E. Tahara

Immuno-histochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non-tumor tissue sections.

Human telomerase is expressed in germ tissues and in the majority of primary tumors. Cell renewal tissues and some pre-cancerous tissues also have weak telomerase activity. Yet, neither the exact location and frequency of telomerase-positive cells nor the changes in telomerase expression during differentiation or carcinogenesis of individual cells are known. This paper reports on the expression of hTERT (telomerase reverse transcriptase) protein in tumor and non-tumor colorectal tissues by Western blotting and tissue sections by immuno-histochemistry using antibodies raised against partial peptides of hTERT. Though telomerase activity and hTERT expression at both mRNA and protein levels were generally higher in tumor part than in non-tumor part, these two were not always correlated: expression of hTERT did not always give rise to high telomerase activity. Colonic carcinoma cell nuclei were stained with anti-hTERT antibodies but not with antigen-preabsorbed antibodies. In normal mucosa, hTERT protein was expressed, though weaker than in carcinoma, in all colonic crypt epithelial cells except those at the tip; the expressing-cell distribution was much wider than that of Ki-67 positive cells which were located at the bottom of the crypt. Isolated crypt contained a significant level of hTERT protein revealed by Western blotting, while having very weak telomerase activity. Telomerase activity was detected in epithelial cells only at the bottom half of the crypt. Specific hTERT-staining was positive in tissue lymphocytes but negative in almost all other stromal cells. It is of interest to see whether a significant level of hTERT expression with low telomerase activity is characteristic of physiologically regenerating tissues containing stem cells. In situ detection of the hTERT protein will permit further analysis of cancer diagnosis and stem cell differentiation.

Transfection of interleukin-8 increases angiogenesis and tumorigenesis of human gastric carcinoma cells in nude mice

The growth and spread of tumour cells depends on adequate vasculature. We have previously reported that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. To provide evidence for a causal role of IL-8 in angiogenesis and tumorigenicity of human gastric cancer, we used the lipofectin method to stably transfect the human TMK-1 gastric carcinoma cells (low endogenous IL-8) with an IL-8 expression vector or control vector. Transfection with IL-8 did not affect the proliferation of cultured cells, yet the culture supernatants of the transfected (but not control) cells stimulated proliferation of human umbilical vein endothelial cells. The IL-8-transfected and control cells were injected into the gastric wall of nude mice. IL-8-transfected cells produced rapidly growing, highly vascular neoplasms as compared to control cells. These results provide direct evidence for the role of IL-8 in the angiogenesis and tumorigenicity of human gastric carcinomas.

DNA hypermethylation at the pS2 promoter region is associated with early stage of stomach carcinogenesis

pS2, a member of the trefoil peptide family, has been suggested to be a gastric-specific tumor suppressor. We examined the expression of pS2 in gastric carcinomas, adenomas and non-neoplastic mucosa and analyzed the DNA methylation in the pS2 promoter. Reduced expression of pS2 was frequently associated with well-differentiated adenocarcinomas. The CpG sites within the promoter region of the pS2 gene were methylated in pS2-negative gastric carcinoma cell lines whereas it was not in pS2-positive cell line. The promoter methylation was detected in gastric carcinoma tissues and intestinal metaplasia with reduced pS2 expression whereas none of the carcinomas with preserved pS2 expression showed the promoter methylation. These findings suggest that reduced expression of pS2 due to the promoter methylation may participate in an early stage of stomach carcinogenesis, especially of well differentiated type.

Expression of telomerase catalytic component, telomerase reverse transcriptase, in human gastric carcinomas

Telomerase activity is believed to be crucial for cellular immortality, which is considered to participate in the development of a majority of human cancers. Human telomerase reverse transcriptase (TERT) has recently been identified as a catalytic subunit of telomerase. We examined the expression of TERT and other telomerase components such as human telomerase RNA component (hTR, encoded by TERC) and human telomerase‐associated protein (TEP1) by reverse transcription‐polymerase chain reaction in human gastric carcinomas and non‐neoplastic mucosa, in addition to measuring the telomerase activity. Of 20 gastric carcinomas examined, 18 (90%) and 18 (90%) showed increased expression of TERT and higher telomerase activity in comparison with corresponding non‐neoplastic mucosa, respectively. Increased expression of hTR/TERC was also observed in 15 (75%) of the gastric carcinomas. Immunohistochemically, strong expression of TERT protein was detected in the nuclei of the tumor cells of all carcinoma tissues, while the expression of TERT in non‐neoplastic mucosal cells as well as stromal elements (except lymphocytes) was weak or negative. These findings suggest that increased TERT expression associated with telomerase activity may serve as a novel marker for the diagnosis of stomach cancer.

Quantitative reevaluation of telomerase activity in cancerous and noncancerous gastrointestinal tissues.

Telomerase activity has been examined extensively in a variety of human cancerous and noncancerous tissues. However, it was sometimes difficult to measure telomerase activity quantitatively with the methods used and in the tissues examined. We examined telomerase activity quantitatively in gastrointestinal tissues by using the hybridization protection assay combined with the telomeric repeat amplification protocol (TRAP) to assess the diagnostic utility of measuring telomerase activity and to determine the relationship between telomerase activity and human telomerase reverse transcriptase (hTERT) expression. We report here that (i) polymerase chain reaction (PCR) inhibitors in the tissue extracts used for the telomerase assay were practically nullified by using tissue extract at 0.1 μg of protein/assay; (ii) RNase activity in tissue extracts should be blocked with 0.5 U of RNase inhibitor/μg tissue protein for the quantitative telomerase assay; (iii) no inhibitors of telomerase were found in tissue extracts other than RNase and PCR inhibitors (iv) higher telomerase activity in cancerous tissue than in noncancerous tissue from the same patients was observed in both gastric and colorectal tissues, but the telomerase activity varied from low to high levels in cancerous tissues, and it was not practical to set a general cut‐off level for cancer diagnosis; (v) hTERT was expressed in both cancerous and noncancerous tissues, and (vi) the telomerase activity levels were generally lower than expected from the hTERT expression levels, suggesting posttranscriptional regulation of expression of telomerase activity. Mol. Carcinog. 26:312–320, 1999.

Expression of telomerase component genes in hepatocellular carcinomas.

The aim of the study was to clarify the role of telomerase component genes in hepatocarcinogenesis and to examine both the relationship between the expression of telomerase component genes and histological differentiation in hepatocellular carcinoma (HCC) and the relationship between expression levels of telomerase component genes and telomerase activity in HCCs. Telomerase is a ribonucleoprotein enzyme composed of a template RNA and several proteins. Recently, three such telomerase component genes have been identified: human telomerase reverse transcriptase (hTERT); human telomerase RNA component (hTERC); and telomerase-associated protein 1 (TEP1). The expression of these components was evaluated in 34 HCCs and 24 non-cancerous liver tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of hTERT mRNA was detected in most HCCs, but not in the non-cancerous tissues (P<0.01). Expression of hTERC was detected in both HCCs and non-cancerous tissues, but the expression level in HCCs was higher than that in non-cancerous tissues (P<0.01) and tended to increase as histological differentiation became less marked. The expression level of hTERT mRNA correlated with relative telomerase activity (P<0.01). These results suggest that telomerase reactivation during hepatocarcinogenesis might be regulated by only hTERT and an increase in telomerase activity level in tumour progression might be regulated by both hTERT and hTERC.

Immunohistochemical Detection of Human Telomerase Reverse Transcriptase in Normal Mucosa and Precancerous Lesions of the Stomach

Telomerase activity confers cell immortality through stabilization of the chromosome, participating in the development of a majority of human cancers. Human telomerase reverse transcriptase (TERT) has been identified as a catalytic subunit of telomerase, and is overexpressed in most gastric carcinomas. We immunohistochemically examined the expression of TERT in normal gastric mucosa and candidate precancerous lesions such as intestinal metaplasia and adenoma. In non‐neoplastic gastric mucosa including intestinal metaplasia and normal fundic mucosa, weak but significant expression of TERT was detected in nuclei of epithelial cells located in the lower two‐thirds of the glands (wider than the proliferative zone). The telomerase activity was found in a half of gastric adenomas, whose levels of the activity were about 10% of those in gastric carcinomas. TERT protein was expressed in the nuclei of the adenoma cells at moderate levels, that were not necessarily comparable with the telomerase activities. These findings overall suggest that TERT expression may be one of the prerequisites for telomerase activation in an early stage of stomach carcinogenesis.

Lysozyme in human gastric carcinoma: a retrospective immunohistochemical study.

A total of 171 gastric carcinomas comprising 69 advanced cancers and 102 early cancers were examined immunohistochemically for lysozyme. Tumour cells containing lysozyme were detected in 65 cases or 38% of the 171 gastric cancer cases. The incidence of these cells did not differ remarkably by histological type and infiltrative growth of gastric carcinoma. Of the foregoing 65 cases, two well‐differentiated adenocarcinomas and three signet ring cell carcinomas had numerous lysozyme‐containing tumour cells, 13 had many argentaffin or argyrophil cells, and 40 had various amounts of several types of mucin. In addition, tumour cells containing both lysozyme and mucin could be identified. No correlation could be observed between lysozyme immunoreactivity in the tumour cells and cellular infiltration of granulocytes or macrophages around the tumour. The lysozyme appeared to be produced by tumour cells. The two year survival rates indicate a tendency for advanced gastric cancers containing lysozyme to have a poor prognosis.

Mucosal Changes of the Gallbladder in Anomalous Union with the Pancreatico-Biliary Duct System

Histological examination of the gallbladder mucosa was made on a total of 39 patients with anomalous union of the pancreatico-biliary duct system. The most characteristic finding was mucosal hyperplasia of the gallbladder. Measurement of the height of the mucosa revealed that 15 cases (38.5%) showed mucosal hyperplasia composed of ordinary gallbladder epithelium without any metaplastic changes, including two cases of primary mucosal hyperplasia of the gallbladder. The other finding was metaplastic changes which were observed in 26 cases in various degrees, of whom 16 cases showed only focal metaplasia. These findings indicate that metaplasia frequently occurred in the gallbladder but the distribution of metaplasia was relatively focal in anomalous union with the pancreatico-biliary duct system. In our present materials there were nine cases of gallbladder adenocarcinoma and based on our classification, they were divided into seven cases of metaplastic type and two cases of non-metaplastic type. This ratio was not different from that of usual gallbladder carcinoma not complicated by this anomaly. These results indicate that the reflux of pancreatic juice into the gallbladder by anomalous union with the pancreatico-biliary duct system may cause two different effects on the gallbladder mucosa, the first being a proliferative effect resulting in mucosal hyperplasia and the other being chronic irritation causing metaplastic changes. The relation between these changes and the pathogenesis of gallbladder carcinoma is briefly discussed.

In situ mRNA Hybridization Technique for Analysis of Human Telomerase RNA in Gastric Precancerous and Cancerous Lesions

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal somatic cells but is reactivated during tumor progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human gastric precancerous and cancerous lesions by using in situ mRNA hybridization (ISH) with avidin‐biotin staining. We also examined telomerase activity in these lesions by using hybridization protection assay connected with a telomeric repeat amplification protocol (TRAP/HPA). Analyzed tissue samples were as follows; 132 cases of chronic atrophic gastritis without intestinal metaplasia, 115 incomplete‐type intestinal metaplasias, 40 complete‐type intestinal metaplasias, 23 hyperplastic polyps, 23 tubular adenomas and 26 adenocarcinomas. In ISH analysis, high levels of hTR expression were observed preferentially in the nuclei at the single‐cell level. hTR‐expressing cells in carcinomas and adenomas were significantly more frequent than those of the other lesions (P<0.001). The expression pattern of hTR in carcinoma and adenoma tissues was heterogeneous and similar intratumor heterogeneity was detected in Ki‐67 immunoreactivity. Infiltrating lymphocytes in tissues also exhibited high levels of hTR expression. In TRAP/HPA analysis, carcinomas had significantly more frequent positivity for telomerase activity and a higher level of telomerase activity than the other lesions (P<0.05). However, the amount of telomerase activity did not parallel the expression level of hTR. Our data suggest that hTR expression increases in the early stages of stomach carcinogenesis and that sufficient synthesis of hTR is a prerequisite for telomerase reactivation in tumorigenesis.