Nobuya Ohishi

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase.

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer†

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non‐small‐cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5‐aza‐CdR treatment. After a detailed DNA methylation analysis, we found that mir‐34b and mir‐126 were silenced by DNA methylation. Mir‐34b was silenced by the DNA methylation of its own promoter, whereas mir‐126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir‐34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir‐34b and mir‐126 decreased the expression of c‐Met and Crk, respectively. The 5‐aza‐CdR treatment of lung cancer cell line resulted in increased mir‐34b expression and decreased c‐Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir‐34b and mir‐126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir‐34b was not associated with c‐Met expression determined by immunohistochemistry, but both mir‐34b methylation (p = 0.007) and c‐Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir‐34b can be used as a biomarker for an invasive phenotype of lung cancer.

Fine Localization of a Major Disease-Susceptibility Locus for Diffuse Panbronchiolitis

Diffuse panbronchiolitis affecting East Asians is strongly associated with the class I human leukocyte antigen (HLA) alleles. Recent observations suggest that a major disease-susceptibility gene may be located between the HLA-B and HLA-A loci in the class I region of the major histocompatibility complex on chromosome 6. To test this possibility, we analyzed 14 polymorphic markers in 92 Japanese patients and 93 healthy controls. Of these, seven marker alleles, including HLA-B54 and HLA-A11, were significantly associated with the disease. Maximum-likelihood haplotype analysis and subsequent direct determination of individual haplotypes identified a group of disease-associated haplotypes, one of which contained all seven disease-associated marker alleles. Another haplotype, containing HLA-B*5504, was also associated with the disease. All these haplotypes seem to have diverged from a common ancestral haplotype in East Asians and share a specific segment containing three consecutive markers between the S and TFIIH loci in the class I region. Furthermore, one of the markers within the candidate region showed the highest delta value, indicating the strongest association. Of 20 Korean patients with diffuse panbronchiolitis, 17 also shared the combination of the disease-associated marker alleles within the candidate region. These results indicate that an HLA-associated major susceptibility gene for diffuse panbronchiolitis is probably located within the 200 kb in the class I region 300 kb telomeric of the HLA-B locus on the chromosome 6p21.3.

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT–PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2′-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Tissue distribution of leukotriene A4 hydrolase activity in guinea pig

The distribution of the activity of leukotriene A4 hydrolase, an enzyme catalyzing hydrolysis of the allylic epoxide leukotriene A4 to the dihydroxy acid leukotriene B4, was determined in various tissues of guinea pig by using the supernatant fraction (100,000 X g, 60 min) of the homogenates. The activity was ubiquitously distributed in all tissues examined, and the highest specific activity was found in small intestine, followed by lung, aorta, colon, and spleen, in this order. The specific activity in these tissues was higher than that of leukocytes. The physiological roles of this compound in these organs deserve reexamination in this context.

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Abstract: Leukotriene B4 [5(S), 12(R)‐dihydroxy‐6,14‐cis‐8,10‐trans‐eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5‐lipoxygenase [arachidonic acid to 5(S)‐hydroperoxy‐6‐trans‐8,11,14‐cis‐eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5‐lipoxygenase was Ca2+‐dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5‐lipoxygenase by four‐ to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.

Leukotriene A4 hydrolase, a bifunctional enzyme Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu‐297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His‐296, His‐300 and Glu‐319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site‐directed mutagenesis experiments were carried out. Glu‐297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu‐297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.

Bronchiolitis obliterans organizing pneumonia after syngeneic bone marrow transplantation for acute lymphoblastic leukemia

We report a patient who developed bronchiolitis obliterans organizing pneumonia (BOOP) after syngeneic BMT for ALL. The patient complained of persistent low-grade fever and non-productive cough after engraftment. Chest CT scan showed patchy infiltration bilaterally in the lower lung fields. Antibiotics were ineffective. Cultures, serological studies and polymerase chain reaction detected no infectious pathogens. We finally made a diagnosis of BOOP by thoracoscopical lung biopsy. The lung lesion disappeared in a month with corticosteroid therapy. While BOOP following allogeneic BMT has been reported, this is the first report after syngeneic transplantation.

Identification of G0S2 as a gene frequently methylated in squamous lung cancer by combination of in silico and experimental approaches

Epigenetic changes can lead to abnormal expression of genes in cancer, and several genes have been reported to have aberrant promoter DNA methylation in non‐small‐cell lung cancer (NSCLC). We identified aberrantly methylated genes in NSCLC by combination of in silico and experimental approaches. We first applied bioinformatics, and from microarray datasets, we selected genes with low expression and having functions related to cancer. Next, combined bisulfite restriction analysis was carried out in 10 pooled resected lung cancer tissues to screen for genes that were aberrantly methylated, and the methylation ratio (the fraction of methylated DNA in extracted DNA from a cancer tissue sample) was quantified using quantitative analysis of methylated alleles. We identified 8 methylated genes (ARPC1B, DNAH9, FLRT2, G0S2, IRS2, PKP1, SPOCK1 and UCHL1) previously unreported in NSCLC. Analyses of methylation profiles of 101 resected lung cancer tissue samples revealed quantitatively low methylation in whole, methylation ratios were almost less than 30% even in the methylated samples, and no significant correlation to prognosis after 2 years of follow‐up using hierarchical clustering. DNA methylation of G0S2 gene was significantly more frequent in squamous lung cancer (n = 18, mean of methylation ratios: 15%) compared with nonsquamous lung cancer (n = 83, mean of methylation ratios: 2.6%) (Mann‐Whitney U test, p < 0.001). DNA methylation of G0S2 can be an important biomarker for squamous lung cancer.

CpG island methylation of microRNAs is associated with tumor size and recurrence of non-small-cell lung cancer

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non‐small‐cell lung cancer. The methylation of mir‐152, ‐9‐3, ‐124‐1, ‐124‐2, and ‐124‐3 was analyzed in 96 non‐small‐cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir‐9‐3, ‐124‐2, and ‐124‐3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression‐free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression. (Cancer Sci 2011; 102: 2126–2131)