Kazutaka Kurokohchi

pp60c-src activation in lung adenocarcinoma

Nine src family members are known including c-Src, c-Yes, c-Lck, c-Fyn, c-Hck, c-Lyn, c-Blk, c-Fgr and c-Yrk. They encode proteins with molecular weights of 55-62 kilodaltons (kDa), which are either cytoplasmic or membrane-associated protein tyrosine kinases. A close correlation exists between an elevated pp60c-src tyrosine kinase activity and cell transformation. However, the level of activation of pp60c-src in non-small cell lung cancers (NSCLC) remains obscure. The aim of this study was to examine the level of activity of pp60c-src in NSCLC. pp60c-src expression and in vitro protein tyrosine kinase activity in lung cancer tissue samples were measured by western blotting and in vitro kinase assays and compared with those in the surrounding non-tumour lung tissue from the same patient. pp60c-src phosphorylation was assessed by two-dimensional tryptic phosphopeptide mapping. The kinase activity of pp60c-src was significantly activated in NSCLC, especially in adenocarcinomas. In addition, the pp60c-src kinase activity increased with the size of the adenocarcinoma. Two-dimensional tryptic phosphopeptide mapping showed dephosphorylation of pp60c-src at Tyr 530 in adenocarcinomas. The proto-oncogene product, pp60c-src, was activated in NSCLC, especially in adenocarcinomas, in part through the dephosphorylation of Tyr 530. Our results suggest that activation of pp60c-src might play an important role in the progression of lung adenocarcinomas.

Combination therapy of bezafibrate and ursodeoxycholic acid in primary biliary cirrhosis: a preliminary study

Combination therapy of bezafibrate and ursodeoxycholic acid in primary biliary cirrhosis: a preliminary study

Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

Characterization of anti-histone antibodies in patients with type 1 autoimmune hepatitis

We have recently found that antibodies to total histones are common in a group of American patients with type 1 autoimmune hepatitis (AIH). In an attempt to determine the profile and clinical association of anti‐histone antibody (AHA), 45 Japanese AIH patients were studied for serum isotypic reactivity with individual histones (H1, H2A, H2B, H3, H4) by enzyme‐linked immunosorbent assay and western blotting. The results revealed that 40% of sera had reactivities with at least one of individual histones and that the antibodies were detected in all three classes of immunoglobulins (IgG, IgM, IgA). Immunoglobulin G type anti‐H3 showed the dominant reactivity and it characterized 72% of sera with AHA. The titre of anti‐H3 decreased significantly (P < 0.0075) after steroid therapy and the index of decrease for anti‐H3 was correlated in individuals with that for serum aminotransferase. In general, patients with AHA showed higher serum level of alanine aminotransferase (P < 0.05), immunoglobulin G (P < 0.025), and higher frequency of A2‐DR4 haplotype (53 vs 17%) than their seronegative counterparts. However, the titre of AHA was low in this disease condition and histone class‐specific antibodies did not distinguish patients with distinctive clinical features, although patients with anti‐H3 tended to be younger than those without AHA.

A novel cytotoxic T-cell epitope presented by HLA-A24 molecule in hepatitis C virus infection.

BACKGROUND/AIMS It has been suggested that cytotoxic T lymphocytes (CTL) have crucial roles for the hepatocellular damage in hepatitis C virus (HCV) infection. A series of CTL epitopes located in the HCV protein have been identified. However, no CTL epitopes restricted by HLA-A24, a common HLA allele in humans, has been identified. METHODS Peripheral blood and liver infiltrating mononuclear cells from the patients with hepatitis C virus infection and healthy controls were stimulated with a series of peptides containing HLA-A24 binding motifs located in HCV protein. RESULTS An immunodominant HLA-A24 restricted CTL epitope (A24-4; AYSQQTRGL, amino acids 1031-1039) presented by HLA-A24 molecule was identified using a series of synthetic peptides containing the HLA-A24 binding motifs. The CTL activity against this peptide was induced both in peripheral blood and liver infiltrating mononuclear cells from HLA-A24-positive chronic hepatitis C patients, not from HLA-A24-negative patients and HLA-A24-positive healthy controls. CTL activity was blocked by anti-HLA-A24 and anti-CD8 antibodies, not by anti-CD4 antibody. Furthermore, the A24-4-specific CTL recognized the HCV gene transfected target cells. CONCLUSIONS Because this peptide is presented by a common HLA class I molecule, it might be useful for protection against hepatocellular damage and vaccine development in large population of the HCV-infected patients.

Reduced expression of cell cycle regulator p18INK4C in human hepatocellular carcinoma

Cyclins, cyclin‐dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18INK4C, a member of the INK4 family of CdkIs, is a potential tumor‐suppressor gene product. However, the expression of p18INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18INK4C‐ positivity. Further, the kinase activity of Cdk4 was found to be higher in p18INK4C‐negative HCCs than in p18INK4C‐ positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18INK4C‐ positive HCCs, p18INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18INK4C ‐ negative than in p18INK4C ‐ positive HCCs. In conclusion, the loss of p18INK4C expression may play a role in the differentiation and development of HCC through the up‐regulation of Cdk4 activity. (HEPATOLOGY 2004;40:677–686.)

Assessment of the presence and severity of esophagogastric varices by splenic index in patients with liver cirrhosis

Purpose To determine whether spleen size is related to the severity of esophageal varices or associated gastric varices and liver functions in patients with cirrhosis. Method The authors retrospectively studied spleen size on CT (splenic index [SI] = length × width × height of the spleen), liver functions, and the results of esophagogastric endoscopy in 110 patients with cirrhosis. They also analyzed SI in 112 controls. Results In controls, body weight, height, and age affected the SI. The SI in patients with uncompensated cirrhosis was greater compared with the SI in those with well-compensated disease (p = 0.0363). The SI in patients with esophageal varices was greater than in patients without esophageal varices (p < 0.0001), but patients with and without gastric varices had similar SI values. The SI in patients with the red color signs (red wale marking, cherry red spot, and hematocystic spot) on esophageal varices or with risky varices (enlarged tortuous varices with beady, nodular, or tumor shape associated with red color signs) was greater than in patients without these signs (p = 0.0029 and p = 0.0030, respectively). Conclusion The SI is a good indicator of the severity of esophageal varices and hepatic functional reserve in patients with cirrhosis.

Intravascular large B-cell lymphoma with FDG accumulation in the lung lacking CT/67gallium scintigraphy abnormality

Intravascular large B‐cell lymphoma (IVLBCL) is a rare lymphoma characterized by the presence of large tumour cells within the blood vessels. It has been considered that IVLBCL is a highly malignant disease with poor prognosis. However, it has been shown that a therapeutic effect resembling that of conventional B‐cell lymphomas may be obtained with the application of systemic chemotherapy at the early stage of this disease. Although involvement in the lung is often detected at autopsy, early diagnosis is quite difficult. In this report, we present a case of IVLBCL with pulmonary involvement where 18‐fluoro‐deoxyglucose positron emission tomography (FDG‐PET) was useful in the early diagnosis. Neither computed tomography (CT) nor 67gallium scintigraphy could reveal the presence of disease in the lung. Histological evidence of IVLBCL was obtained by TBLB after FDG uptake in the lung was confirmed by FDG‐PET. The patient exhibited a good response to the subsequent combination chemotherapy. We propose that FDG‐PET is a powerful tool for the early diagnosis of IVLBCL with pulmonary involvement, if the possibility of this disease presents in the patient with respiratory symptoms without abnormal findings by CT and 67gallium scintigraphy. Copyright

Repeated adenoviral administration into the biliary tract can induce repeated expression of the original gene construct in rat livers without immunosuppressive strategies

Background: Systemic adenoviral readministration appears to be limited by immunogenicity. Aims: We examined the feasibility of repeated adenovirus mediated gene transfer into the liver via the biliary tract. Methods: Recombinant adenoviruses carrying a reporter lacZ gene were infused retrogradely into the common bile duct of rats. Transduction efficiency of the lacZ gene was estimated histochemically and quantitatively. Results: Retrograde administration of recombinant adenoviruses into the common bile duct of rats resulted in efficient transgene expression in the liver, specifically in hepatocytes, but not in biliary epithelia. Transduction efficiency induced by intrabiliary adenoviral administration was not substantially different from that induced by intraportal adenoviral infusion. Transgene expression in the liver was however transient, and development of neutralising antibodies against adenovirus was observed in serum but not in bile. When adenoviruses were readministered into the common bile duct, successful re-expression of the transgene in the liver was achieved despite the existence of neutralising antibodies in serum. Interestingly, although proliferation of adenovirus specific T cells in response to adenoviral readministration was suppressed significantly by immunosuppressive FK506 treatment, levels of transgene expression in the liver achieved by intrabiliary adenoviral readministration were not significantly different between animals treated with and without FK506. Furthermore, third adenoviral administration into the common bile duct also induced successful transgene expression in the liver. Conclusions: These results suggest that adenovirus mediated gene transfer into the liver may be repeatable without immunosuppressive strategies in clinical settings by means of endoscopic retrograde cholangiography.

CD28-negative CD8-positive cytotoxic T lymphocytes mediate hepatocellular damage in hepatitis C virus infection.

The pathogenic mechanism for hepatocellular damage in hepatitis C virus (HCV) infection has not been clearly understood. Analysis of costimulatory molecules on lymphocytes may give us insight into the pathogenic mechanism of hepatocellular damage in HCV infection. Peripheral blood mononuclear cells (PBMCs) and liver infiltrating mononuclear cells (LIMCs) isolated from the HCV-infected patients were analyzed with antibodies directed against a variety of costimulatory molecules by flow cytometry. Blocking experiment against HLA-A24-restricted HCV-specific CTLs and immunohistochemical analysis were also performed. PBMCs expressing CD8, CD28, CD80, or CD154 were significantly reduced in HCV-infected patients compared with the healthy controls. CD28(+)CD8(+) PBMCs in the patients inversely correlated with ALT levels. Conversely, levels of CD28(−)CD8(+) LIMCs correlated with ALT levels. HCV-specific CTL activity was blocked by the treatment with anti-CD8 antibody, but not with anti-CD4 or anti-CD28 antibody. Immunohistochemical analysis revealed the accumulation of CD28(+) cells around the portal area in the liver of a patient with chronic active hepatitis C. These results suggest that CD28(+)CD8(+) T cells leave the circulation, move to the livers, and are activated in the portal area in proportion to the extent of liver diseases. CD28(−)CD8(+) T cells may finally function as effector T cells causing the hepatocellular damage in HCV infection.