Interferon alfa receptor expression and growth inhibition by interferon alfa in human liver cancer cell lines.

Type I interferon (IFN) receptor consists of two chains (Hu-IFN-alphaR1 and Hu-IFN-alphaR2), and Hu-IFN-alphaR2 takes a soluble (Hu-IFN-alphaR2a), short (Hu-IFN-alphaR2b), or long (Hu-IFN-alphaR2c) form. We examined the expression of type I IFN receptor, the growth-suppression effect of IFN-alpha, and their relationship in 13 liver cancer cell lines. With reverse-transcription polymerase chain reaction (RT-PCR) analysis, the expressions of Hu-IFN-alphaR1, Hu-IFN-alphaR2a, and Hu-IFN-alphaR2c were confirmed in all cell lines, and that of Hu-IFN-alphaR2b in 12 cell lines. All cell lines expressed mRNAs of a transcriptional activator, interferon regulatory factor (IRF)-1, and its antagonistic repressor (IRF-2). Flow cytometry revealed weak expression of Hu-IFN-alphaR2 on the cell surface in 12 cell lines. The soluble-form protein of Hu-IFN-alphaR2 was detected at varying levels in culture supernatants of all cell lines with enzyme-linked immunosorbent assay (ELISA). Cell proliferation was suppressed in proportion to the dose of human natural IFN-alpha at 96 hours of culture, but it was not clearly related to the expression of Hu-IFN-alphaR2 protein on the cell surface. Investigations on the morphology, DNA, and cell cycle presented four growth suppression patterns as a result of IFN-alpha: 1) induction of apoptosis and blockage of cell cycle at the S phase (9 cell lines); 2) blockage at the S phase (2 cell lines); 3) induction of apoptosis and blockage at the G2/M phase (1 cell line); and 4) blockage at the G1 phase (1 cell line). There was no evidence showing that changes in the expressions of Bcl-2, Bcl-xL, Bak, and Bax lead directly to IFN-alpha-mediated apoptosis. Our findings demonstrated that IFN-alpha would express growth-suppression effects at varying degrees by inducing inhibition of cell-cycle progression with or without apoptosis, regardless of the expression level of Hu-IFN-alphaR2 protein on the cell surface.

Expressions of epidermal growth factor family and its receptor in hepatocellular carcinoma cell lines: relationship to cell proliferation.

In 6 HCC cell lines, clear expressions of EGFR and TGF-alpha were found in flow cytometry, while expressions of EGF, HB-EGF and AR were quite low. TGF-alpha secretion into culture supernatants became measurable when TPA 0.5 microM was added. TPA accelerated the proliferation of KYN-3 cells, and anti-TGF-alpha neutralizing antibody suppressed this proliferation in a dose-dependent manner. Addition of exogenous TGF-alpha, EGF, AR, or HB-EGF with heparin accelerated cell proliferation. In non-stimulated cultures, cell proliferation was suppressed by anti-EGFR neutralizing antibody, but not by the antibodies for EGF, TGF-alpha, AR and HB-EGF. HCC may possess a paracrine system regulated by these 4 ligands, and an autocrine system, under a certain condition, via TGF-alpha and EGFR.

Expression of vascular endothelial growth factor in human hepatocellular carcinoma.

Vascular endothelial growth factor (VEGF) is thought to take an important role in tumor angiogenesis. The present study examined VEGF expression immunohistochemically in hepatocellular carcinomas (HCCs) in various histological grades and sizes. In HCCs that were composed of cancerous tissues of single histological grade, VEGF expression was the highest in well-differentiated HCCs, followed by moderately differentiated HCCs, and then poorly differentiated HCCs. VEGF positivity gradually decreased with the increase in tumor size. In the nodules larger than 3.0 cm, 36.8% were VEGF-negative. In HCCs consisting of cancerous tissues of two different histological grades, the expression was less intensive in the higher-grade HCC component. VEGF was not expressed in sarcomatous areas, while VEGF was expressed in the surrounding HCC tissues. The expression was also remarkable in the noncancerous tissues in which inflammatory cell infiltration was apparent. VEGF expression was also examined in six HCC cell lines. In reverse-transcription polymerase chain reaction (RT-PCR) analysis, expressions of the two secretion types (VEGF121 and VEGF165) were the highest. Thus, VEGF protein in culture supernatant was measured by using enzyme-linked immunosorbent assay (ELISA) with or without inflammatory cytokines, i.e., interleukin (IL)-1beta, interferon (IFN)-alpha, IFN-gamma, and tumor necrosis factor (TNF)-alpha; and growth factors, i.e., epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)-alpha. As a result, secretion of VEGF from the cell lines was upregulated at various degrees. Based on these findings, VEGF expression in HCC tissues was thought to be related to the histological grade. The findings also indicate that various cytokines and growth factors could cooperatively act to enhance VEGF expressions in HCC.

A new human hepatocellular carcinoma cell line (HAK-2) forms various structures in collagen gel matrices.

We established a new human hepatocellular carcinoma (HCC) cell line, designated HAK-2, from a surgically resected HCC of a 57-yr-old Japanese man. The patient's tumor consisted of 5 different histological features in a single nodule: well-differentiated HCC with trabecular pattern; and moderately differentiated HCC with 4 different patterns (i.e., trabecular, pseudoglandular, solid, and scirrhous). Morphologically, HAK-2 cells on a plastic dish showed oval-shaped nuclei and large flat, polygonal eosinophilic cytoplasm and proliferated in a monolayered sheet with a population doubling time of 36.8 hours. Meanwhile, various structures, such as compact, trabecular, and tubular arrangements, were induced in HAK-2 cells cultured in type I collagen gel matrix. Also, HAK-2 cells in vitro underwent spontaneous apoptosis more frequently than other HCC cell lines examined. HAK-2 cells secreted various plasma proteins including albumin into the culture medium. Chromosome and flow cytometric analyses revealed that HAK-2 had many structural abnormalities with human karyotype and a single aneuploid cell population with a DNA index of 3.7, respectively. These findings suggest that HAK-2 is a new human HCC cell line representing two morphological characteristics; (1) formation of various structures in the presence of extracellular matrix and (2) frequent spontaneous apoptosis in vitro.

Expression of Fas and anti-Fas-mediated apoptosis in human hepatocellular carcinoma cell lines.

BACKGROUND/AIMS: Fas transduces apoptotic signals upon cross-linking with the Fas ligand, which is experimentally replaced by anti-Fas antibodies. Because little is known about Fas expression and function in hepatocellular carcinoma, these issues are addressed in the current article. METHODS: We examined Fas expressions at protein and mRNA levels, and susceptibility to anti-Fas-mediated apoptosis, on six hepatocellular carcinoma cell lines. RESULTS: Two cell lines constitutively expressed high levels of Fas both on their cell surface and in their cytoplasm, whereas the other four cell lines expressed Fas mainly in their cytoplasm. Fas mRNA of normal size was detected in all cell lines in reverse transcriptase-polymerase chain reaction analyses. Although a Fas mRNA variant, suggesting a soluble Fas molecule, was detected in the two cell lines expressing high levels of Fas, its amount was very small compared to that of normal-sized Fas transcript. Anti-Fas dose-dependently induced apoptosis exclusively in the two cell lines which constitutively express high levels of cell surface Fas. However, after preincubation with interferon-gamma, one cell line with low surface Fas expression became anti-Fas sensitive equivalent to the two cell lines expressing surface Fas at high levels. Studies of two clonally related cell lines showed that dedifferentiated clones had lower Fas expression and resistance to anti-Fas, suggesting deterioration of Fas system after clonal cell dedifferentiation. CONCLUSIONS: These findings suggest sensitivity to anti-Fas is virtually relevant to cell surface Fas, but not to cytoplasmic Fas expression. However, its expression level does not correlate to sensitivity to anti-Fas.

A human combined hepatocellular and cholangiocarcinoma cell line (KMCH-2) that shows the features of hepatocellular carcinoma or cholangiocarcinoma under different growth conditions.

BACKGROUND/AIMS: Combined hepatocellular and cholangiocarcinoma is a rare tumor of the liver, and its histogenesis remains unclear. The authors addressed this issue in the current article. METHODS: A specimen aseptically obtained from the surgically resected combined hepatocellular and cholangiocarcinoma was processed for primary culture. The morphologic features of the established cell line cultured on a plastic dish and in type I collagen gel matrix, and transplanted in nude mice were examined. RESULTS: The authors established a new human combined hepatocellular and cholangiocarcinoma cell line, designated KMCH-2, from a 40-year-old Japanese man. KMCH-2 cells on a plastic dish proliferated in a monolayered sheet with a population doubling time of 32 to 44 h. KMCH-2 expressed functional characteristics of hepatocellular carcinoma, such as albumin synthesis at protein and mRNA levels, but were poorly differentiated in morphology, showing an overlap of features with cholangiocarcinoma. KMCH-2 cells cultured within type I collagen gel matrix proliferated, forming compact to vaguely trabecular and pseudoglandular arrangements, and differentiated to show morphological characteristics of hepatocellular carcinoma unlike the cells on a plastic dish. Mucin production was not detected in KMCH-2 cells in vitro. Subcutaneous tumors which developed in nude mice injected with KMCH-2 cells represented features of adenocarcinoma with mucin production. CONCLUSIONS: The present results revealed the presence of an albumin-producing human hepatic neoplastic cell, such as KMCH-2, that can differentiate to show not only the features of hepatocellular carcinoma but also those of cholangiocarcinoma under certain growth conditions.

Expression of CD44 in human hepatocellular carcinoma cell lines.

CD44 is a glycosylated cell surface adhesion molecule expressed on a diverse range of cells and has several variant forms, some of which are involved in metastasis of cancer cells. Because little is known about CD44 in human hepatocellular carcinoma (HCC), we investigated its expression in tissue specimens from primary lesions (12 cases), in smear specimens from peritoneal effusions (2 cases), and in cell lines (HCC cell lines, KIM-1, KYN-1, KYN-2, KYN-3, HAK-1A, and HAK-1B; combined hepatocholangiocarcinoma cell lines, KMCH-1 and KMCH-2; and bile duct carcinoma cell lines, KMC-1 and KMBC). Immunohistochemical studies using monoclonal antibody recognizing epitope Group 1 of human CD44 molecule showed that HCC cells in all tissue specimens, including the original tumors of one smear specimen and HAK-1A, were negative for CD44; whereas, HCC cells in two-smear specimens, KIM-1, KYN-2, KYN-3, HAK-1A, HAK-1B, KMCH-1, KMC-1, and KMBC, showed positive reactions on the cell membrane. Immunostain-positive cell lines showed a positive cell rate of 51.9% to 99.8% by flow cytometric analysis. Western blotting detected CD44 protein of hemopoietic type in KIM-1, KYN-3, HAK-1A, and HAK-B and epithelial type in KMC-1 and KMBC. Southern blotting of complementary DNA amplified after reverse transcriptase-polymerase chain reaction (RT-PCR) detected hemopoietic type and some variant forms with longer insertion in all cell lines but KMCH-2, whereas hemopoietic type and variants with minor insertion were only detectable in tissue specimens. These findings suggest that HCC cells in ascites and in culture often express CD44, but those in tissue do not at protein level.

The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase.

Water-soluble ingredients of the herbal medicine sho-saiko-to dose-dependently inhibited the proliferation of a human hepatocellular carcinoma cell line (KIM-1) and a cholangiocarcinoma cell line (KMC-1). Fifty % effective doses on day 3 of exposure to sho-saiko-to were 353.5 +/- 32.4 micrograms/ml for KIM-1 and 236.3 +/- 26.5 micrograms/ml for KMC-1. However, almost no suppressive effects were detected in normal human peripheral blood lymphocytes or normal rat hepatocytes. Sho-saiko-to suppressed the proliferation of the carcinoma cell lines significantly more strongly than did each of its major ingredients, i.e., saikosaponin a, c, and d, ginsenoside Rb1 and Rg1, glycyrrhizin, baicalin, baicalein, and wogonin, or another herbal medicine, juzen-taiho-to (P < 0.05 or 0.005). Because such ingredients are barely soluble in water, there could be synergistic or additive effects of the ingredients in sho-saiko-to. Morphological, DNA, and cell cycle analyses revealed two possible modes of action of sho-saiko-to to suppress the proliferation of carcinoma cells; (a) it induces apoptosis in the early period of exposure and (b) it induces arrest at the G0/G1 phase in the late period of exposure.

Morphological and biological changes of a hepatocellular carcinoma cell line cultured in a three-dimensional matrix of collagen.

A human hepatocellular carcinoma (HCC) cell line, KIM-1, was cultured in three different concentrations (0.1, 0.2 and 0.3%) of type-I collagen gel matrix and its morphologic features, growth kinetics and alpha-fetoprotein and albumin productions were compared with each other or with those of cells growing on a plastic dish. KIM-1 cells in any concentration of collagen gel matrix formed various sized three-dimensional colonies with compact to trabecular cell arrangement. Larger colonies with a more definitive trabecular cell arrangement, resembling the in vivo structure of HCC, tended to form in the collagen gel matrix of a low concentration. The prolongation of doubling time was identified as the collagen concentration in the gel became higher. The cells on a plastic dish proliferated in a monolayered sheet with a shorter doubling time than others. Ultrastructurally, the cells in collagen gel matrix have more distinct cell membranes, junctional complexes and bile canaliculus-like structures, and less cytoskeletons than those on plastic dishes, similar to those in vivo. The productions of alpha-fetoprotein per 10(4) cells and albumin per 10(5) cells were much higher in the collagen gel matrix culture than on a plastic dish in a stationary phase. These data suggest that collagen gel matrix culture is suitable to monitor the morphologic features and protein production of the tumor cells in similar conditions to those in vivo, and tht the three-dimensional presence of an extracellular matrix is important in cellular proliferation and differentiation.

Establishment of two distinct human hepatocellular carcinoma cell lines from a single nodule showing clonal dedifferentiation of cancer cells.

Hepatocellular carcinomas often contain tumor cells of more than one histological grade. The clonal relationship and biological behavior of hepatocellular carcinoma cells in histologically heterogeneous areas have not been fully explored. We established two distinct human hepatocellular carcinoma cell lines (HAK-1A and 1B) from a single nodule showing a three-layered structure with a different histological grade in each layer. Morphologically, HAK-1A and 1B resembled well-differentiated hepatocellular carcinoma cells in the outer layer of the original tumor and poorly differentiated ones in the inner layer, respectively. HAK-1B appeared less differentiated morphologically and more aggressive biologically than HAK-1A; HAK-1B had a shorter doubling time, higher tumorigenicity and an aneuploid DNA index. Chromosome analysis revealed many different abnormalities in the two cell lines, in which, however, two identical structural abnormalities (2q+ and 17p+) were identified. Moreover, sequence analysis of the p53 gene showed identical mutations at codon 242 in both cell lines. These findings suggest that the two cell lines are of clonal origin and that hepatocellular carcinomas consisting of cancerous tissues of more than one histological grade may reflect clonal dedifferentiation in the tumor. Furthermore, we predict that a clonal, morphologically less differentiated subpopulation such as HAK-1B is more aggressive in proliferation and may be closely related to subsequent tumor progression in hepatocellular carcinoma.

Ultrastructural studies of the bile duct in alcoholic liver disease.

The fine structural characteristics of the bile duct in patients with alcoholic disease are described. Dark cell metamorphosis, edematous microvilli, and increased number of pinocytotic vesicles on the basal wall surface of the duct epithelium were observed. These alterations may be interpreted as evidence of disordered water metabolism, probably reflecting secretion and reabsorption hyperfunction in the duct epithelium. In addition, widened intercellular spaces in the basal half of the epithelium suggested retention of fluid following reverse pinocytosis along the lateral cell surface. Although no alterations of the duct epithelium distinct from those in patients with other liver diseases were apparent in patients with alcoholic liver disease, the basement membrane of the bile duct exhibited unusual duplication with multiple layers and occasional loop-formation in lacunae on the basal surface.

Incidence of hepatitis D virus infection in Japanese patients with hepatocellular carcinoma--immunohistochemical investigation of the delta antigen.

In order to clarify the incidence of the delta agent among hepatocellular carcinomas (HCCs) in Kurume, where the hepatitis B infection and its related HCC are most prevalent in Japan, liver tissues from sero hepatitis B surface antigen-positive autopsy cases, with or without HCC, were immunohistochemically investigated for detection of the delta antigen. Only one patient (1.7%) among 58 patients with HCC was found to have delta-antigen in the nuclei in the hepatocytes, which were diffusely distributed throughout the non-cancerous liver. None of 26 patients with liver cirrhosis showed delta-antigen in the liver tissue. The incidence is so low that the delta agent is unlikely to have a role in the development of HCC in our areas.