Gen Tamura

Inducible Nitric Oxide Synthase and Tumor Necrosis Factor-Alpha in Myocardium in Human Dilated Cardiomyopathy

OBJECTIVES We examined the mRNA expression and protein localization of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in myocardial tissue obtained from patients with dilated cardiomyopathy (DCM). BACKGROUND The etiology of DCM is unknown, but viral infection or autoimmune abnormalities that induce cytokine expression have been proposed as pathogenetic factors. Nitric oxide (NO), synthesized by nitric oxide synthase (NOS), has negative inotropic and cytotoxic effects on cardiomyocytes. Cytokines such as TNF-alpha are potent stimulators of iNOS expression. Expression of iNOS leads to excessive production of NO in the myocardium and may modulate cardiac contractility and ventricular morphology. METHODS We examined the mRNA expression and protein localization of iNOS and TNF-alpha in myocardial tissue obtained from 24 patients with DCM, 20 patients with hypertrophic cardiomyopathy (HCM) and 15 control subjects, using the reverse transcriptase-polymerase chain reaction method and immunohistochemical studies. We then compared the differences in clinical characteristics between DCM patient subgroups with and without myocardial iNOS expression. RESULTS Messenger RNA expression of iNOS and TNF-alpha was observed, respectively, in 13 (54%) and 18 (75%) patients with DCM. Gene expression of TNF-alpha was consistently detected in endomyocardial tissue from patients with DCM and INOS expression. Inducible NOS protein was evident only in cardiomyocytes, whereas TNF-alpha was apparent in both cardiomyocytes and endomyocardial endothelium. Neither mRNA expression nor protein localization of iNOS or TNF-alpha was observed in cardiac tissue obtained from patients with HCM or control subjects. Patients with DCM and iNOS mRNA showed a lower left ventricular ejection fraction (p < 0.01) and a higher left ventricular volume (p < 0.05) than the negative DCM group. CONCLUSIONS Inducible NOS was consistently coexpressed with TNF-alpha in myocardial tissue obtained from a subgroup of patients with DCM and advanced left ventricular dysfunction.

Inactivation of the E-Cadherin Gene in Primary Gastric Carcinomas and Gastric Carcinoma Cell Lines

We investigated the E (epithelial)‐cadherin gene for mutations and loss of heterozygosity (LOH) in 24 primary gastric carcinomas (12 differentiated and 12 undifferentiated types, including 3 signet‐ring cell carcinomas), as well as 4 gastric carcinoma cell lines of the undifferentiated type (MKN‐45, GCIY, HGC‐27 and GT3TKB). We utilized PCR‐SSCP and RT‐PCR followed hy direct sequencing to detect gene mutations and skipped exons, and RT‐PCR‐SSCP to examine LOH. In primary carcinomas, gene mutations or skipped exons, were detected in 4 of 9 (44%) undifferentiated carcinomas of the scattered type, including 2 signet‐ring cell carcinomas, and in none of the 3 undifferentiated carcinomas of the adherent type and 12 differentiated carcinomas. Demonstrated mutations of the E‐cadherin gene included an 18 bp deletion (codon 418‐423) and a 3 bp deletion (codon 400, calcium‐binding domain), both located in exon 9. Skipping of exon 9 with a 1 bp insertion at codon 337, and skipping of exon 8 with a 1 bp deletion at codon 336, also were detected. LOH was confirmed in all of the carcinomas in which gene mutations or skipped exons (3/3 informative cases) were demonstrated. The MKN‐45 cell line exhibited an 18 bp deletion at the exon 6‐intron 6 boundary with loss of the wild‐type allele, and 2 of the remaining 3 cell lines (HGC‐27 and GT3TKB) had lost expression without detectable structural alteration of the E‐cadherin gene. These data provide support for classic two‐hit inactivation of the E‐cadherin gene in a high percentage of undifferentiated carcinomas of the scattered type.

ALLELOTYPE OF ADENOMA AND DIFFERENTIATED ADENOCARCINOMA OF THE STOMACH

The molecular mechanism of gastric tumourigenesis has not yet been clarified, although investigators have postulated that differentiated adenocarcinoma may arise from pre‐existing adenoma, similarly to the colorectal adenoma–carcinoma sequence. An allelotype analysis has been performed to identify chromosomal regions which are frequently deleted in gastric tumours and to examine the significance of the adenoma–carcinoma sequence in gastric tumourigenesis. Forty‐five gastric tumours, 20 adenomas, and 25 differentiated adenocarcinomas were examined for loss of heterozygosity (LOH) using 39 microsatellite markers covering each non‐acrocentric chromosome arm. Frequent LOH in the adenocarcinomas was observed on chromosomes 2q (33 per cent), 4p (33 per cent), 5q (50 per cent), 6p (33 per cent), 7q (43 per cent), 11q (36 per cent), 14q (38 per cent), 17p (45 per cent), 18q (36 per cent), and 21q (40 per cent). In contrast, the incidence of LOH in adenomas did not exceed 10 per cent at any of the loci examined. In addition to the p53 gene on 17p and the DCC gene on 18q, which are known to be frequently deleted in differentiated adenocarcinomas of the stomach, other unknown tumour suppressor genes on the above‐mentioned chromosomes may also be inactivated. These observations suggest that the adenoma–carcinoma sequence is not a major pathway in gastric tumourigenesis.

Loss of heterozygosity during the development and progression of differentiated adenocarcinoma of the stomach

In a recent allelotypic analysis of differentiated adenocarcinoma of the stomach, loss of heterozygosity (LOH) was found frequently on chromosomes 2q, 4p, 5q, 6p, 7q, 11q, 14q, 17p, 18q, and 21q. To clarify the sequence of these chromosomal losses during gastric carcinogenesis, microsatellite analysis of the chromosome arms described above was performed in 25 early and 29 advanced differentiated adenocarcinomas of the stomach. LOH on these chromosome arms fell within a range of 20–50 per cent. On 4p, 7q, 14q, 17p, and 21q, LOH was detected at a similar frequency in both early and advanced carcinomas, while LOH on 2q, 5q, 6p, 11q, and 18q was observed more than twice as frequently in advanced than in early lesions. Mean fractional allelic losses (FALs) were 0·221 in early and 0·413 in advanced carcinomas, representing a significant difference (P<0·05). These results suggest that LOH on 4p, 7q, 14q, 17p, and 21q is a relatively early event, while LOH on 2q, 5q, 6p, 11q, and 18q typically accumulates during the progression of gastric carcinogenesis.

Functionally inactivating point mutation in the tumor‐suppressor IRF‐1 gene identified in human gastric cancer

Loss of heterozygosity (LOH) observed in human tumors strongly suggests the existence of (a) tumor‐suppressor gene(s) at the concerned locus. A series of studies has revealed that LOH on the long arm of chromosome 5 (5q) frequently occurs in differentiated gastric adenocarcinomas. Furthermore, it has been shown that the interferon regulatory factor‐1 (IRF‐1) locus on chromosome 5q31.1 is one of the common minimal regions of LOH in these cancers. IRF‐1 is a transcriptional activator that shows tumor‐suppressor activity in the mouse. In the present study, we examined the sequence of the IRF‐1 gene in 9 cases of histologically differentiated gastric adenocarcinomas, all of which exhibited LOH at the IRF‐1 locus. We identified a mis‐sense mutation in the residual allele in one case. This mutated form of IRF‐1 showed markedly reduced transcriptional activity. In addition, over‐expression of wild‐type IRF‐1 induced cell‐cycle arrest, whereas such activity was attenuated in the mutant IRF‐1. These results suggest that the loss of functional IRF‐1 is critical for the development of human gastric cancers. Int. J. Cancer 77:522–527, 1998.

Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy.

Viral infection, especially by enteroviruses, has been considered to be the most common cause of myocarditis, which may progress to dilated cardiomyopathy (DCM). Although the mechanism of progression remains uncertain, a cytokine-associated injury of myocytes has been proposed. Using reverse transcriptase polymerase chain reaction (RT-PCR), we examined the expression of interleukin 1β (IL-1β), IL-6, IL-8 and tumour necrosis factor alpha (TNF-α) and the presence of enteroviral genomic RNA in endomyocardial biopsy tissues obtained from patients with myocarditis and DCM. We examined endomyocardial biopsy tissues obtained from 6 patients with myocarditis, 21 with DCM and 15 with non-infectious cardiac diseases as controls. In patients with myocarditis, endomyocardial biopsy was performed twice at an interval of 1 month to 8 years after the onset of myocarditis. We used RT-PCR to detect IL-1β, IL-6, IL-8 and TNF-α genes expression and nested RT-PCR (nRT-PCR) to detect enteroviral genomic RNA. IL-1β, IL-6, IL-8 and TNF-α genes were expressed in 100% (6/6) and enteroviral genomic RNA in 67% (4/6) of myocarditis patients at the first biopsy. At the second biopsy, IL-1β, IL-6, IL-8 and TNF-α genes were expressed in none, 50% (3/6), 67% (4/6) and 67% (4/6), respectively, and enteroviral genomic RNA in 67% (4/6). Four patients with myocarditis, in whom IL-8 and TNF-α genes and enteroviral genomic RNA were detected, progressed to DCM at the second biopsy. IL-1β, IL-6, IL-8 and TNF-α genes were expressed in none, 24% (5/21), 38% (8/21), 57% (12/21) of DCM patients, respectively. Enteroviral genomic RNA was detected in 43% (9/21) of DCM. Neither cytokine expression nor enteroviral genomic RNA were detected in the controls. The high incidence of cytokines, especially IL-6, IL-8 and TNF-α, expression in myocarditis and DCM, which might be induced by enteroviral infection, suggests that cytokines play an important role in myocytic damage leading to DCM.

Commonly deleted regions on the long arm of chromosome 21 in differentiated adenocarcinoma of the stomach

During an allelotype analysis of differentiated adenocarcinoma of the stomach, we observed frequent loss of heterozygosity (LOH) on several chromosomes including the long arm of chromosome 21 (21q). Therefore, we analyzed DNA isolated from 45 tumors for LOH at 10 loci on 21q by using polymorphic microsatellite markers. In 20 (44%) of 45 tumors, we detected LOH at single or multiple loci on 21q. Deletion mapping of these 20 tumors revealed two separate commonly deleted regions. Our findings suggest that 21q contains at least two potential tumor suppressor genes which play crucial roles in the development of differentiated adenocarcinoma of the stomach. Genes Chromosom. Cancer 18:318–321, 1997.

Aberrations of theAPC gene in primary breast carcinoma

Aberrations of theAPC gene, which plays an important role in the genesis of familial adenomatous polyposis and colorectal carcinoma, were investigated in 31 surgical specimens of primary breast carcinoma. These studies utilized the polymerase chain reaction followed by restriction-fragment-length polymorphism and single-strand-conformation polymorphism analyses combined with tumor cell enrichment by cell sorting. Loss of heterozygosity at theAPC locus was detected in 8 (38%) of 21 informative cases, but only 2 (6%) of 31 tumors carried a mutatedAPC gene. Direct DNA sequencing analysis confirmed mutations at codon 1081 (AGC to ATC) resulting in an amino acid substitution of serine for isoleucine, and at codon 1096 (CAG to CAT) resulting in a substitution of glutamine for histidine. There were no significant correlations between the loss of heterozygosity or mutation at theAPC locus and any clinicopathological characteristics. Our present observations suggest that the mutations of theAPC gene may play an important role in the genesis of certain breast carcinomas, and that another tumor-suppressor gene, which is the true target of frequent loss of heterozygosity, may exist near theAPC gene.

Mutations in the human homologue of the Drosophila patched gene in esophageal squamous cell carcinoma

The human homologue (PTCH) of the Drosophila segment polarity gene patchedhas recently been identified as a tumor‐suppressor gene for nevoid basal cell carcinoma syndrome and for sporadic basal cell carcinomas of the skin. We analyzed 30 esophageal squamous cell carcinomas (ESCC) for intrageneic mutations of the PTCH gene by polymerase chain reaction–single‐strand conformation polymorphism analysis followed by DNA sequencing. We identified two somatic PTCH mutations (7%) in 30 ESCC. These were a nonsense mutation (CAG to TAG at codon 361) in exon 8 and a missense mutation (CAG to CTG, Gln to Leu at codon 816) in exon 14. These tumors exhibited loss of heterozygosity at the polymorphic site of the PTCH gene. These results indicate that inactivation of the PTCH gene via a two‐hit mechanism occurs in a subset of ESCC. Genes Chromosomes Cancer 21:276–279, 1998.

Tylosis esophageal cancer locus on chromosome 17q25.1 is commonly deleted in sporadic human esophageal cancer

BACKGROUND & AIMS Tumor-suppressor genes found in inherited cancer predisposition syndromes are also responsible for sporadic cancers of the same type. Recently, the tylosis oesophageal cancer (TOC) gene locus has been mapped to 17q25 by linkage analyses of pedigrees with focal nonepidermolytic palmoplantar keratoderma associated with a high risk of esophageal cancer development. The aim of this study was to clarify whether the TOC locus is affected in sporadic esophageal cancers. METHODS We investigated loss of heterozygosity (LOH) on 17q in 58 sporadic esophageal squamous cell carcinomas (ESCs) using 20 microsatellite markers focusing on the TOC locus. RESULTS LOH on 17q was observed in 37 of 52 (71%) informative cases at one or more loci, 80% (33/37) of which included the TOC locus. The smallest common deleted region was at D17S1839 within the TOC locus. CONCLUSIONS The constructed deletion map revealed that the TOC locus is commonly deleted in sporadic ESCs, suggesting that a tumor-suppressor gene responsible for ESC is contained within this locus.