotecan toxicity. Accumulating evidence provides support to the idea that determination of UGT1A1 polymorphisms before irinotecan treatment is clinically useful and important for predicting and avoiding related toxicities. On the basis of these backgrounds, the irinotecan label was updated in 2005 in the United States to provide pharmacogenetic information, and a dose reduction of irinotecan should be considered for patients known to be homozygous for the UGT1A1*28 allele when administered in combination with other agents or a single agent. The irinotecan/UGT1A1 issue and the development of molecular diagnostic testing are now to be translated into clinical practice.

1. Although the clinical effects of the tulobuterol patch have been reported to include an increase in morning peak expiratory flow (PEF) values and a decrease of symptoms and the frequency of the rescue use of inhaled short-acting beta2-adrenoceptor agonists, no trials comparing the efficacy of the tulobuterol patch to other standard inhaled long-acting beta2-adrenoceptor agonists have yet been conducted. The aim of the present study was to compare the clinical effects of the patch formulation of tulobuterol with those of inhaled salmeterol in moderate to severe asthma. 2. Fifty-four patients with moderate to severe asthma, whose conditions were suboptimally controlled despite receiving inhaled corticosteroids, were recruited. The study was a prospective, randomized trial of cross-over design comparing the effects of 4 weeks treatment with tulobuterol patch, 2 mg once daily, and salmeterol, 50 mg twice daily. The mean prebronchodilator morning PEF during the last 14 days of each treatment period and health-related quality of life (HRQoL) were the primary outcome variables. The HRQoL was assessed using the St George's Respiratory Questionnaire. 3. Forty-four patients (81.5%) completed the trial and were included in the analysis. The mean morning PEF and HRQoL score were significantly improved in both the salmeterol (P < 0.0001 and P < 0.05, respectively) and the tulobuterol patch (P < 0.01 and P < 0.05, respectively) treatment periods compared with the run-in period. Although the mean morning PEF was significantly higher in the salmeterol-treated group than in the tulobuterol-treated group (P < 0.001), the HRQoL scores were comparable. 4. The tulobuterol patch may be useful as a controller medication in addition to inhaled corticosteroids in moderate to severe asthma.

We report the case of a woman with gefitinib-sensitive lung adenocarcinoma, who was successfully re-treated with gefitinib for carcinomatous meningitis as the disease recurrence. The good response to gefitinib treatment was supported in part by the existence of epidermal growth factor receptor mutation in carcinoma cells in the specimen obtained from transbronchial lung biopsy, in which E709G in exon 18 and L858R in exon 21 were shown. Although carcinomatous meningitis had been well controlled by the treatment with gefitinib, serum carcinoembryonic antigen (CEA) level increased with re-growth of primary tumor and development of lymphangitic carcinomatosis. Immunohistochemical findings revealed de novo emergence of CEA-producing carcinoma cells in the biopsy specimen taken after recurrence of pulmonary lesion during re-treatment of gefitinib, but revealed little or no CEA expression in the specimen obtained at first presentation. These facts may suggest the possibility of oligo clonality of carcinoma cells in this case.

Accumulating evidence provides support for the idea that determination of UGT1A1 polymorphisms before irinotecan (CPT-11) treatment is clinically useful and important for predicting and avoiding related toxicities. In 2005, the irinotecan label was updated in the USA in order to provide pharmacogenetic information. A dose reduction of irinotecan should be considered for patients known to be homozygous for the UGT1A1*28 allele when administered in combination with other agents or as a single agent. The feasibility of genotyping for DNA polymorphisms prior to treatment depends on the availability of rapid, accurate and efficient genotyping methods. Genotyping of UGT1A1*28, *6 and *27 by the newly developed Invader UGT1A1 Molecular Assay agreed completely with the genotyping results obtained with established methods, and this molecular assay provides rapid detection of polymorphisms. This newly developed method for detecting UGT1A1 polymorphisms is feasible and has the potential to be widely used for rapid and accurate screening before irinotecan treatment.

The viscoelastic and dynamic nonlinear properties of guinea pig tracheal smooth muscle tissues were investigated by measuring the storage (G′) and loss (G") moduli using pseudorandom small-amplitude length oscillations between 0.12 and 3.5 Hz superimposed on static strains of either 10 or 20% of initial length. The G" and G′ spectra were interpreted using a linear viscoelastic model incorporating damping (G) and stiffness (H), respectively. Both G and H were elevated following an increase in strain from 10 to 20%. There was no change in harmonic distortion ( Kd), an index of dynamic nonlinearity, between 10 and 20% strains. Application of methacholine at 10% strain significantly increased G and H while it decreased Kd. Cytochalasin D, isoproterenol, and HA-1077, a Rho-kinase inhibitor, significantly decreased both G and H but increased Kd. Following cytochalasin D, G, H, and Kd were all elevated when mean strain increased from 10 to 20%. There were no changes in hysteresivity, G/H, under any condition. We conclude that not all aspects of the viscoelastic properties of tracheal smooth muscle strips are similar to those previously observed in cultured cells. We attribute these differences to the contribution of the extracellular matrix. Additionally, using a network model, we show that the dynamic nonlinear behavior, which has not been observed in cell culture, is associated with the state of the contractile stress and may derive from active polymerization within the cytoskeleton.

The aim of this study was to evaluate the efficacy and tolerability of gefitinib ('IRESSA') in Japanese patients with previously untreated stage IV non-small-cell lung cancer (NSCLC). This was a multi-institutional phase II study. Thirty-four patients with previously untreated stage IV NSCLC were enrolled between May 2003 and September 2004. Gefitinib was administered orally 250 mg once a day and was continued until there was either disease progression or severe toxicity. Objective tumour response rate was 26.5% (95% confidence interval, 11.7-41.3%). Adverse events were generally mild (National Cancer Institute-Common Toxicity Criteria grade 1 or 2) and consisted mainly of skin rash, fatigue and liver dysfunction. No pulmonary toxicity was observed. The global health status revealed that there was no change in quality of life during the study. This study found that single-agent gefitinib is active and well tolerated in chemo-naive Japanese patients with advanced NSCLC.

SummaryAlthough pulmonary toxicity associated with gefitinib, an epidermal growth factor receptor inhibitor, has been reported recently, the accumulation of clinical information and the underlying mechanisms of gefitinib-induced interstitial lung disease (ILD) remain insufficient and unclear. After retrospectively reviewing the clinical records and chest X-rays of 489 lung cancer patients who were treated with gefitinib, we diagnosed four cases of gefitinib-induced ILD who underwent fiberoptic bronchoscopy and bronchoalveolar lavage (BAL). We found that the period of time from starting gefitinib to the onset of ILD was short, and concluded that a careful and close observation of a chest imaging study and a collection of respiratory symptoms was recommended. All four patients were treated with a high dose of corticosteroids, and ILD was resolved. We detected high levels of interferon-inducible protein-10 in BAL fluid, although we could not demonstrate the characteristic features of laboratory findings or BAL fluid cell analysis. We speculated that a Th1 type of lung tissue inflammation or lung injury might be involved as a part of mechanisms underlying gefitinib-induced ILD.

Summary Background It is well known that β-adrenoceptor agonists (β-agonists) cause relaxation in airway smooth muscle mediated by a reduction in the concentration of intracellular Ca2+ ([Ca2+]i). However, little is currently known regarding whether reduced sensitization to Ca2+ is involved in the β-adrenergic relaxation. Objective This study was designed to determine the intracellular mechanisms underlying suppression of Ca2+ sensitization in β-adrenergic relaxation (Ca2+-independent relaxation by β-agonists). Methods Isometric tension and [Ca2+]i were simultaneously measured in fura-2-loaded strips isolated from guinea-pig tracheal smooth muscles. The relationships between tension and [Ca2+]i were examined in the inhibitory action of isoprenaline (ISO) and other cAMP-related agents against methacholine-induced contraction. Results The concentration-inhibition curve for ISO against methacholine in tension was significantly dissociated from the curve for ISO in [Ca2+]i. In ISO-induced relaxation, a reduction in tension was significantly greater than that in [Ca2+]i. This phenomenon was mimicked by other cAMP-related agents: forskolin and dibutyryl-cAMP. In contrast, the inhibitory action of SKF-96365, a non-selective inhibitor of Ca2+ channels, was associated with that in [Ca2+]i. In the presence of Rp-cAMPS, an inhibitor of protein kinase A (PKA), ISO caused an equivalent relaxation with less reduction in [Ca2+]i. The effects of ISO were not affected by Y-27632, an inhibitor of Rho-kinase, or by bisindolylmaleimide, an inhibitor of protein kinase C. ISO failed to inhibit contraction elicited by calyculin A, an inhibitor of myosin phosphatase. Conclusion β-Adrenergic action antagonizes not only Ca2+ mobilization but also Ca2+ sensitization in methacholine-induced contraction. The cAMP/PKA-independent, Gs-direct action is more potent in Ca2+-independent relaxation by β-agonists than the cAMP/PKA-dependent pathway. Moreover, myosin phosphatase is a fundamentally affected protein in the reduced response to Ca2+ mediated by β-agonist. Our results may provide evidence that this Ca2+ desensitization is a novel target for a reliever medication using rapid-acting β-agonists in acute asthma management.

As many asthma patients suffer from asthma for long periods of time, we considered what pharmacists can do to help them lead a life free of asthma attacks in which they have a high QOL and autonomy.With this in mind, we established Japan's first “Asthma Pharmaceutical Care Clinic for Outpatients”. Our subjects for counseling at the clinic were new patients with bronchial asthma who were scheduled to begin treatment using a metered dose inhaler or dry powdered inhaler and patients who were undergoing treatment but in whom there had been no improvement in symptoms. At the clinic, the pharmacist identifies problems in the inhalation treatment by questioning the patient and based on the responses, provides instruction on the proper use inhalers so that such problems are avoided.More than 70% of the 116 patients who visited our pharmaceutical care clinic between July 2001 and March 2004 did not understand what asthma is, or why it is treated with an inhaler. In addition, they were not using inhalers correctly. Initial counseling significantly increased their level of understanding and it was further increased through repeated counseling. Our counseling maintained PEF scores at high levels for over two years.The counseling pharmacists provided at the bronchial asthma pharmaceutical care clinic for outpatients not only ensured that patients were following the proper inhalation technique, it also gave them useful information on the pathophysiology of asthma and the pharmacology and toxicity of the medicines they use. We are convinced of its effectiveness and necessity.

Study objectives: Malignant and tuberculous pleurisies are two major causes of lymphocyte-dominant pleurisy. Several studies have already reported that tuberculous pleurisy is a T-helper type 1(Th1)-dominant disease. In this study, we sought to examine the Th1/T-helper type 2 (Th2) balance, especially focusing on the polarizing status of T-cells to Th1/Th2 in malignant pleural effusions by measuring cytokines in pleural effusions and by evaluating the polarizing status of T-cells on the point of stimulation with interleukin (IL)-12 and/or IL-18. Furthermore, we evaluated inhibitors of interferon (IFN)-γ production in effusions to rule out the possibility of direct inhibition of T-cell polarization. Patients: Effusion samples were collected from 19 patients with malignant pleurisy caused by lung cancer and from 7 patients with tuberculous pleurisy. Measurements: Concentrations of pleural fluid IFN-γ, IL-12, and IL-4 were measured. IFN-γ production of T-cells enriched from malignant pleural effusions in the presence of IL-12 and/or IL-18 was also examined. We further compared the inhibitory activity of malignant pleural effusions against IFN-γ production and analyzed the expression of T-cell immunoglobulin mucin, mucin domain (Tim-3), a Th1-specific molecule in pleural fluid T-cells. Results: Although malignant pleural effusions showed low levels of Th1 and Th2 cytokines and ratios of IFN-γ and IL-12 to IL-4 were low, isolated T-cells produced a significant level of IFN-γ in the presence of IL-12 and IL-18. Soluble factors were not found to inhibit IFN-γ production in malignant pleural effusions. In tuberculous pleural effusion, ratios of IFN-γ and IL-12 to IL-4 were significantly higher, and T-cells showed the expression of Tim-3 messenger RNA. Conclusions: We confirmed that T-cells in the malignant pleural effusions are mainly nai¨ve or not definitely polarized to Th1. Moreover, malignant tumor does not actively distort the cytokine condition through production of soluble inhibitors within effusions. The present study indicates that antitumor immunity may be enhanced by restored IFN-γ activity through combination of IL-12 and IL-18, and that it will lead to new therapies for malignant effusion.

We examined the mechanisms underlying anion secretion mediated by protease-activated receptor 2 (PAR2) and its role in the regulation of ion transport, using polarized human airway Calu-3 cells. PAR2 stimulation by trypsin and a PAR2-activating peptide (PAR2AP), especially from the basolateral aspect, caused transient Cl− secretion due to cytosolic Ca2+ mobilization. Antagonists of PI-PLC (U73122, ET-18-OCH3) and inositol 1,4,5-triphosphate (xestospongin C (Xest C)) were without effect on the PAR2AP-mediated Cl− secretion, whereas it was attenuated by D609 (a PC-PLC inhibitor) and phorbol 12-myristate 13 acetate (PMA, a PKC activator). Even 30 min after removal of PAR2AP after a 10-min-exposure, cells were still poorly responsive to PAR2 stimulation, but the reduced responsiveness was upregulated by a PKC inhibitor, GF109203X (GFX). Pretreatment with PAR2AP did not affect responses to anion secretagogues, such as isoproterenol, forskolin, thapsigargin, 1-ethyl-2-benzimdazolinone, and adenosine, but ATP-induced responses were significantly reduced. Nystatin permeabilization studies revealed that the presence of PAR2AP prevented ATP-induced increments in basolateral membrane K+ conductance without affecting apical membrane Cl− conductance. ATP-elicited Ca2+ mobilization, which was sensitive to D609 and PMA, was inhibited by the pretreatment with PAR2AP, and this inhibition was blunted by the presence of GFX. Collectively, stimulation of PAR2 generates a brief response of Cl− secretion through PC-PLC-mediated pathway, followed by not only auto-desensitization of PAR2 itself but also cross-desensitization of a PC-PLC-coupled purinoceptor. The two types of desensitization seem likely to have PKC-mediated downregulation of PC-PLC in common. British Journal of Pharmacology (2005) 146, 397–407. doi:10.1038/sj.bjp.0706330

Secreted frizzled related protein 1 (SFRP 1) is an antagonist of the transmembrane frizzled receptor, a component of the Wnt signaling pathway, and has been suggested to be a candidate tumor suppressor in several human malignancies. Since SFRP 1 is located at chromosome 8 p 11, where lung cancers also exhibit frequent allelic loss, we hypothesized that the inactivation of SFRP 1 is also involved in lung carcinogenesis. To substantiate this, we performed mutational analysis of SFRP 1 for 29 non-small-cell lung cancer (NSCLC) and 25 small-cell lung cancer (SCLC) cell lines, and expression analysis for the same cell lines. Although somatic mutations were not detected in the coding sequence, downregulation of SFRP 1 was observed in 14 (48%) NSCLC and nine (36%) SCLC cell lines. We analysed epigenetic alteration of the SFRP 1 promoter region and detected hypermethylation in 15 (52%) of 29 NSCLC cell lines, two (8%) of 25 SCLC cell lines, and 44 (55%) of 80 primary lung tumors. By comparing the methylation status with SFRP 1 expression, we found a significant correlation between them. We also performed loss of heterozygosity (LOH) analysis and found that 15 (38%) of 40 informative surgical specimens had LOH in the SFRP 1 gene locus. Furthermore, we performed colony formation assay of two NSCLC cell lines (NCI-H 460 and NCI-H 2009) and found the reduction of colony formation with SFRP 1 transfection. In addition, we also detected that SFRP 1 inhibits the transcriptional activity of beta-catenin, which is thought to be a downstream molecule of SFRP 1, with luciferase reporter assay. Our current studies demonstrated that the SFRP 1 gene is frequently downregulated by promoter hypermethylation and suppresses tumor growth activity of lung cancer cells, which suggests that SFRP 1 is a candidate tumor suppressor gene for lung cancer.

Tuberculous and malignant pleural effusions are representative of lymphocytic pleural effusions. In tuberculous pleurisy, especially, T-helper type 1 (Th1) cytokines are dominant, containing, for example, high concentrations of interferon (IFN)-gamma. We focused on cytokines that induce expression of IFN-gamma and Th1 cell-specific CXC chemokines induced by IFN-gamma. We also evaluated the diagnostic utility of these markers in tuberculous pleural effusions. Forty-three patients with pleural effusions (11 with tuberculous pleuritis, 32 with malignant pleuritis) were studied. We measured the pleural concentrations of IFN-gamma, IFN-gamma-inducing cytokines (interleukin IL-12 and IL-18), and IFN-gamma-inducible chemokines (interferon-gamma-inducible protein of 10-kD [IP-10], monokine induced by interferon-gamma [Mig], and interferon-inducible T-cell alpha chemoattractant [I-TAC]). Our results demonstrate that the concentrations of IFN-gamma, IFN-gamma-inducing cytokines, and IFN-gamma-inducible chemokines were all higher in tuberculous pleural effusions than in malignant pleural effusions. Also, IFN-gamma was significantly correlated with IL-12, Mig, and I-TAC. Moreover, receiver-operator-characteristic (ROC) analysis demonstrated that IFN-gamma produced a greater area under the ROC curve than any other factor. We conclude that the concentrations of IFN-gamma, cytokines that induce expression of IFN-gamma, and chemokines induced by IFN-gamma in tuberculous pleural effusion were all increased. The Th1 chemokines we examined, especially IP-10, are comparable to IFN-gamma as diagnostic markers of tuberculous and malignant pleural effusions, although IFN-gamma is the most valuable.

The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (Isc) measurement revealed that isoproterenol (ISO, 0.1 microM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca2+-activated K+ channel opener)-induced Isc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 microM) or 8-bromo-cAMP (2 mM). C2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.

The reliability of varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) was evaluated for rapid diagnosis of viral infection. VZV-specific primers only amplified VZV DNA; no LAMP products were observed in reactions performed with other viral DNA templates. The specificity of this method was confirmed by two independent determinations, agarose gel electrophoresis and a turbidity assay. The sensitivity of VZV LAMP, determined by agarose gel electrophoresis, were 500 copies/tube. Detection using the turbidity assay, however, gave a sensitivity of 1,000 copies/tube. After these initial validation studies, reliability of VZV LAMP was evaluated for the detection of viral DNA in clinical specimens. Thirty-two swab samples collected from patients with vesicular skin eruptions were tested for VZV DNA. VZV was confirmed in sample numbers 10-32 by VZV real-time PCR, a previously established technique. VZV LAMP products were detected using turbidity from samples 13 to 32 (sensitivity; 87.0%, specificity; 100%, positive predictive value; 100%, negative predictive value; 75%). Although low levels of VZV DNA could be detected in the three samples exhibiting divergent results (samples numbers 10-12), no VZV LAMP product was detected in these samples, indicating a higher detection limit for this assay. Requirement of a DNA extraction step in the VZV LAMP method was examined in next experiment. The turbidity assay detected a VZV LAMP product in all of the 20 positive swab samples (samples numbers 13-32), regardless of DNA extraction.

It has been suggested that attenuation of the decatenation G2 checkpoint function, which ensures sufficient chromatid decatenation by topoisomerase II before entering into mitosis, may contribute to the acquisition of genetic instability in cancer cells. To date, however, very little information is available on this type of checkpoint defect in human cancers. In this study, we report for the first time that a proportion of human lung cancer cell lines did not properly arrest before entering mitosis in the presence of a catalytic, circular cramp-forming topoisomerase II inhibitor ICRF-193, whereas the decatenation G2 checkpoint impairment was present independently of the impaired DNA damage G2 checkpoint. In addition, the presence of decatenation G2 checkpoint dysfunction was found to be associated with diminished activation of ataxia-telangiectasia mutated in response to ICRF-193, suggesting the potential involvement of an upstream pathway sensing incompletely catenated chromatids. Interestingly, hypersensitivity to ICRF-193 was observed in cell lines with decatenation G2 checkpoint impairment and negligible activation of ataxia-telangiectasia mutated. These findings suggest the possible involvement of decatenation G2 checkpoint impairment in the development of human lung cancers, as well as the potential clinical implication of selective killing of lung cancer cells with such defects by this type of topoisomerase II inhibitor.

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcriptional factor, which has been suggested to be a candidate tumor suppressor gene in prostate cancer and astrocytic glioma. Because KLF6 is located at chromosome 10p15, where non-small cell lung cancers (NSCLCs) also exhibit frequent allelic loss, we hypothesized that the inactivation of KLF6 is also involved in the development of NSCLC. To determine this, we performed mutational analysis for 105 NSCLCs, including 9 cell lines and 96 primary tumors, and Northern blot analysis for 74 NSCLCs, including the 9 cell lines and 65 primary tumors. Although somatic mutations were not detected in the coding sequence of KLF6, expression of KLF6 mRNA was down-regulated in the 9 cell lines and in 55 (85%) of the 65 primary tumors compared with normal lung tissue. Treatment of two cell lines expressing KLF6 at low levels with 5-azacytidine did not induce KLF6 expression, suggesting that KLF6 down-regulation is not due to promoter hypermethylation. We also performed loss of heterozygosity (LOH) analysis using the laser capture microdissection technique, and found that 21 of 62 (34%) informative samples had LOH in the KLF6 gene locus. Comparing the LOH status with mRNA expression of KLF6, we found that 14 of the 14 (100%) samples with LOH showed KLF6 down-regulation, and that even 23 of 31 (74%) samples without LOH also showed this down-regulation. We also studied the expression of the WAF1 gene, a possible downstream gene of KLF6, and detected simultaneous down-regulation of WAF1 and KLF6 mRNA in 6 of 9 (67%) cell lines and 48 of the 55 (87%) primary tumors, although there was not a significant association between loss of KLF6 and WAF1 expression. Furthermore, colony formation assay of two NSCLC cell lines (NCI-H1299 and NCI-H2009) induced a markedly reduced colony formation by KLF6 transfection, and Annexin V staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that KLF6 induced apoptosis. Our present studies demonstrated that KLF6 is frequently down-regulated in NSCLC and suppresses tumor growth via induction of apoptosis in NSCLC, which may suggest that KLF6 is a tumor suppressor for NSCLC.

The present study investigated mechanisms underlying apical and basolateral P2Y(1)-mediated Cl(-) secretion in human airway epithelial cells. Apical and basolateral ATP induced short-circuit currents (I(sc)) with different properties via P2Y(1) receptors. The former comprised an immediate rise followed by a slow attenuation, whereas the latter was a transient rise with a higher peak and shorter duration (2 min). The actions of ATP were simulated by those of ADP, ADPbetaS, and ATPgammaS. Antagonists of phosphatidylinositol-phospholipase C (U73122, ET-18-OCH(3)) were without any effect on the bilateral ATP-induced I(sc), which were, in contrast, attenuated by a phosphatidylcholine-phospholipase C inhibitor (D609) and an adenylate cyclase inhibitor (SQ22536). The responses to ATP from either aspect were also sensitive to an intracellular Ca(2+) chelator, 1,2-bis (o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester, or a Ca(2+)-activated K(+) channel inhibitor, charybdotoxin, although differential Ca(2+) signals were concomitant with each reaction. Nystatin permeabilization studies revealed a good correlation between the I(sc) and the basolateral K(+) current rather than the apical Cl(-) current under ATP-stimulated conditions. In conclusion, apical and basolateral P2Y(1) receptors couple with both phosphatidylcholine-phospholipase C and adenylate cyclase, leading to Cl(-) secretion, whose rate is essentially regulated by the Ca(2+)-activated K(+) channel-mediated K(+) conductance. This suggests the importance of this channel in airway mucociliary clearance.

Objective: The purpose of this study was to assess the high-resolution computed tomography (CT) findings of familial idiopathic pulmonary fibrosis (IPF). Methods: High-resolution CT scans obtained in 9 consecutive patients with biopsy-proven familial IPF were reviewed. The presence, distribution, and extent of various CT findings were evaluated. Results: All 9 patients had ground-glass opacities, intralobular reticular opacities, and irregular thickening of the interlobular septa; 7 (78%) had traction bronchiectasis, 7 (78%) had small foci of consolidation; and 3 (33%) had honeycombing. The abnormalities involved mainly the lower lung zones in 6 patients and the upper lung zones in 2 patients and had no zonal predominance in 1 patient. Follow-up CT showed increased extent of disease in 8 patients. Conclusions: The high-resolution CT findings of familial IPF resemble those ofnonfamilial IPF. Familial IPF has a lower prevalence of honeycombing and a lower prevalence of predominant lower lung zone distribution than nonfamilial IPF, however.

Beta-catenin is an essential element for the transcriptional activation of target genes in the Wnt signaling cascade and is also a cell adhesion molecule that couples with cadherins. Although plakoglobin (gamma-catenin), a closely related homologue of beta-catenin, is also known to be a cell adhesion molecule, its function as a transcriptional factor has not been revealed in detail. Using a human malignant mesothelioma cell line, NCI-H28, in which we have identified a homozygous deletion of the beta-catenin gene, we studied whether plakoglobin has a T-cell factor/lymphocyte enhancer factor (TCF/LEF) family-dependent transcriptional activity. Transfection with the wild-type plakoglobin expression vector induced accumulation of plakoglobin in the nucleus. Immunoprecipitation assay with cotransfection of plakoglobin and either TCF-4 or LEF-1 detected binding of plakoglobin to TCF-4 or LEF-1. Luciferase reporter assay demonstrated transcriptional activity of the wild-type plakoglobin when transfected with TCF/LEF, although plakoglobin showed less activity than beta-catenin. Exogenous plakoglobin was also shown to promote entrance of exogenous beta-catenin into the nuclei. Furthermore, small interfering RNA directed against plakoglobin suppressed expression of endogenous plakoglobin and its transcriptional activity, suggesting that endogenous plakoglobin has a weak transcriptional activity. These results suggest that plakoglobin can activate the Wnt signaling cascade directly without interaction of beta-catenin, and that plakoglobin has multiple functions as a transcriptional activator and a cell adhesion molecule like beta-catenin.

Mi-2 beta is the main component of the nucleosome remodeling and deacetylase complex and plays an important role in epigenetic transcriptional repression. Here we show that the amino-terminal and carboxyl-terminal regions of Mi-2 beta have distinct transcriptional activities and bind to BRG1, a component of the SWI/SNF complex, and the RET finger protein (RFP), respectively. Analysis by luciferase reporter assay revealed that the amino-terminal region of Mi-2 beta has a strong transactivating ability, whereas its carboxyl-terminal region has transcriptional repressive activity. Co-localization and association of Mi-2, RFP, and histone deacetylase 1 suggested that these proteins cooperate in transcriptional repression. Furthermore, the functional importance of the association of Mi-2 beta and RFP was confirmed by using Rfp-/- fibroblasts. On the other hand, we demonstrated that Mi-2 and BRG1 were associated with each other and that the bromodomain region of BRG1 strongly suppressed transactivation by the amino-terminal region of Mi-2 beta. The findings that Mi-2 beta interacts with both transactivating and repressing proteins and directly associates with another chromatin remodeling protein, BRG1, provide new insight into the formation of multiprotein supercomplex involved in transcriptional regulation.

Recently, others and we have shown that one of the functions of GADD34 is a recovery from a shutoff of protein synthesis induced by endoplasmic reticulum stress. GADD34 has been shown to induce growth arrest and apoptosis. Main protein of apoptosis is p53, especially phosphorylation of p53. And one of the main proteins of growth arrest is p21/WAF1. Here we analyzed the effects of GADD34 on p53 phosphorylation and p21/WAF1 transcription. Transfected Myc-tagged p53 was dose-dependently phosphorylated at Ser15 by increasing the amount of GADD34. Transfection of GADD34 also induced the endogenous phosphorylation of p53 and enhanced p21 protein expression. Transfection of GADD34 induced p21/WAF1 promoter activity. This activity was dependent on p53, because GADD34 transfection to p53-deficient cells produced only a slight increase of p21/WAF1 promoter activity. The p21/WAF1 promoter activity was greatly enhanced by the transfection of p53. Both GADD34 and p53 transfection induced much higher p21/WAF1 promoter activity. The promoter activity of p21/WAF1 was very low in GADD34 deficient MEF. The transfection of GADD34 increased the p21/WAF1 promoter activity in GADD34 deficient MEF.

We have found that a malignant mesothelioma cell line, NCI-H28, had a chromosome 3p21.3 homozygous deletion containing the beta-catenin gene (CTNNB1), which suggested that the deletion of beta-catenin might have a growth advantage in the development of this tumor. To determine whether beta-catenin has a growth-inhibitory activity, we transfected wild-type beta-catenin, Ser37Cys mutant beta-catenin as an activated type, and C-terminus deletion mutant beta-catenin that lacks the transcription activity, into the NCI-H28 cells. A non-small cell lung cancer cell line, NCI-H1299, which expressed endogenous beta-catenin, was also studied. We tested the localization of exogenous beta-catenin in the NCI-H28 cells with immunofluorescence, and found that the wild-type beta-catenin and the C-terminus deletion mutant were more strongly expressed in the plasma membrane and cytoplasm than in the nucleus, while the Ser37Cys mutant was more in the nucleus than in the cytoplasm. By using luciferase-reporter assay, the beta-catenin/T-cell factor 4-mediated transactivity of the Ser37Cys mutant was shown to be higher than that of the wild-type beta-catenin in both cell lines. However, the transactivity of the C-terminus deletion mutant was strongly reduced in both. Colony formation of the NCI-H28 cells was reduced by 50% after transfection with the wild-type beta-catenin, and 60% with the Ser37Cys mutant, but only 20% with the C-terminus deletion mutant compared to the vector control. Inhibition of colony formation in NCI-H28 cells was because of apoptosis, manifested by positive staining of Annexin V and TUNEL assays in transfected cells. In contrast, when transfected with the wild-type beta-catenin, no significant reduction in colony formation was seen in beta-catenin wild-type NCI-H1299 cells. In conclusion, our data indicate that inactivation of beta-catenin by a 3p21.3 homozygous deletion might be a crucial event in the development of the mesothelioma NCI-H28 cells. Thus, while beta-catenin is well known to be a positive growth-stimulating factor for many human cancers, it can also act as a potential growth suppressor in some types of human cancer cells.