Functional coupling of chloride-proton exchanger ClC-5 to gastric H+,K+-ATPase.

It has been reported that chloride-proton exchanger ClC-5 and vacuolar-type H(+)-ATPase are essential for endosomal acidification in the renal proximal cells. Here, we found that ClC-5 is expressed in the gastric parietal cells which secrete actively hydrochloric acid at the luminal region of the gland, and that it is partially localized in the intracellular tubulovesicles in which gastric H(+),K(+)-ATPase is abundantly expressed. ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase in the lysate of tubulovesicles. The ATP-dependent uptake of (36)Cl(-) into the vesicles was abolished by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH28080), an inhibitor of H(+),K(+)-ATPase, suggesting functional expression of ClC-5. In the tetracycline-regulated expression system of ClC-5 in the HEK293 cells stably expressing gastric H(+),K(+)-ATPase, ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase, but not with endogenous Na(+),K(+)-ATPase. The SCH28080-sensitive (36)Cl(-) transporting activity was observed in the ClC-5-expressing cells, but not in the ClC-5-non-expressing cells. The mutant (E211A-ClC-5), which has no H(+) transport activity, did not show the SCH28080-sensitive (36)Cl(-) transport. On the other hand, both ClC-5 and its mutant (E211A) significantly increased the activity of H(+),K(+)-ATPase. Our results suggest that ClC-5 and H(+),K(+)-ATPase are functionally associated and that they may contribute to gastric acid secretion.

High level of expression of alpha-fetoprotein receptor in gastric cancers.

The expression of the receptor for alpha-fetoprotein (AFP-R) was examined immunohistochemically in 47 cancer and 14 benign human gastric tissues. Rabbit polyclonal antibody against human AFP-R was used for immunohistochemical staining. Thirty-four of the 47 cancer tissues expressed AFP-R showing granular or reticular staining on the cancer cell surface, while only 2 of 61 control cases (14 benign gastric tissues and 47 nonmalignant tissues adjacent to cancer) showed faint and homogeneous staining in the cytoplasm of noncancerous cells. There was a significant difference in staining intensity between the cancerous and noncancerous groups. However, no statistically significant difference in staining intensity was found among the groups of well-differentiated, moderately differentiated and poorly differentiated adenocarcinomas. On the other hand, the staining intensity of signet ring cell carcinoma was significantly weaker than that of the three adenocarcinoma groups. The high level of AFP-R expression in gastric cancers may allow the use of AFP-R as a new clinically useful marker of gastric cancer in the tissue level.

Risk factors for hepatocellular carcinoma in Hepatitis C patients with sustained virologic response to interferon therapy.

BACKGROUND: Although a variety of papers demonstrated inhibited hepatocarcinogenesis with interferon (IFN) therapy for chronic hepatitis C, a small number of hepatocellular carcinomas (HCCs) were still observed even in sustained virologic responders. AIMS: To clarify factors affecting the development of HCC, we analyzed the frequency of HCC in sustained virologic responders over a long-term observation period. METHODS: Seven hundred and ninety-two out of the 2623 IFN-treated hepatitis C patients who had undergone liver biopsy showed sustained virologic response. Screening for development of HCC was performed periodically during an average follow-up of 5.1 years. Fibrosis of the pretreatment liver biopsy sample was graded. Risk factors for HCC were analyzed by using Cox proportional hazards regression. RESULTS: Of 792 patients, 23 developed HCC. Univariate analysis showed that stage of hepatic fibrosis, age, and alcohol consumption were significantly associated with a risk of HCC (P<0.001). There was a significant difference in the cumulative incidence between patients stratified according to these variables (P<0.001). CONCLUSIONS: Pretreatment hepatic fibrosis score, age, and alcohol consumption may affect development of HCC even in sustained virologic responders. Thus, patients with these factors should be carefully followed even after eradication of the virus.

Anti-endostatin monoclonal antibody enhances growth of human hepatocellular carcinoma cells by inhibiting activity of endostatin secreted by the transplanted cells in nude mice.

Endostatin, a fragment of collagen XVIII, inhibits angiogenesis in tumors, and is expected to become a new anticancer drug. However, its effectiveness is still controversial, because some researchers failed to reproduce the same marked regression of tumors by the peptide. We gave anti-endostatin monoclonal antibody, designated as CH18B, to nude mice transplanted with human hepatocellular carcinoma cells (JHH-1 line) that endogenously produced endostatin from collagen XVIII secreted by the cells themselves. As a result, CH18B promoted tumor angiogenesis by inhibiting endostatin activity in the tumor and subsequently increased tumor mass by preventing cancer cells from undergoing apoptosis. But the antibody itself did not stimulate proliferation of the tumor cells. Our present experimental procedure, the use of anti-endostatin antibody, definitely solved the question whether endostatin might exert its anticancer activity.

Distribution of collagen type IV alpha1-6 chains in human normal colorectum and colorectal cancer demonstrated by immunofluorescence staining using chain-specific epitope-defined monoclonal antibodies.

BACKGROUND AND AIM: Loss of basement membrane (BM) components, such as type IV collagen, has been demonstrated in colorectal cancer, but the fine diversity of the assembly of alpha (IV) chains, the composition of type IV collagen, and alterations in the collagen have not been fully analyzed. Here, we defined immunohistochemically the expression of alpha1-6 (IV) chains in colorectal cancer tissues and adjacent normal mucosa by the use of chain-specific monoclonal antibodies. METHODS: Tissue samples of tumor and adjacent normal mucosa obtained from patients with colorectal adenocarcinoma were stained with chain-specific monoclonal antibodies raised against synthetic peptides of individual alpha (IV) chains using an indirect immunofluorescence method. RESULTS: In the normal mucosa, alpha1 (IV), alpha2 (IV), alpha5 (IV), and alpha6 (IV) were found in the BM-delineating mucosal epithelium and the gland crypts, whereas alpha3 (IV) and alpha4 (IV) were limited to the BM of the luminal surface epithelium. In contrast, staining of alpha3-6 (IV) was rarely observed in the BM of cancer cells. Staining of alpha1 (IV) and alpha2 (IV) was reduced or lost from the cancer BM in relation to the degree of tumor differentiation: continuous staining in well-differentiated portions, discontinuous staining in moderately differentiated portions, and absence of staining in poorly differentiated portions. CONCLUSIONS: Our findings indicate that type IV collagen expression is altered in the BM of colorectal cancer as a result of changes of alpha (IV) chain expression, particularly alpha1 (IV) and alpha2 (IV), in relation to the degree of tumor differentiation.