Established Hepatoma Cell Line Research Articles

Acquired Resistance to Antiangiogenic Therapies in Hepatocellular Carcinoma Is Mediated by Yes-Associated Protein 1 Activation and Transient Expansion of Stem-Like Cancer Cells.

Induction of neoangiogenesis is a hallmark feature during disease progression of hepatocellular carcinoma (HCC). Antiangiogenetic compounds represent a mainstay of therapeutic approaches; however, development of chemoresistance is observed in the majority of patients. Recent findings suggest that tumor‐initiating cells (TICs) may play a key role in acquisition of resistance, but the exact relevance for HCC in this process remains to be defined. Primary and established hepatoma cell lines were exposed to long‐term sorafenib treatment to model acquisition of resistance. Treatment effects on TICs were estimated by sphere‐forming capacity in vitro, tumorigenicity in vivo, and flow cytometry. Adaptive molecular changes were assessed by whole transcriptome analyses. Compensatory mechanisms of resistance were identified and directly evaluated. Sustained antiproliferative effect following sorafenib treatment was observed in three of six HCC cell lines and was followed by rapid regrowth, thereby mimicking responses observed in patients. Resistant cells showed induction in sphere forming in vitro and tumor‐initiating capacity in vivo as well as increased number of side population and epithelial cell adhesion molecule‐positive cells. Conversely, sensitive cell lines showed consistent reduction of TIC properties. Gene sets associated with resistance and poor prognosis, including Hippo/yes‐associated protein (YAP), were identified. Western blot and immunohistochemistry confirmed increased levels of YAP. Combined treatment of sorafenib and specific YAP inhibitor consistently revealed synergistic antioncogenic effects in resistant cell lines. Conclusion: Resistance to antiangiogenic therapy might be driven by transient expansion of TICs and activation of compensatory pro‐oncogenic signaling pathways, including YAP. Specific targeting of TICs might be an effective therapeutic strategy to overcome resistance in HCC.

Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel™) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBPα, HNF4α, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

Study of the endocytosis and intracellular localization of subviral particles of hepatitis B virus in primary hepatocytes.

AbstractAlthough our knowledge of the hepatitis B virus (HBV) life cycle has increased tremendously during the past 20 yr, the early steps of human HBV infection are still poorly understood. This is partly caused by the lack of a suitable cell culture system. Established hepatoma cell lines (e.g., HepG2 or Huh7) are not susceptible at all and proposed receptors for HBV have failed to support HBV entry to date. Because HBV is strictly hepatotropic, cultures of primary human hepatocytes are suitable for elucidation of the entry process of HBV, but very difficult to obtain and handle. Many insights into hepadnaviral uptake have come from the study of duck hepatitis B virus (DHBV) infection in primary duck hepatocytes. The characterization of a primary receptor of DHBV (1) and recombinant expression in nonsusceptible cell lines has shown that DHBV is taken up via endocytic vesicles (2). Use of labeled DHBV virions from sera of infected ducks for uptake experiments using confocal microscopy has greatly improved the possibilities of studying the endocytic distribution of DHBV virions during infection.KeywordsPrimary HepatocytePrimary Human HepatocyteSucrose Density Gradient CentrifugationCsCl SolutionSubviral ParticleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Use of established hepatoma cell lines in biotoxicology

In conclusion, hepatoma cell lines displaying multiple combinations of bioactivating and inactivating enzymes, even if do not seem to represent liver-specific bio-indicators of toxicity, are a polyvalent liver system, suitable to evaluate particular end-points.

Altered levels of phosphoinositide metabolites and activation of guanine-nucleotide dependent phospholipase C in rat hepatic tumors.

The metabolism of phosphatidylinositol was studied in normal quiescent hepatocytes, hepatocellular carcinomas induced by single dose of diethylnitrosamine, followed by 2-acetylaminofluorene and partial hepatectomy (Solt-Farber model), and in an established hepatoma cell line, JB1. The JB1 hepatoma cell line and hepatocellular carcinomas demonstrated a 4- to 5-fold higher rate of turnover of [3H]-inositol and [3H]-glycerol than the control hepatocytes. Significantly, elevated levels of second messengers inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol were noted in hepatic tumor cells within 4 hr of labeling with precursor molecules, whereas no detectable level of 3H-labeled inositol trisphosphate was noted in quiescent hepatocytes, even after incubation with 10 mM LiCl for 30 min. Approximately 2.5-fold higher specific activities of a guanine nucleotide and Ca+2 dependent phosphatidylinositol 4,5-bisphosphate specific phospholipase C were detected in the hepatocellular carcinoma cells. The cellular location of the phospholipase C activity was also different, being membrane bound in hepatocytes and equally distributed between cytosolic and membrane factions in the hepatomas. These data are consistent with the hypothesis that the enhanced production of diacylglycerol and inositol 1,4,5-trisphosphate in hepatocellular carcinomas may be due to the activation of a guanine nucleotide dependent phosphatidylinositol 4,5-bisphosphate specific phospholipase C. These data are the first to compare phosphoinositide turnover in normal liver and hepatic tumor cells and suggest that the sustained levels of second messengers is closely associated with the transformation and enhanced growth rate in hepatic tumor cells.

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line.

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzo[<italic>a</italic>]pyrene- and Aldrin-Metabolizing Activities in Cultured Human and Rat Hepatoma Cell Lines<xref ref-type="fn" rid="FN2">2</xref>

Five established hepatoma cell lines, 1 of rat origin and 4 derived from human liver carcinoma, were compared for their capacity to perform metabolic activation of one polycyclic hydrocarbon, benzo[a]pyrene (BP), and one cyclodiene chlorinated insecticide, aldrin. The results of these investigations indicated both species and individual differences among these cell lines. Aldrin was found to be converted into dieldrin much more efficiently by the rat hepatoma cell line than by any human cell lines, whereas 2 human lines displayed the highest BP-metabolizing activity whether measured as the amounts of water-soluble products or estimated by cytotoxic cell-mediated assays. Results also showed that one particular human cell line can replace, advantageously, V79 hamster cells as target in a cell-mediated assay owing to its incapability to metabolize BP and its high sensitivity to BP metabolites.

Primary cultured cells and an established cell line of human hepatocellular carcinomas.

Cancer cells obtained from human hepatocellular carcinoma nodules were subjected to primary culture, and a hepatoma cell line was established. The cell clumps obtained by needle puncture were plated directly in plastic tissue culture flasks without any cell dissociation procedures. Cell clusters became attached to flasks in 24 h with an efficiency of about 90%. No fibroblast outgrowth was observed. Primary cultured cells were composed of polygonally shaped epithelial cells with dense cytoplasm and one or more large nuclei. They excreted plasma protein biosynthetic markers of hepatocytes into the culture medium. Plasma protein synthesis of primary cultured hepatoma cells decreased as the age of the primary cultures increased. Cells seeded in September 1980 started to grow continuously after 5 months of cultivation. A new hepatocellular carcinoma cell line (designated as KG55T) was established from these growing cells. KG55T cells have been subcultured for more than 20 passages and form a monolayer of polygonal epithelial cells which pile up after they reach confluence. The cells had a doubling time of 50-60 h and a plating efficiency of 60-65%. Albumin, alpha 1-antitrypsin and alpha 2-macroglobulin syntheses and tyrosine aminotransferase activity were detected. At the 10th passage, KG55T cells were pseudotriploid (mode, 69), and 8q+ and 15q+ translocations were distinctive of this cell line. The morphological characteristics and the capacity for plasma protein synthesis of the primary cultured hepatoma cells and cells of the established hepatoma cell line were compared.

Depressed natural killer cell activity in patients with hepatocellular carcinoma. In vitro effects of interferon and levamisole.

Enriched lymphocytes from patients with primary hepatocellular carcinoma showed considerably reduced natural cytotoxicity against two established hepatoma cell lines and against the myelogenous derived cell line K562, as compared to lymphocytes from normal volunteers or from patients with various nonmalignant liver diseases. The serum of hepatoma patients did not effect normal or patient NK cell activity. When lymphocytes from patients or controls were treated for three hours with human leukocyte interferon, NK cell cell activity was significantly improved. Levamisole did not produce a significant increase in normal NK cytotoxicity but did improve the killing of lymphocytes from patients with hepatocellular carcinoma.

Action of insulin on glycogen metabolism in cultured hepatoma cells

ZHC cells, an established hepatoma cell line characterized by its capacity to synthesize and store glycogen, retain responsiveness to insulin. Sensitivity to insulin is correlated with culture development and is maximal in the confluent monolayer cultures. Insulin induces, within 2–3 h, an increase of glycogen content by stimulating the net synthesis of new glycogen molecules and without affecting their breakdown. Insulin directly acts on glycogen metabolism, and does not modify total cell protein or DNA synthesis. The ZHC cell line can provide a new model for the study of insulin regulation of glycogen metabolism, in the absence of other hormones that modulate the same pathway.

In vitro characteristics of two established hepatoma cell lines

The in vitro behaviours of a fibroblast-like (DENA-RH 13) and an epithelial-like (DENA-RH 13A) cell line, derived from a chemically induced hepatocarcinoma of rat are described. The DENA-RH 13A can be maintained and studied either in monolayer as a transplantable solid tumor or in fluid-suspension culture as ascites tumor in Wistar rats. The cytomorphology and growth pattern of the clones of the cell lines are described. While DENA-RH 13 cells produced undifferentiated tumors with mainly sarcoma-like structures in allogeneic hosts the epithelial-like cell line (DENA-RH 13A) grew in a carcinoma-like pattern in animals.

Comparison of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in hepatoma cells grown in vivo and in vitro.

Unlike the normal liver, numerous transplantable rodent and human hepatomas are unable to alter their rate of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-GoA) reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] activity in response to a dietary cholesterol challenge. It has been suggested that this metabolic defect is linked to the process of malignant transformation. Hepatoma 7288C "lacks" feedback regulation of cholesterol synthesis when grown in vivo but expresses this regulatory property when grown in vitro (then called HTC). Therefore, it was used as a model system to answer whether an established hepatoma cell line that modulates its rate of cholesterol synthesis in vitro can express this property when grown in vivo, and whether cells reisolated from the tumor mass have the same regulatory phenotype as before transplantation. Our results show that long-term growth of hepatoma 7288C in tissue culture has not caused a biotransformation that permits feedback regulation of HMG-CoA reductase when the cells are transplanted back into host animals. In addition, HTC cells reisolated from the tumor mass and established in tissue culture continue to have the ability to regulate HMG-CoA reductase activity. Therefore, malignant transformation is not categorically linked to the loss of the cellular components necessary to regulate sterol synthesis and HMG-CoA reductase activity.

Differences between Liver and Hepatoma Cells in Their Complements of Adenosine 3′:5′-Monophosphatebinding Proteins and Protein Kinases

Abstract The soluble fractions from both rat liver and an established hepatoma cell line (HTC cells) contain protein kinases and proteins which bind adenosine 3':5'-monophosphate (cyclic AMP). Liver cytosol exhibits somewhat more binding activity and less protein kinase activity than HTC cells. Protein kinase activity from both sources is stimulated by cyclic AMP. Scatchard plots for the binding of cyclic AMP have been compared at pH 4 and 6.5. At both pH values, liver shows nonlinear plots which are increased in curvature and apparent affinities by prior dialysis of the samples, mostly because of the removal of endogenous cyclic AMP. For HTC cells the Scatchard plot at pH 4 is also nonlinear and exhibits high binding affinity (Kd l 0.5 nm), but prior dialysis is unnecessary and the shape of the curve differs from that of liver. At pH 6.5, the binding affinity observed in HTC extracts (Kd g 10 nm) is lower than seen with liver and is insensitive to dialysis for a day or 2. However, this binding can be partially converted to higher affinity by aging or by overnight treatment at pH 4. The binding activity present in liver extracts is considerably less stable than that from HTC cells at pH 4 in the absence of cyclic AMP. Increasing the pH and adding cyclic AMP stabilize the binding activities from both sources. Much of the nonlinearity observed in the Scatchard binding plots is in the opposite direction from that caused by the instability of binding components at low concentrations of cyclic AMP. Chromatography on DEAE-cellulose shows that HTC extracts lack one of two major cyclic AMP-stimulated protein kinase fractions which are present in liver. The deficiency seems to be due primarily to the absence of a cyclic AMP-binding fraction. Chromatographically analogous protein kinase peaks are present in both HTC and liver extracts, although their proportions differ. The cyclic AMP-binding fraction which is present only in liver is very labile at pH 4 and accounts for the greater instability of binding activity in liver extracts compared with HTC samples. HTC cells and liver also differ in the properties of the cyclic AMP-stimulated protein kinase fraction which is common to both. The activity of this chromatographic fraction from HTC cells is less responsive to cyclic AMP and has a lower binding affinity than that from liver. This lower HTC binding affinity can be increased by the same treatments which are effective with unfractionated extracts.