Ephrin-A1 expression contributes to the malignant characteristics of {alpha}-fetoprotein producing hepatocellular carcinoma.

BACKGROUND AND AIMS: alpha-Fetoprotein (AFP), a tumour marker for hepatocellular carcinoma (HCC), is associated with poor prognosis. Using cDNA microarray analysis, we previously found that ephrin-A1, an angiogenic factor, is the most differentially overexpressed gene in AFP producing hepatoma cell lines. In the present study, we investigated the significance of ephrin-A1 expression in HCC. METHODS: We examined ephrin-A1 expression and its effect on cell proliferation and gene expression in five AFP producing hepatoma cell lines, three AFP negative hepatoma cell lines, and 20 human HCC specimens. RESULTS: Ephrin-A1 expression levels were lowest in normal liver tissue, elevated in cirrhotic tissue, and further elevated in HCC specimens. Ephrin-A1 expression was strongly correlated with AFP expression (r = 0.866). We showed that ephrin-A1 induced expression of AFP. This finding implicates ephrin-A1 in the mechanism of AFP induction in HCC. Ephrin-A1 promoted the proliferation of ephrin-A1 underexpressing HLE cells, and an ephrin-A1 antisense oligonucleotide inhibited the proliferation of ephrin-A1 overexpressing Huh7 cells. Thus ephrin-A1 affects hepatoma cell growth. cDNA microarray analysis showed that ephrin-A1 induced expression of genes related to the cell cycle (p21), angiogenesis (angiopoietin 1 and thrombospondin 1), and cell-cell interactions (Rho, integrin, and matrix metalloproteinases) in cultured hepatoma cells. These ephrin-A1 induced genes are also activated in HCC tissues that overexpress AFP. CONCLUSION: These findings suggest that the poor prognosis of patients with AFP producing HCC is partially caused by ephrin-A1 expression, which induces expression of genes related to tumour cell growth, angiogenesis, invasion, and metastasis.

Identification of differentially expressed genes in hepatocellular carcinoma with cDNA microarrays.

Genes expressed in hepatocellular carcinoma (HCC) were analyzed using cDNA microarrays to clarify gene abnormalities in HCC. mRNA was extracted from cancerous and noncancerous tissues of 10 patients with HCC, cDNA labeled with Cy5 and Cy3 fluorescence was prepared, and it was hybridized for each patient with a cDNA microarray consisting of 1,080 elements (930 unique genes). The mRNA expression rate of each element in HCC was evaluated using the level of mRNA expression in noncancerous tissue in each patient as a reference. The expression of 10 genes was enhanced 2 times or more in HCC cancerous tissue compared with noncancerous tissue in 5 or more of the 10 patients. In contrast, 9 genes were expressed at half the level or less in HCC cancerous tissue compared with noncancerous tissue. When hierarchical clustering was performed to identify genes related to clinical phenotypes of the patients, 22 genes showed changes associated with the degree of differentiation of HCC. Thirteen of these genes were transcriptional factors or tissue-specific expression proteins related to cell differentiation or development. Our present analysis clarified a number of genes that characterize HCC. This information based on examination of clinical samples is considered to be useful for clarification of the mechanism of hepatocarcinogenesis and the diagnosis and treatment of HCC.

alpha-fetoprotein-producing hepatoma cell lines share common expression profiles of genes in various categories demonstrated by cDNA microarray analysis.

Liver carcinogenesis is a multistep process involving various genetic alterations. cDNA microarray containing 1,080 elements (930 unique genes) was used to comprehensively analyze the genetic alterations in hepatoma cell lines, and clustering analysis was used to analyze the relatedness of the gene-expression profiles. Among 7 hepatoma cell lines analyzed, 5-alpha-fetoprotein (AFP)-producing hepatoma cell lines (HepG2, Huh7, Hep3B, PLC/PRF/5, and Huh6) were shown to have common gene-expression profiles compared with those of AFP-negative hepatoma cell lines (HLE and SK-Hep1) and cancer cell lines of nonhepatocyte origin (HeLa and KMBC). Furthermore, HepG2, Huh7, and Hep3B had higher expressions of AFP and shared a common gene-expression profile even when compared with other AFP-producing cells. Analysis of the genes with a common expression profile among these 3 AFP-positive cells revealed 254 genes across various categories. We found that 18 of these genes consistently showed altered levels of expression (more than 3-fold changes) in the 3 AFP-producing hepatoma cell lines (11 up-regulated and 7 down-regulated). In these 18 genes, 5 genes, including that for AFP, were previously reported to be involved in HCC and 6 genes involved only in other types of cancer. Our study showed that AFP-producing hepatoma cell lines shared a distinct expression profile of genes in various categories. An understanding of a causal relationship of this particular expression profile of genes to AFP-positive and AFP-negative hepatocellular carcinoma (HCC) may contribute to more rational therapy in future.

Shift of hepatitis E virus RNA from hepatocytes to biliary epithelial cells during acute infection of rhesus monkey.

Hepatitis E virus (HEV) has been considered to be the major cause of enterically transmitted non-A, non-B hepatitis in developing countries. However, little is known about viral replication and localization in the liver. The aim of this study was to examine the distribution of HEV-infected cells in experimentally infected animals. Seven captured wild rhesus monkeys were inoculated intravenously with faecal extract derived from a Myanmar strain of HEV. Animals were killed at different time-points of clinical illness: during early infection, during prehepatitis with viral-like particles in bile, during acute hepatitis and during convalescence. Intrahepatic localization of HEV was analysed using non-isotopic thymine dimer in situ hybridization (NITDISH). Both plus and minus strands of HEV RNA were found in hepatocytes during the early infection period. Staining in the submembranous cytoplasmic region of hepatocytes was observed. In the prehepatitis period, both plus and minus strand HEV RNAs appeared in the canalicular side of isolated bile epithelial cells. Subsequently, HEV RNA became universally distributed in the cytoplasm of medium-size bile epithelial cells. After recovery, HEV RNA disappeared.