Protocadherin-1 is a glucocorticoid-responsive critical regulator of airway epithelial barrier function.

BACKGROUND: Impaired epithelial barrier function renders the airway vulnerable to environmental triggers associated with the pathogenesis of bronchial asthma. We investigated the influence of protocadherin-1 (PCDH1), a susceptibility gene for bronchial hyperresponsiveness, on airway epithelial barrier function. METHODS: We applied transepithelial electric resistance and dextran permeability testing to evaluate the barrier function of cultured airway epithelial cells. We studied PCDH1 function by siRNA-mediated knockdown and analyzed nasal or bronchial tissues from 16 patients with chronic rhinosinusitis (CRS) and nine patients with bronchial asthma for PCDH1 expression. RESULTS: PCDH1 was upregulated with the development of epithelial barrier function in cultured airway epithelial cells. Immunocytochemical analysis revealed that PCDH localized to cell-cell contact sites and colocalized with E-cadherin at the apical site of airway epithelial cells. PCDH1 gene knockdown disrupted both tight and adhesion junctions. Immunohistochemical analysis revealed strong PCDH1 expression in nasal and bronchial epithelial cells; however, expression decreased in inflamed tissues sampled from patients with CRS or bronchial asthma. Dexamethasone (Dex) increased the barrier function of airway epithelial cells and increased PCDH1 expression. PCDH1 gene knockdown eradicated the effect of Dex on barrier function. CONCLUSION: These results suggest that PCDH1 is important for airway function as a physical barrier, and its dysfunction is involved in the pathogenesis of allergic airway inflammation. We also suggest that glucocorticoids promotes epithelial barrier integrity by inducing PCDH1.

Non-CpG sites preference in G:C > A:T transition of TP53 in gastric cancer of Eastern Europe (Poland, Romania and Hungary) compared to East Asian countries (China and Japan).

AIM: Mutation spectrum of TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions. SUBJECTS AND METHOD: In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations (25 from Hungary, 71 from Poland and 192 from Romania), 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C > A:T mutations the mutations in CpG and non-CpG sites were divided. The Cancer Genome Atlas data (TCGA, ver.R20, July, 2019) having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data. RESULTS: The most frequent base substitutions were G:C > A:T transition in all the areas investigated. The G:C > A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C > A:T transition in CpG sites and A:T > G:C mutation was more prevalent in Asian countries. CONCLUSION: The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia.

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier.

BACKGROUND: Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs). METHODS: Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1. RESULTS: HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRG's effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA. CONCLUSION: These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.

The Rac1/JNK pathway is critical for EGFR-dependent barrier formation in human airway epithelial cells.

The airway epithelial barrier provides defenses against inhaled antigens and pathogens, and alterations of epithelial barrier function have been proposed to play a significant role in the pathogenesis of chronic airway diseases. Although the epidermal growth factor receptor (EGFR) plays roles in various physiological and pathological processes on the airway epithelium, the role of EGFR on barrier function in the airway remains largely unknown. In the present study, we assessed the effects of EGFR activation on paracellular permeability in airway epithelial cells (AECs). EGFR activation induced by the addition of EGF increased transepithelial electrical resistance (TER) in AECs. An EGFR-blocking antibody eradicated the development of TER, paracellular influx of dextran, and spatial organization of tight junction. Moreover, the effects of EGFR activation on paracellular permeability were eradicated by knockdown of occludin. To identify the EGFR signaling pathway that regulates permeability barrier development, we investigated the effects of several MAP kinase inhibitors on permeability barrier function. Pretreatment with a JNK-specific inhibitor, but not an ERK- or p38-specific inhibitor, attenuated the development of TER induced by EGFR activation. Rac1 is one of the upstream activators for JNK in EGFR signaling. Rac1 knockdown attenuated the phosphorylation of JNK activation and EGFR-mediated TER development. These results suggest that EGFR positively regulates permeability barrier development through the Rac1/JNK-dependent pathway.

Identification of colorectal cancer patients with tumors carrying the TP53 mutation on the codon 72 proline allele that benefited most from 5-fluorouracil (5-FU) based postoperative chemotherapy.

BACKGROUND: Although postoperative chemotherapy is widely accepted as the standard modality for Dukes' stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous in vitro and clinical investigations reported that CRC patients with wild-type p53 gene (TP53)-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated TP53-tumors do not. However, these studies evaluated the mutation-status of TP53 by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined. METHODS: To evaluate the significance of the TP53 mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy, we examined the mutation by direct sequencing from fresh CRC tumors and evaluated the R72P polymorphism of the mutated TP53 by a combined mutant allele- and polymorphic allele-specific polymerase chain reaction (PCR). RESULTS: The TP53 mutation occurred in 147 (70%) of 211 Japanese CRC tumors. The mutation was observed in 93 (63%) tumors on the R72 allele and in 54 (37%) tumors on the P72 allele. Although the alterations to TP53 have no prognostic significance for CRC patients overall, we found that Dukes' stage C CRC patients who did not receive postoperative chemotherapy and carried the mutated TP53-R72 showed significantly longer survival times than those with the mutated TP53-P72 when evaluated by overall survival (p = 0.012). CONCLUSION: Using a combined mutant allele- and polymorphic allele-specific PCR, we defined the codon 72 polymorphic status of the TP53 mutated allele in Japanese CRC patients. We raised a possibility that Dukes' stage C colorectal cancer patients with tumors carrying TP53 mutation, especially the P72 allele, benefited from 5-FU based postoperative chemotherapy.

Genome-wide cDNA microarray screening of genes related to the benefits of paclitaxel and irinotecan chemotherapy in patients with advanced non-small cell lung cancer.

Previous studies have demonstrated that not only the benefits but also the toxicities of chemotherapy can be predicted by cDNA microarray analysis of tumor specimens obtained before chemotherapy against non-small cell lung cancer (NSCLC). We conducted a study of cDNA microarray analysis to determine whether the gene expression in peripheral blood taken from patients prior to chemotherapy were correlated with the outcome of chemotherapy with paclitaxel (Pac) and irinotecan (CPT) against advanced NSCLC. Thirty-one patients with stage IIIB or IV NSCLC were treated with CPT at 60 mg/m2 and Pac at 160 mg/m2 every 2 weeks. Seventeen of 31 patients achieved PR and the overall RR was 54.8%. The median survival time was 426 days and the 1-year survival rate was 58.1%. The expression levels of 1176 genes were analyzed in 31 patients with the AtlasTM Human Cancer 1.2 Array. Stepwise multivariate analysis revealed that the genes encoding protein phosphatase, IL-1alpha and IgA were independent predictive factors for chemosensitivity. Stepwise regression analysis revealed that the thyrotropin-releasing hormone receptor and alkylation repair genes were independent prognostic factors. In conclusion, the expression of certain genes was able to predict the benefits of this Pac and CPT chemotherapy regimen.

Genome-wide cDNA microarray screening of genes related to survival in patients after curative resection of non-small cell lung cancer.

We conducted a study to determine whether the expression levels of genes in tumors were correlated with the survival of patients after complete resection of non-small cell lung cancer (NSCLC). The expression levels of 1176 genes in resected tumor specimens from 28 patients were analyzed using the Atlastrade mark Human Cancer 1.2 Array. The pathological stages of the resected tumors were I, II and III in 14, 5 and 9 patients, respectively. We compared the differences of gene expression between patients who survived (n=12) and those who died (n=16). The expression levels of cyclin-dependent kinase 8, phosphoinositide-3-kinase, interferon regulatory factor 3 and tubulin were significantly higher in the tumors of surviving patients with stage I lung cancer (p<0.01). The expression levels of 12 genes, including the interferon- stimulated genes, were significantly higher in surviving patients with stage II or III lung cancer (p<0.01). Stepwise multivariate regression analysis revealed that 4 and 12 genes in stage I and stage II/III cancers, respectively, were independent prognostic factors (p<0.01). In conclusion, these survival-related genes are considered to be possible targets of adjuvant therapy after surgical resection of NSCLC.

Genomic-wide cDNA microarray screening to correlate gene expression profile with chemoresistance in patients with advanced lung cancer.

We conducted a study using cDNA microarray analysis to determine whether expression levels of genes in tumors were correlated with tumor response to chemotherapy. Between September 2000 and December 2001, 47 patients were registered in the study. Eighteen patients had small cell lung cancer (SCLC), and others had non-small cell lung cancer (NSCLC). All patients except three received platinum-based chemotherapy. Sixteen of the 18 patients with SCLC (89%) and 13 of the 29 patients with NSCLC (45%) responded to chemotherapy, respectively. Transbroncheal biopsy specimens of tumors were obtained before chemotherapy. The expression levels of 1176 genes in tumor specimens were analyzed using the Atlas Human Cancer 1.2 Array. When we analyzed the data for correlations between gene expression levels and tumor response to chemotherapy, there was a significant increase in the expression of nine genes in non-responders compared with responders to chemotherapy (p< 0.01). Multivariate regression analysis revealed that allogenic inflammatory factor, HLA-DR antigen associated invariant subunit and MHC class II HLA-DR-beta precursor were independent chemo-resistant factors (p<0.0001). When we analyzed the differences in gene expression levels between patients with SCLC and NSCLC, expression levels of one or more resistant genes were increased in comparison with the mean expression of control house keeping genes in five of 18 SCLC patients and 19 of 29 NSCLC patients, respectively (p=0.012). In conclusion, some chemo-resistant genes were detected in the tumor tissue of lung cancer patients using cDNA microarray analysis. A prospective study is required to confirm whether expression levels of these genes reflect chemosensitivity.

Detection of occult tumor cells in peripheral blood from patients with small cell lung cancer by promoter methylation and silencing of the retinoic acid receptor-beta.

In order to investigate the possibility of detecting occult tumor cells of small cell lung cancer (SCLC), we used the methylation-specific PCR assay to detect methylation of retinoic acid receptor-beta (RARbeta) in peripheral mononuclear cells (PMNC). The methylation-specific PCR assay can detect one tumor cell per 10(3) normal cells. Seventy-two patients participated in this study. They received an initial full-dose of combination chemotherapy for SCLC between October 1995 and June 2000. The patients were classified according to the clinical stage of their tumors as limited disease (LD) in 31 patients and extensive disease (ED) in 41. PMNC were obtained before each patient underwent chemotherapy. We detected RARbeta methylation in PMNC from 42 of the 72 SCLC patients (58.3%). Nineteen of 31 patients with LD-SCLC (61.3%) and 23 of 41 patients with ED-SCLC (56.1%) were positive for RARbeta methylation. The overall survival for patients who were positive and negative for RARbeta methylation was not significantly different in both the LD- and ED-SCLC groups (log-rank test, p=0.13 and p=0.38, respectively); however, 2-year survival was significantly greater in the RARbeta methylation-positive LD-SCLC patients (chi(2) test, p=0.044). In conclusion, occult tumor cells can be detected in the peripheral blood of patients with SCLC using methylation of RARbeta, and long-term survival appears to be better in LD-SCLC patients with occult tumor cells in their blood.

Genome-wide cDNA microarray screening to correlate gene expression profile with survival in patients with advanced lung cancer.

We conducted a study using cDNA microarray analysis to determine whether expression levels of genes in tumors were correlated with survival after chemotherapy. Between September 2000 and December 2001, 47 patients were registered in the study. Eighteen patients had small cell lung cancer (SCLC), and the others had non-small cell lung cancer (NSCLC). All patients except three received platinum-based chemotherapy. Transbroncheal biopsy specimens of tumors were obtained before chemotherapy. The expression levels of 1176 genes in tumor specimens were analyzed using the Atlas Human Cancer 1.2 Array. The expression levels of three genes, G1/S-specific cyclin, type II cGMP-dependent protein kinase and hepatocyte growth factor-like protein, were significantly correlated with survival (p<0.01). Ten of the 47 patients who showed an elevated expression level of one or more of the three genes had a significantly increased chance of survival (p=0.0056). In conclusion, some survival-related genes were detected in the tumor tissue of lung cancer patients using cDNA microarray analysis. A prospective study is required to confirm whether expression levels of these genes can be used for prognosis.

Colorectal small cell carcinoma in ulcerative colitis with identical rare p53 gene mutation to associated adenocarcinoma and dysplasia.

Colorectal small cell carcinomas (SCCs) are rare tumors and are infrequently associated with ulcerative colitis (UC). We report a case of primary rectal SCC combined with adenocarcinoma arising in left-sided UC. Immunohistochemically, tumor cells were positive for chromogranin A, synaptophysin, and CD56 in the SCC but not in the adenocarcinoma. The patient simultaneously developed multiple lesions of adenocarcinoma and high-grade dysplasia in the sigmoid colon and rectum. To elucidate whether SCC might evolve from multipotential cells in dysplasia and/or adenocarcinoma, we examined the mutational status of TP53 and KRAS. The same clonality of these lesions including SCC was confirmed by the presence of an identical single nucleotide point mutation in TP53. KRAS mutation was not observed in these lesions. Thus, these lesions seem to have developed from the same origin. Long-standing inflammation leading to dysplasia might be responsible for the development of some SCCs in UC particularly when they are combined with dysplasia and/or adenocarcinoma.

Glucocorticoids enhance airway epithelial barrier integrity.

Asthma is a chronic inflammatory disorder of the airways, but its pathogenesis is incompletely understood. While asthma is a complex disease caused by multiple factors, epithelial barrier damage is a cardinal feature. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs in the treatment of asthma. However, the effects of GCs on the airway epithelial barrier have not been evaluated. Epithelial barrier functions were evaluated in cultured human airway epithelial cell monolayers, Calu-3 and 16HBE. Then, the cells were treated with dexamethasone (Dex), fulticasone propionate (FP), or budesonide (BD) for 5 days. Permeability measured by transepithelial electrical resistance was increased by treatment with Dex, FP, and BD in a dose-dependent manner. Permeability to fluorescein isothiocyanate-labeled dextran was markedly reduced by these treatments. Immunocytostaining revealed that Dex treatment potentiated tight junction formation in these polarized epithelial cells. Knockdown of epidermal growth factor receptor (EGFR) by small interference RNA blunted the effects of Dex on barrier integrity. Although EGFR expression was not affected by Dex treatment, EGFR phosphorylation was enhanced in Dex-treated cells. This is suggesting that EGFR are important for this phenomenon. These findings suggest that GC inhalation therapy can improve epithelial barrier integrity and might contribute to the therapeutic effects of GCs for treating asthma.

Comparison of the chemosensitivity of the primary lesion and a pancreatic metastasis of colon cancer: a case report.

Pancreatic metastasis from colorectal cancer is rare, and accounts for less than 2% of all pancreatic metastases. There have been no studies that have reported the differences in the sensitivity to chemotherapy between the primary lesion and the pancreatic metastasis in colorectal cancer. We experienced a rare example of pancreatic metastasis from colorectal cancer, and report here the difference in the sensitivity to the antitumor drug. A 68-year-old female underwent colectomy for rectal carcinoma with a mass in the pancreatic tail and the liver. The patient also underwent a distal pancreatectomy and a segmental liver resection at the same time. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and tumor protein 53 (TP53) gene mutation analyses, in addition to the histopathological examinations, revealed tumors of the liver and the pancreatic tail as being metastases from the primary carcinoma. We employed a collagen gel droplet-embedded culture drug sensitivity test for both the primary lesion and the pancreatic metastasis. The sensitivity to oxaliplatin and FOLFOX (5-flurouracil, folinic acid and oxaliplatin) were lower in the pancreatic metastasis compared to the primary lesion. In conclusion, pancreatic metastasis from colorectal malignancy is rare, and the present results suggest that there are potential differences in the sensitivity to chemotherapy between the primary colorectal tumor and its pancreatic metastasis.

Acute myocardial infarction associated with small-size lung cancer in a young woman.

A 34-year-old woman visited our hospital with chest pain and was diagnosed with acute myocardial infarction (AMI) on admission. Echocardiography imaging revealed the presence of complex masses in the aortic valve. As serum tumor marker CA19-9 was elevated, she was screened for malignant disease. A computed tomography (CT) scan revealed a solitary pulmonary nodule, but because the nodule was small and non-specific, CT follow-up was considered appropriate. However, she developed hemorrhagic stroke in the short term and was subsequently diagnosed with lung adenocarcinoma. Clinicians should be on alert for the occurrence of AMI in patients with small-size lung cancer.

Examination of stability of bone marrow blood RNA in the PAXgene tube.

The PAXgene RNA blood collection tube is used for RNA of peripheral blood (PB) and the stability of PB RNA in this tube has already been reported. However, the stability of bone marrow blood (BM) RNA in the PAXgene tube is unknown. Thus, we examined the stability of BM RNA in the PAXgene tubes. BM from leukemia patients was collected into PAXgene and EDTA tubes and stored at 4 degrees C for 5 days. RNA isolated from both tubes was analyzed by a quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Porphobilinogen deaminase (PBGD) mRNA of BM (as high-expression mRNA) and Wilms tumor suppressor (WT1) mRNA of BM (as low-expression mRNA) and very low copies of major BCR-ABL mRNA (as minimal residual disease of leukemia) of leukemia of BM were quantified by a LightCycler system. RNA yield from the PAXgene tubes and the intensity of 28S rRNA bands on RNA electrophoresis showed a degradation trend. However, the intensity of 18S rRNA bands from the PAXgene tubes remained. The expression of PBGD and WT1 of BM in the PAXgene tubes did not decrease for 5 days. The very low copies of major BCR-ABL mRNA in the PAXgene tubes were detectable on day 5 but those in the EDTA tubes were not detectable. Therefore, the PAXgene tube can be used for BM samples in a quantitative RT-PCR of the fusion gene transcripts of leukemia.

Genomewide cDNA microarray screening of genes related to benefits and toxicities of platinum-based chemotherapy in patients with advanced lung cancer.

The authors conducted a study using cDNA microarray analysis to determine whether expression levels of genes in tumors were correlated with the outcome of chemotherapy. Forty-seven patients were studied, and all except 3 received platinum-based chemotherapy. The expression levels of 1176 genes in transbronchial biopsy specimens of tumors that were obtained before chemotherapy were analyzed using the Atlas Human Cancer 1.2 Array. Multivariate regression analysis revealed that 3 genes were each independent factors related to tumor resistance to chemotherapy and patient survival (P < 0.01). Among various chemotherapy-related toxicities, 1, 3, 3, 1, and 1 genes were also revealed to be independent factors that were correlated with neutropenia, anemia, diarrhea, infection, and increased serum creatinine respectively (P < 0.01). It is concluded that not only the benefits but also the toxicities of chemotherapy can be predicted by cDNA microarray using tumor specimens obtained before chemotherapy.