近藤 征史

Background The release of adenosine triphosphate (ATP) from the airway epithelial cells during the inflammatory process is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. Objective This study was designed to determine whether extracellular ATP is involved in the bronchial hyperresponsiveness as an interaction between epithelium and smooth muscle in the airways. Methods We examined the contractile response to methacholine (MCh) before and after exposure to low concentrations (<= 10 mu m) of ATP in isolated, epithelium-denuded guinea-pig tracheal smooth muscle by measuring isometric tension. Intracellular Ca2+ concentrations ([Ca2+](i)) were assessed by fluorescent intensities of fura-2. Results MCh-induced contractile force was increased with no change in [Ca2+](i) after exposure to 10 pm ATP for 15 min. The ability of ATP to enhance the MCh-induced contraction was markedly attenuated by suramin, a non-selective P2 receptor inhibitor. Pre-incubation with ATP gamma S, a non-hydrolysable analogue of ATP and alpha,beta-meATP, a P2X agonist, also enhanced the MCh-induced contraction. In contrast, uracil triphosphate, a P2Y agonist, did not affect the MCh-induced contraction. Y-27632, a Rho-kinase inhibitor, suppressed the ability of ATP to enhance the MCh-induced contraction. Moreover, PP1 and PP2, Src tyrosin kinase inhibitors, suppressed the enhancement of MCh-induced contraction by ATP. Conclusion Pre-treatment with ATP induces hyperresponsiveness to MCh mediated by Ca2+ sensitization via the P2X receptor in airway smooth muscle. The present findings suggest the possible involvement of both the Rho-kinase and Src pathways in the intracellular mechanism of this phenomenon.

(第90回 日本肺癌学会中部支部会,支部活動)

Identification of a homozygous deletion in cancer cells provides strong evidence for the location of a tumor suppressor gene (TSG). We analyzed the 2p24 homozygous deletion of a non-small-cell lung cancer (NSCLC) cell line, NCI-H2882, and found that the deletion size was 3.7 Mbp. Since RhoB, which has been suggested to be a candidate TSG, was located in this region, we analyzed RhoB for alterations in NSCLC. Although we found no mutations in 48 cell lines including 20 NSCLCs, a loss of heterozygosity (LOH) analysis in 128 primary NSCLCs showed that 25 of 62 informative samples had LOH at the RhoB locus. Northern blot analysis of 28 cell lines (including 15 NSCLCs) indicated that RhoB expression was downregulated in 27. We analyzed RhoB expression in 112 primary NSCLCs with immunohistochemistrv and found no or a weak RhoB expression in 33 (42%) of 78 adenocarcinomas, whereas we found it in 29 (94%) of 31 squamous cell carcinomas. No or a weak expression of RhoB was more frequently observed in poorly- or moderately-differentiated adenocarcinomas than in well-differentiated ones (p = 0.0014). Furthermore, no or a weak expression of RhoB indicated a tendency to poor patient prognosis. Although hypermethylation was not to found at the promoter region, the RhoB expression in NSCLC cell lines was induced by histone deacetylase inhibition, suggesting that RhoB downregulation may be due to histone modification. The present study demonstrates that RhoB expression is frequently downregulated in NSCLCs by multiple mechanisms, suggesting that RhoB is a candidate TSG or NSCLC. (c) 2006 Wiley-Liss, Inc.

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyperreactivity 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)-(+)-transN-(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 Ca2+ mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (< 100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (< 100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca2+ sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.

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 (H2O2), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca2+ concentrations ([Ca2+](i)), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H2O2 (0-01-1 mM) caused contraction with an augmentation of [Ca2+](i) in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca2+ concentrations. The contractile force and [Ca2+](i) by H2O2 (I mM) were approximately half of those in response to 1 mu M methacholine. However, contraction by H2O2 was not generated under the condition that extracellular Ca2+ concentrations were less than 0.15 mM. Verapamil (10 mu M), an inhibitor of voltage-operated Ca2+ channels, partially but significantly inhibited the H2O2-induced contraction. In contrast, SKF-96365 (1-beta-[3-(4-methoxyphenyl)propoxy]-4methoxyphenethyl)-1H-imidazole hydrochloride) (100 mu M), a non-selective inhibitor of Ca2+ channels, completely abolished both the contraction and the increase in [Ca2+](i) elicited by H2O2. Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03-10 mu M), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H2O2-induced contraction. In conclusion, both the Ca2+ influx from the extracellular side and the Ca2+ sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H2O2. An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress. (c) 2006 Elsevier B.V All rights reserved.

Enhanced proliferation of smooth muscle cells contributes to airway remodeling of bronchial asthma. Recently, statins, inhibitors of 3-hydroxy-3-methylglutaryi-coenzyme A reductase, have been shown to inhibit proliferation of both vascular and airway smooth muscle cells independently of lowering cholesterol. However, the mechanisms remain to be elucidated. The purpose of this study was to determine molecular processes by which statins inhibit proliferation of human bronchial smooth muscle cells. Sirrivastatin (0.1-1.0 mu M) significantly inhibited cell proliferation and DNA synthesis induced by FBS in a concentration-dependent manner. The inhibitory effects of simvastatin were antagonized by mevalonate and geranylgeranylpyrophosphate, whereas the effects were not affected by squalene and farnesylpyrophosphate. The antiproliferative effects of simvastatin were mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, and Y-27632, an inhibitor of Rho-kinase, a target protein of RhoA. Western blot analysis showed that the level of membrane localization of RhoA (active Rho) was markedly increased by FBS, and that the level of active RhoA increased by FBS was reduced by simvastatin. Moreover, the inhibitory effect of simvastatin on FBS-induced RhoA activation was also antagonized by geranylgeranylpyrophosphate, but not by farnesylpyrophosphate. Because these isoprenoids are required for prenylation of small G proteins RhoA and Ras, respectively, the present results demonstrate that an inhibition in airway smooth muscle cell proliferation by simvastatin is due to prevention of geranylgeranylation of RhoA, not farnesylation of Ras. Therefore, statins may have therapeutic potential for prohibiting airway remodeling in bronchial asthma.

In guinea pigs, it is well-known that mechanical stretch of airway smooth muscle exhibits spontaneous tone which is mediated by cyclooxygenase (COX) activation. We tested the hypothesis that this spontaneous contraction of airway smooth muscle is mediated by stretch-activated non-selective cation channels and the Rho/Rho-kinase pathway, as well as COX-2 using a pharmacological approach. Isometric force and intracellular Ca2+ concentrations ([Ca2+](i)) were assessed in isolated guinea pig tracheal smooth muscle tissues. The samples were stretched to a given level and the muscle behavior was monitored under isometric conditions. We observed an increase in [Ca2+](i) and subsequent force generation over a 15-min period. The augmented [Ca2+](i) and spontaneous contraction due to the stretch were markedly attenuated by application of Gd3+, an inhibitor of stretch-activated channels, and removal of extracellular Ca2+. In contrast, nifedipine only had a mild inhibitory effect on the contraction. (R)-(+)trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide (Y-27632; a Rho-kinase inhibitor) abolished the spontaneous contraction with no changes in [Ca2+](i). Simvastatin, which down-regulates Rho activity, also significantly inhibited the contraction. Moreover, indomethacin, an inhibitor of COX-1 and -2, and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; a COX-2 inhibitor) abolished the stretch-induced contraction without affecting [Ca2+](i), whereas the inhibitory effect of 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC560; a COX-1 inhibitor) on the contraction was much less. These findings demonstrated that Ca2+ entry via stretch-activated channels, the Rho/Rho-kinase pathway, and COX-2 are involved in the mechanotransduction in guinea pig tracheal smooth muscle. Additionally, while the Rho/Rho-kinase pathway and COX-2 regulate the spontaneous contraction independently of [Ca2+](i), COX-1 is not involved in the stretch-induced force generation. (c) 2006 Elsevier B.V. All rights reserved.

(症例6, 第47回日本肺癌学会総会)

Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription-polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild-type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second-hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light-smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC.

The Hedgehog (Hh) signaling pathway and the Wnt signaling pathway are known to play important roles in carcinogenesis and the progression of various human malignant tumors. Although a relationship between these two pathways has recently been reported, the mechanism by which β-catenin, one of the key molecules of the Wnt signaling pathway, influences the Hh pathway has not yet been revealed in detail. To clarify the role of β-catenin in relation to the Hh signaling pathway, we transfected GLI1 and β-catenin expression constructs into human malignant cells, including stomach, colon, and lung cancers, and evaluated the luciferase activity of GLI-responsive reporter constructs. While exogenous GLI1 increased the luciferase activity, exogenous β-catenin also enhanced the activity under overexpression of GLI1. However, co-transfection with T-cell factor (TCF)-4 or lymphocyte enhancer factor (LEF)-1 did not influence the activity, indicating that the enhancement of β-catenin in relation to the Hh signaling pathway is not TCF/LEF-dependent. Our results suggest that β-catenin might be involved in the Hh signaling pathway via enhancement of the transcriptional activity of GLI.

Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D) from Japanese patients, with the latter two from the same patient with biphasic-like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI-1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC-MESO-1. We detected homozygous deletion of p16(INK4A)/p14(ARF) in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16(INK4A)/p14(ARF). To characterize the biological differences between Y-MESO-8A and Y-MESO-8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies.

The epidermal growth factor receptor (EGFR) gene has recently been reported to be mutated in a subset of non-small cell lung cancers (NSCLC), with the mutations being correlated with the patients' drug sensitivity to gefitinib, an EGFR kinase inhibitor. In this study, we searched for EGFR mutations in patients with lung cancer using primary tumor specimens obtained at initial surgery and examined whether their recurrent tumors showed a response to gefitinib depending on the presence of the activating mutation. Among 12 lung cancers that were treated with gefitinib after recurrence, we found that all four tumors which showed a response to gefitinib had an activating mutation in EGFR, whereas none of the remaining eight tumors had a mutation. Southern blot analysis showed that two of the four responsive tumors had the EGFR gene amplification. We also examined another 73 NSCLC specimens (47 mates and 26 females; 53 adenocarcinomas and 20 non-adenocarcinomas) which were not treated with gefitinib to determine whether NSCLCs with an EGFR mutation have different clinicopathological properties and/or unique genetic alterations of the other cancer-associated genes. We found that 13 (18%) of 73 tumors had a mutation of the EGFR gene, with the most being detected in female adenocarcinomas. Comparing the alterations in KRAS and P53 with the EGFR mutation, we found that 10 tumors with the KRAS mutation did not have an EGFR mutation, suggesting that each mutation occurs exclusively during the development of lung cancer. These results suggest that the mutation analysis of the EGFR gene using the specimens obtained at surgery might be useful in selecting the appropriate treatment(s) for recurrent Lung cancer patients. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

1 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. 2 PAR2 stimulation by trypsin and a PAR2-activating peptide (PAR2AP), especially from the basolateral aspect, caused transient Cl- secretion due to cytosolic Ca2+ mobilization. 3 Antagonists of PI-PLC (U73122, ET-18-OCH3) and inositol 1,4,5-triphosphate (xestospongin C (Xest C)) were without effect on thePAR2AP- mediated Cl- secretion, whereas it was attenuated by D609 ( a PC-PLC inhibitor) and phorbol 12-myristate 13 acetate (PMA, a PKC activator). 4 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). 5 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. 6 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. 7 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.

Secreted frizzled related protein 1 (SFRP1) 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 SFRP1 is located at chromosome 8p11, where lung cancers also exhibit frequent allelic loss, we hypothesized that the inactivation of SFRP1 is also involved in lung carcinogenesis. To substantiate this, we performed mutational analysis of SFRP1 for 29 nonsmall-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 SFRP1 was observed in 14 (48%) NSCLC and nine (36%) SCLC cell lines. We analysed epigenetic alteration of the SFRP1 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 SFRP1 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 SFRP1 gene locus. Furthermore, we performed colony formation assay of two NSCLC cell lines (NCI-H460 and NCI-H2009) and found the reduction of colony formation with SFRP1 transfection. In addition, we also detected that SFRP1 inhibits the transcriptional activity of beta-catenin, which is thought to be a downstream molecule of SFRP1, with luciferase reporter assay. Our current studies demonstrated that the SFRP1 gene is frequently downregulated by promoter hypermethylation and suppresses tumor growth activity of lung cancer cells, which suggests that SFRP1 is a candidate tumor suppressor gene for lung cancer.

Non-small cell lung cancer frequently shows loss of heterozygosity of the chromosome 3p21.3 region and several genes such as RASSF1A, BLU, and SEMA3B have been identified as candidate tumor suppressor genes at this region since their downregulation and hypermethylation at their promoter regions were frequently detected in lung cancer. To determine whether these three genes are simultaneously inactivated during lung cancer development, we studied 138 primary non-small cell lung cancers for the promoter methylation status of these genes and allelic loss of the chromosome 3p21.3 region. We found promoter hypermethylation at 32% in RASSF1A, 30% in BLU, and 47% in SEMA3B. Allelic loss of 3p21.3 was detected in 54 (58%) of 93 informative tumors. Despite the weak association of methylation status among these three genes, there was no correlation between the methylation status of each gene and loss of heterozygosity. We also studied possible genes downstream of RASSF1A in 16 primary non-small cell lung cancers and found that the expressions of SM22 and SPARC were significantly downregulated in RASSF1A-hypermethylated tumors. Our results showed that, while candidate tumor suppressor genes at this locus can be simultaneously inactivated by epigenetic alterations, loss of heterozygosity without any hypermethylation of the three genes can also occur in some cases, suggesting that just one allelic loss might also be sufficient for the inactivation of any of these genes for lung cancer development. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Background : The Fas/Fas ligand (FasL) system is a major regulator of apoptosis. Chemotherapeutic drugs have been shown to induce Fas expression on the surface of lung cancer cells, and cancer cell apoptosis. However, this mechanism is not considered to be associated with Fas expressed on lung cancer cells. Soluble Fas and FasL concentrations are reportedly elevated in the peripheral blood of patients with lung cancer, but the roles of circulating soluble Fas and FasL in that disease have not been clarified. Materials and methods : We measured the circulating soluble Fas and FasL levels in 21 patients with small cell lung cancer (SCLC), and 12 healthy matched controls, in order to examine whether such ligands could provide any important information and/or reveal any new clinical features of SCLC. Results : In the CR patients, the neuronal specific enolase (NSE), soluble Fas and soluble FasL concentrations were 21.26 +/- 3.65 ng/ml, 3.58 +/- 0.19 ng/ml and 0.50 +/- 0.15 ng/ml, while in the partial response (PR)/no change (NC)/progressive disease (PD) group of patients they were 33.96 +/- 7.86 ng/ml, 5.29 +/- 0.29 ng/ml and 0.59 +/- 0.07 ng/ml, respectively. The NSE, soluble Fas and soluble FasL concentrations were all elevated in the PR/NC/PD patients, however, significant differences were only seen in Fas concentration between CR and PR/NC/PD patients and CR patients and the controls (p < 0.001). Conclusions : Serum soluble Fas and FasL play important roles in the proliferation and metastasis of SCLC, as well as in the cytotoxic reaction and apoptosis induced by anticancer drugs in SCLC. Further study of the mechanisms and participation of circulating soluble Fas and FasL is necessary to develop treatment strategies for SCLC. (c) 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.

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.

FUS1 is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region that is deleted in many cancers. Using surface-enhanced laser desorption/ionization mass spectrometric analysis on an anti-Fus1-antibody-capture ProteinChip array, we identified wild-type Fus1 as an N-myristoylated protein. N-myristoylation is a protein modification process in which a 14-carbon myristoyl group is cotranslationally and covalently added to the NH2-terminal glycine residue of the nascent polypeptide. Loss of expression or a defect of myristoylation of the Fus1 protein was observed in human primary lung cancer and cancer cell lines. A myristoylation-deficient mutant of the Fus1 protein abrogated its ability to inhibit tumor cell-induced clonogenicity in vitro, to induce apoptosis in lung tumor cells, and to suppress the growth of tumor xenografts and lung metastases in vivo and rendered it susceptible to rapid proteasome-dependent degradation. Our results show that myristoylation is required for Fus1-mediated tumor-suppressing activity and suggest a novel mechanism for the inactivation of tumor suppressors in lung cancer and a role for deficient posttranslational modification in tumor suppressor-gene-mediated carcinogenesis.

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 ATRgammaS. 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-phospholipaseC inhibitor (13609) and an adenylate cyclase inhibitor (SQ22536). The responses to ATP from either aspect were also sensitive to an intracellular Call 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 Call 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.

Airway mucociliary clearance is subject to the autocrine/paracrine regulation of extracellular nucleotides released from the airway epithelial cells. The present study was performed in pursuit of effective modulators of ATP release under physiologic conditions in polarized human airway epithelial cells (Calu-3). Neither isoproterenol, forskolin, nor ionomycin augmented extracellular ATP release detected by luciferase assay. However, direct activation of the human intermediate conductance, Ca2+-activated K+ channel (hlK-1) by 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM) and chlorzoxazone (CZ, 1 mM) increased ATP release predominantly in the apical compartment. Measurement of fluo-3 signals revealed that I-EBIO-and CZ-stimulated cytosolic Ca2+ mobilization was suppressed by the presence of MRS-2179, a specific P2Y(1) receptor antagonist. The hlK-1-mediated ATP release was inhibited by a hlk-1 blocker (charybdotoxin), and an Na+-K+-2CI(-) cotransport blocker (bumetanide) without interruption by GdCl3, an inhibitor of stretch-activated nonselective cation (SA) channels, or glybenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR). These results suggest that a cell volume decrease via the hlk-1-mediated KCl loss and the resultant induction of a regulatory volume increase via the Nal(+)-K+-2Cl(-) transporter may trigger release of ATP, which causes P2Y(1)-mediated Ca2+ mobilization, through mechanisms unrelated to the CFTR and SA channels.

1. Lysophosphatidylcholine (Lyso-PC), which is synthesized by phospholipase A(2), is generally considered to induce adhesion molecules. However, little is known about the involvement of Lyso-PC in the pathogenesis of bronchial asthma. The present study was designed to examine whether pre-exposure to Lyso-PC causes eosinophil recruitment and an increase in resistance in airways. 2. Eosinophils in bronchoalveolar lavage fluid (BALF) and the airway walls were enumerated after inhalation of 0.5 mg/mL Lyso-PC to guinea-pigs for 10 min. Respiratory resistance (R-rs) was recorded continuously over 6 h after inhalation of an equi-dose of Lyso-PC for an equivalent period. 3. The proportion of eosinophils was increased from 10.7 +/- 3.3 to 27.5 +/- 3.1% (P < 0.0001) in BALF 6 h after inhalation of Lyso-PC, whereas the proportion of neutrophils and lymphocytes was not increased. Histological examination also showed uniform distribution of eosinophils in the airway wall of bronchi and bronchioles 6 h after inhalation of Lyso-PC. The number of eosinophils (/10 h.p.f.) in the bronchi and bronchioles was increased from 43.5 ± 16.8 to 154.8 ± 21.7 (P < 0.0001) and from 34.8 +/- 0.7 to 106.0 +/- 26.6 (P < 0.01), respectively. This eosinophil infiltration was similarly observed 24 h later. 4. Next, we examined the effects of eosinophil infiltration induced by Lyso-PC on R-rs. Inhalation of Lyso-PC caused a slow increase in R, and the percentage increase in R-rs was 19.8 ± 1.9% (P < 0.0001) 6 h later. Eosinophil infiltration and an increase in R. did not occur after inhalation of physiological saline. These phenomena induced by Lyso-PC were diminished by pretreatment with dexamethasone (6 mug/kg per day for 3 days). 5. Lysophosphatidylcholine causes eosinophil infiltration and a subsequent increase in resistance in airways. Our results indicate that Lyso-PC may be involved in the pathophysiology of bronchial asthma.

The human respiratory tract is constantly exposed to polycyclic aromatic hydrocarbons (PAHs) through inhalation of atmospheric pollutants. We examined the effects of three PAHs (benzo[a] pyrene, anthracene, and fluoranthene) on the airway ion transport, which is essential for lung defense and normal airway function, using human airway epithelia (Calu-3). These three PAHs had no significant effect on the basal short-circuit current (I-sc). However, fluoranthene (1-100 muM) applied in the apical compartment potentiated I-sc in response to cAMP-related agents (isoproterenol, forskolin, and 8-bromo-cAMP). The effects of fluoranthene were unaffected by ellipticine, a PAH receptor antagonist. Estimation of the anionic composition of I-sc revealed that isoproterenol increased both HCO3- and Cl- transport in the control, whereas it potentiated only Cl- transport in the presence of fluoranthene. The fluoranthene-induced modulations of these anion transporters were counteracted by charybdotoxin (ChTx, a hIK-1 channel blocker). Fluoranthene gradually augmented the ChTx-sensitive K+ current (I-K) across the basolateral membrane, accompanied by a sustained increase in the cytosolic Ca2+ concentration ([Ca2+](i)). In the presence of fluoranthene, however, a much larger hIK-1-dependent I-K was identified by the application of 8-bromo-cAMP without concomitant elevation of [Ca2+](i). These results suggest that fluoranthene switches from cAMP-dependent HCO3- secretion to Cl- secretion through the hIK-1 channel, whose sensitivity to protein kinase A may be up-regulated by the sustained [Ca2+](i) elevation produced by this chemical.

β-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 (γ-catenin), a closely related homologue of β-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 β-catenin gene, we studied whether plakoglobin has a T-cell factor/ lymphocyte enhancer factor (TCF/LEF) family-dependent transcriptional activity. Transaction 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 β-catenin. Exogenous plakoglobin was also shown to promote entrance of exogenous β-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 β-catenin, and that plakoglobin has multiple functions as a transcriptional activator and a cell adhesion molecule like β-catenin.

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild-type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild-type RAS, indicating that wild-type RAS may have onco-suppressive properties. To determine whether loss of wildtype KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non-small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild-type KRAS allele. Among the cell lines with heterozygosity of mutant and wild-type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild-type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild-type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild-type KRAS. Our findings indicate that the wild-type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild-type allele in human NSCLC development. (C) 2003 Wiley-Liss, Inc.

Background: Transforming growth factor-β1 (TGF-β1) is generally considered to play an important role in the pathogenesis of chronic inflammation and fibrosis. Objective and methods: This study was designed to determine mechanisms of reduced responsiveness of guinea-pig tracheal smooth muscle to β-adrenoceptor agonists by TGF-β1, using isometric tension records and tissue cAMP measurement. Moreover, we examined the involvement of the signal transduction processes of TGF-β superfamily in the desensitization of β-adrenoceptors. Results: After exposure to 0.2-2000 pM TGF-β1 for 4-8 h, the inhibitory effects of 1 μM isoprenaline (ISO) and 10 μM forskolin on 1 μM MCh-induced contraction were markedly reduced in a concentration-dependent fashion. The desensitization by TGF-β1 was greater against ISO than for forskolin. The values of EC75 for the curves for ISO after exposure to the normal bathing solution and TGF-β1 were 0.039 ± 0.02 and 0.38 ± 0.28 μM, respectively. The values of EC50 for the curves for forskolin under these conditions were 0.50 ± 0.12 and 0.89 ± 0.21 μM, respectively. On the other hand, the inhibitory effects of phosphodiesterase inhibitors such as theophylline and rolipram were not attenuated after exposure to TGF-β1. Concentration-inhibition curve for ISO was shifted to the right after exposure to 2000 pM TGF-β1 for 8 h more than that curve for forskolin. In contrast, the curve for theophylline was not shifted to the right by TGF-β1. When the tissues were incubated with TGF-β1 in the presence of IFN-γ, an intracellular antagonist of TGF-β signalling, IFN-γ inhibited the reduced response to ISO and forskolin after exposure to TGF-β1 in a concentration-dependent fashion. After exposure to TGF-β1, the effects of cAMP accumulation of ISO was significantly reduced, however, neither forskolin-nor theophylline-induced cAMP accumulation was affected. IFN-γ had no significant effect on cAMP accumulation either to ISO or forskolin. Conclusions: Impairment of the β-adrenoceptors/adenylyl cyclase pathway are involved in heterologous desensitization of β-adrenoceptors induced by TGF-β1 iin airway smooth muscle. IFN-γ functionally suppresses this phenomenon via cAMP-independent processes. Phosphodiesterase is still intact under this condition.