Comparison study of the expressions of myristoylated alanine-rich C kinase substrate in hepatocellular carcinoma, liver cirrhosis, chronic hepatitis, and normal liver.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent substrate for protein kinase C (PKC) in a variety of cells. The aim of this study was not only to evaluate the expression and localization of MARCKS in various pathological liver tissues, including HCC, but also to analyze the difference in MARCKS expression between hepatitis virus-induced HCC and cirrhosis. The level of MARCKS and its phosphorylated proteins, as well as its localization, were determined using Western blot and/or immunohistochemistry in HCC and other pathological liver tissues. We also analyzed the change of MARCKS localization on the influence of MARCKS phosphorylation in the HLF cancer cell line by phosphorylation study. In addition, the relationship between MARCKS expression and proliferative activity was studied in HCC. In the immunohistochemical study, a very small amount of MARCKS protein was found along the contour of the hepatocellular membrane in normal liver and in cases of chronic hepatitis. MARCKS was up-regulated in liver cirrhosis tissue and was localized in the cytoplasm of hepatocytes. The expression of MARCKS was down-regulated in HCC tissues, as compared with non-tumorous liver cirrhosis tissues from the same patients. Furthermore, MARCKS was serine-phosphorylated in liver cirrhosis and HCC, and phosphorylated MARCKS was detected in a cytosolic fraction of these tissues. In a phosphorylation study using the HLF HCC cell line, MARCKS was displaced from the plasma membrane to the cytosol following the activation of protein kinase C (PKC) by phorbol 12-myristrate 13-acetate (PMA). Furthermore, the activity of cyclin D1 and cyclin E kinases was found to be higher in HCCs with low MARCKS expression than in HCCs with high MARCKS expression. These results suggest that up-regulation of MARCKS might be essential in the generation of cirrhotic nodules through chronic hepatitis from normal liver, and that the phosphorylation and/or down-regulation of MARCKS might play an important role in the development and progression of HCC from liver cirrhosis.

Effect of an aldose reductase inhibitor on esophageal dysfunction in diabetic patients.

BACKGROUND/AIMS: Disorders of the digestive tract in diabetic patients are mainly ascribed to disorders of the vagus nerve. Although aldose reductase inhibitors (ARIs) have been shown to be effective against diabetic peripheral neuropathy, their effectiveness on diabetic digestive neuropathy remains to be evaluated. The aim of the present study is to examine the effect of an ARI on the esophageal dysfunction in diabetic patients by monitoring pH and motility of the esophagus. METHODOLOGY: Eight type 2 diabetic patients with peripheral neuropathy were administered with the ARI epalrestat (150 mg/day) for 90 days, and esophageal pH and motility were monitored before and after the ARI treatment. RESULTS: Parameters related to the gastroesophageal acid reflux and the esophageal motility, such as % time of pH<4, DeMeester score, duration of the longest reflux episode, reflux episodes longer than 5 min, ratios of peristaltic waves with the amplitude greater than 25 mmHg and ratios of effective peristalsis were remarkably improved by the ARI treatment. CONCLUSIONS: Because the present study clearly demonstrated the effectiveness of an ARI on the esophageal dysfunction in diabetic patients, ARI may be useful for the treatment of diabetic digestive disorders.

Expression of G1 phase-related cell cycle molecules in naturally developing hepatocellular carcinoma of Long-Evans Cinnamon rats.

It has been shown that a variety of cell cycle-related proteins play important roles in the process of carcinogenesis including hepatocarcinogenesis. In the present study, we evaluated mRNA and protein expression of G1 phase-related cell cycle molecules in the process of hepatocarcinogenesis, using Long-Evans Cinnamon (LEC) rats, an animal model of hepatocellular carcinoma (HCC). The expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6 was measured quantitatively by real-time polymerase chain reaction. Cyclin D1 mRNA expression was increased significantly in chronic hepatitis liver compared with normal liver, and then decreased in HCC and the surrounding precancerous liver of LEC rats. Levels of Cdk4 mRNA were increased significantly in HCC compared to precancerous and chronic hepatitis livers. In contrast, mRNA levels of Cdk6 did not change significantly during hepatocarcinogenesis. We also evaluated the protein levels of these G1 phase-related cell cycle molecules by Western blot analyses and confirmed similar results. Total amounts of retinoblastoma protein (pRb) in the liver did not change significantly in the process of hepatocarcinogenesis in LEC rats. However, levels of phosphorylated pRb were increased markedly in the process of hepatocarcinogenesis, and the highest in HCC compared to precancerous, chronic hepatitis and normal livers. These results indicate that cyclin D1 may be involved in the regeneration of hepatocytes rather than hepatocarcinogenesis, while Cdk4 but not Cdk6 may play an important role in the development of HCC.

Enhancing and suppressing effects of dexamethasone on transgene expression in vitro.

Gene therapy is becoming an important treatment modality against various diseases including cancer, genetic disorders, infectious diseases and inflammatory diseases. For achieving successful gene therapy, it is extremely important to control the expression of a transduced therapeutic gene. However, there have been few studies examining the effect of glucocorticoid on transgene expression. We demonstrate here that the synthetic glucocorticoid dexamethasone significantly affected transgene expression in vitro. Bone marrow cells freshly prepared from rats and murine fibroblasts were infected with retroviruses carrying the neomycin phosphotransferase gene under the transcriptional control of the retroviral long terminal repeat promoter and the reporter lacZ gene under the transcriptional control of the simian virus 40 (SV40) early promoter. Retrovirus-infected cells were selected by G418. When retrovirus-infected bone marrow cells were cultured in the presence of dexamethasone, lacZ expression was markedly decreased. This suppressive effect of dexamethasone on transgene expression in bone marrow cells appeared to be mediated by a methylation-independent mechanism, because the suppressive effect was also observed in bone marrow cells that were supposed to contain a considerable number of methylated cells. In marked contrast, when retrovirus-infected fibroblasts were cultured in the presence of dexamethasone, lacZ expression was significantly increased. The enhancing and suppressing effects of dexamethasone on transgene expression were considered to be independent on promoter types, because the SV40 early promoter used to control lacZ expression does not contain a glucocorticoid-responsive element. Therefore, the different effects of dexamethasone on transgene expression appeared to be dependent on cell types. These results indicate that dexamethasone may play an important role for achieving successful gene therapy. Furthermore, these observations may also have important implications for future clinical applications of gene therapy.

Proliferative capability of hepatocytes and expression of G1-related cell cycle molecules in the development of liver cirrhosis in rats.

Liver cirrhosis is the end stage of various chronic liver diseases and its prognosis is very poor. One of the most important causes of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage. Cell cycle-related molecules have been shown to play essential roles in cell proliferation. Specifically, G1-related cell cycle molecules are important, because they are requisite for the entry into the cell cycle from the quiescent state. However, the role of these cell cycle molecules during the development of liver cirrhosis remains to be examined. In the present study, liver cirrhosis was produced in rats by intraperitoneally administering dimethylnitrosamine (DMN). Proliferative capability of hepatocytes estimated immunohistochemically by proliferating cell nuclear antigen staining was markedly increased at an early stage of cirrhosis development. However, it was gradually decreased thereafter and suppressed substantially at the time of cirrhosis manifestation. Cyclin D1 expression estimated by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method was also increased markedly at an early stage of cirrhosis development but decreased substantially thereafter. mRNA levels of catalytic subunits of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6, did not show significant changes during the development of liver cirrhosis. Among G1-specific Cdk inhibitors, expression of p15INK4b and p16INK4a estimated by an RT-PCR method was increased according to the progression of cirrhosis and reached a peak at the time of cirrhosis manifestation. Conversely, p18INK4c expression did not change significantly during the development of liver cirrhosis. These results suggest that cyclin D1 plays an essential role in hepatocyte proliferation in response to hepatic damage. However, with the decrease of cyclin D1 expression and increase of p15INK4b and p16INK4a expression, proliferative capability of hepatocytes is severely impaired and extracellular matrix components are deposited to retrieve space lost by the destruction of hepatic parenchyma, resulting in establishment of liver cirrhosis.

Aberrant cytokeratin expression and high susceptibility to apoptosis in autoimmune hepatitis.

Immunopathological differences between autoimmune hepatitis (AIH) and chronic hepatitis C (CH-C) have not been well investigated. Therefore, we immunohistochemically examined the expression of various cytokeratins (CKs) not only in liver tissues of AIH but also in those of CH-C at the active stage. Furthermore, to evaluate the immune surveillance system and the susceptibility to apoptosis, immunohistochemical staining of human leukocyte antigen (HLA)-DRalpha, cathepsin D, B cell leukemia-2 (bcl-2), bcl-2-associated X protein (bax) and caspase 3 was also performed. Heterogeneous expression of CK 8 and CK 18 was observed in hepatocytes of AIH, while homogeneous expression was observed in hepatocytes of CH-C. Aberrant expression of CK 7 and CK 19 was observed in hepatocytes of AIH, while it was not in hepatocytes of CH-C. Expression of HLA-DRalpha was observed in hepatocytes of AIH but not in those of CH-C. Furthermore, expression of cathepsin D, bax and caspase 3 was much stronger in hepatocytes of AIH than in those of CH-C. These results indicate that cytoskeletal alterations of hepatocytes in AIH may increase the susceptibility to apoptosis and induce hepatocyte destruction.

Reduced expression of cell cycle regulator p18(INK4C) 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.