Expression of liver phenotypes in cultured mouse hepatoma cells: synthesis and secretion of serum albumin

A permanent cell line, designated Hepa, has been isolated from a mouse hepatoma, BW 7756. The cell line synthesizes and secretes albumin at rates appreciably higher than previously reported hepatomas adapted to in vitro conditions. Monospecific antimouse serum albumin was produced in rabbits, and mouse serum albumin secreted by the hepatoma cells was identified by double diffusion, immunoelectrophoresis, and radioimmunodiffusion. A quantitative immunoassay was used to measure albumin secretion and to study the effects of culture conditions on albumin secretion. A subclonal analysis was performed to study the homogeneity and stability of cloned hepatoma lines in respect to albumin secretion. Different secretion rates were observed during the culture cycle. Significant clonal variation in respect to albumin secretion was found among ten subclones.The significance of clonal variation is discussed in relation to the study of epigenetic control of albumin expression in somatic hybrid cells.     

Cetaben-induced changes on the morphology and peroxisomal enzymes in MH1C1 rat hepatoma cells and HepG2 human hepatoblastoma cells

Human HepG2 and rat MH1C1 hepatoma cell lines were examined for their response to cetaben, an exceptional type of peroxisome proliferator. Shape change and proliferation of peroxisomes as well as induction of selected peroxisomal enzymes catalase, acyl-CoA oxidase, and peroxisomal bifunctional enzyme, were assessed in response to cetaben. In MH1C1 cells, peroxisomes were seen in clusters displaying typical features of microperoxisomes. Cetaben caused little but reversible proliferation and morphological heterogeneity with the occurrence of dumbbell-shaped and cup-shaped peroxisomal profiles. Peroxisomes in HepG2 cells showed marked variation in size and shape. Cetaben treatment of HepG2 cells caused disintegration of Golgi regions and augmented mitochondrial matrix. Interestingly, MH1C1 cells showed different subunit composition of acyl-CoA oxidase in immunoblot analysis: only subunit A at 72 kDa was detected but not the cleavage products. In situ hybridization underlined the marked morphological heterogeneity observed, and both cell lines revealed different stages of gene expression. Our results indicate that cetaben represents an extraordinary type of peroxisomal proliferator with pleiotropic effects on human and rat hepatoma cells, and, at least in the human hepatoma cell line HepG2, these effects are not restricted to peroxisome proliferation.     

Analysis of discontinuous variation in albumin production by hepatoma cells at the cellular level

The clonal variation in the rate of albumin production in cultured rat hepatoma cells has been studied on a cellular basis by immunoperoxidase techniques using specific antisera against rat serum albumin. Previously, it has been shown that an array of clonal hepatoma cell populations that produce serum albumin at different rates can be isolated simply by subcloning a single clonal hepatoma cell line (Fu5). The present study demonstrates conclusively that this phenotypic variation is the result of quantal shifts in the rate of albumin production in the individual cells and is not due to changes in the percentage of albumin-producing cells. Also, by analyzing individual colonies as they develop from single cells, it was possible to establish that the rate of variation in albumin content in several hepatoma cell clones is on the order of 0.5-1.4 10(-2) per cell per generation. This variation in albumin content probably reflects shifts in the rate of albumin synthesis. Even after several sequential subclonings, the same clonal variation persists. The variants are not the result of fluctuations in albumin synthesis with different phases of the cell cycle.     

人原发性肝癌和肝癌细胞株的N-ras癌基因的产物p21

以原发肝癌及肝癌7402细胞株DNA转染NIH/3T3得到的转化细胞与材料,经~(35)S-甲硫氨酸参入,应用放射免疫吸附,聚丙烯酰胺凝胶电泳技术,发现原发性肝癌的转化细胞株中N-ras基因的表达产物p21明显高于非转化的细胞。7402细胞株和7402转化的细胞株可能存在有二个ras基因,一个是ras~N;另一个是ras~H。本文结果为证明N-ras是人肝癌的一种转化基因提供了证据。     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions

Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.     

Phenotyping human leukemic T-Cell lines: Enzyme markers; surface antigens,and cytogenetics

We have compared phenotypic markers for a series of established human leukemic T-cell lines collected from different laboratories. Cell lines were tested first for genetic markers using polymorphic enzymes and then for expression of T lymphoid cell surface differentiation antigens using monoclonal antibodies. Chromosomal analysis was used as an additional method for identification of selected cell lines. On the basis of enzyme markers, it was possible to assign each of the cell lines examined to one of nine different groups. With two exceptions, surface antigen phenotypes for each of 12 cell lines were clearly distinctive. Thus, some groups of cell lines indistinguishable by enzyme markers could be further subdivided by surface antigen phenotyping. However, significant quantitative variation in expression of individual antigens was observed. In addition, surface antigen expression was not uniform in different subcultures of one cell line studied in detail. These results indicate that leukemic T-cell lines cannot be used generally as simple models of surface antigen expression in normal T-cell differentiation.     

Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation

Hepatoma cells show alterations in the response to oxidative stress (decreased lipid peroxidation) and in xenobiotic metabolism enzymes (decreased P450, increased GST and ALDH3). This study examined the effect of lipid peroxidation on the expression of the above enzymes in two rat hepatoma cell lines (MH(1)C(1) and 7777). To induce oxidative stress, cells were exposed to arachidonic acid (to increase lipid peroxidation substrate) and/or to beta-naphthoflavone (to increase CYP450), and treated with one dose of iron/histidine. The cells, that were still viable after the challenge, were refed with the culture medium and CYP1A1, GST, and ALDH3 enzymes monitored for 1, 6, 12, and 24 h. Treatments that increased markers indicative of lipid peroxidation are associated with a decrease in enzyme activities, which was permanent for CYP1A1 and transient for the other enzymes. We speculate from these data that aldehydic byproducts of lipid peroxidation may be responsible for these effects. Thus, restoration of lipid peroxidation in hepatoma cells seems to induce a rapid adaptation to oxidative stress, which is achieved by a simultaneous decrease of reactive oxygen species production and an increase in the two main enzymes involved in the removal of the aldehydic products of lipid peroxidation.     

The glucocorticoid domain: steroid-mediated changes in the rate of synthesis of rat hepatoma proteins.

Changes in the rate of synthesis of rat hepatoma proteins in response to the synthetic glucocorticoid, dexamethasone, have been measured by two-dimensional gel electrophoresis (O'Farrell, 1975a). In the most extensively analyzed cell line (HTC cells), two classes of steroid-responsive gene products are found: seven Class I proteins, which are induced essentially every time they are detected on two-dimensional gel autoradiograms, and about sixteen Class II proteins, which are induced and/or repressed infrequently even though they are detected in almost every experiment. Contained in Class I is the steroid-inducible enzyme tyrosine aminotransferase as identified by its specific immunoprecipitation with antiTAT serum. Also in Class I are two series of proteins, termed Belts I and II, which are tentatively deduced to be sialoglycoproteins from their electrophoretic properties in this gel system. Belt I is localized on the HTC cell surface as shown by its labeling following radioiodination of whole cells. Class I polypeptides respond quite uniformly to dexamethasone in that they were induced with the same apparent kinetics and their induction ratios fell in the range of 3–10. The number of Class I proteins, <1% of the observed proteins, provides a quantitative measure of the specificity of the observed dexamethasone response.Class II proteins behave as if their hormonal sensitivity varied between experiments. All proteins having decreased rates of synthesis after administration of dexamethasone fall into Class II. Their repression ratios are small, never exceeding 3, and only one (r₁) repressed with any consistency (9 of 16 experiments).In three experiments with another rat hepatoma line (FAZA cells), eight proteins were induced by the steroid and one repressed in all three experiments. Except for tyrosine aminotransferase, the inducible gene products from FAZA cells were electrophoretically different from the HTC set. Two of the inducible proteins in FAZA cells, however, were detectable in HTC cells: one was unaffected by dexamethasone in HTC cells and the other (r₁) repressed quite frequently. Thus inducibility of a gene product may be lost and even reversed in different hepatoma cell lines.     

Continuous culture of reuber hepatoma cells in serum-free,arginine-, glutamine-and tyrosine-deprived chemically defined medium

Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic “marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle. This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry of Education, Science and Culture, Japan.     

Continuous culture of Reuber hepatoma cells in serum-free arginine-, glutamine- and tyrosine-deprived chemically defined medium

The rat hepatoma cell line, H4-II-E, was grown serially over a 1-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's minimum essential medium with no supplements other than biotin. The adapted cell line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic "marker" enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle.     

5'-Nucleotide phosphodiesterase and alkaline phosphatase in tumor cells: evidence for existence of novel species in the cytosol

Characteristics of 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) and alkaline phosphatase (EC 3.1.3.1) activities in tumor cell lines of human and murine origin were examined. Of the 15 cell lines tested, 5'-nucleotide phosphodiesterase activity in 13 cell lines and alkaline phosphatase activity in 10 cell lines were inhibited by N-ethylmaleimide and activated by dithiothreitol (N-ethylmaleimide-sensitive), and suggested to be SH-enzymes. In contrast, the two phosphohydrolases from normal tissues were inactivated by dithiothreitol, but not by N-ethylmaleimide (dithiothreitol-sensitive). There was only one tumor cell line in which both activities were dithiothreitol-sensitive. Human hepatoma PLC/PRF/5 cells appear to possess both types of 5'-nucleotide phosphodiesterase and alkaline phosphatase, and the subcellular distribution of these enzymes in this cell line was investigated. Dithiothreitol-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase of PLC/PRF/5 cells were localized in the plasma membrane as in normal tissues, but N-ethylmaleimide-sensitive phosphohydrolases were soluble cytosolic proteins. N-Ethylmaleimide-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase activities from other cell lines were also recovered in the cytosol. Molecular masses of cytosolic N-ethylmaleimide-sensitive phosphohydrolases were apparently smaller than their membrane-bound dithiothreitol-sensitive counterparts, as judged from gel filtration. It was concluded that many tumor cell lines lack plasma membrane 5'-nucleotide phosphodiesterase and alkaline phosphatase, but express enzymes with similar activities in the cytosol, with properties clearly distinguishable from enzymes so far characterized.     

Activation of human alpha 1-antitrypsin gene in rat hepatoma x human fetal liver cell hybrids depends on presence of human chromosome 14

In order to study the involvement of human chromosomes in the expression of liver-specific functions, we have produced somatic cell hybrids between a rat hepatoma (7777) cell line and human diploid skin fibroblasts (series XIX) or human fetal liver cells (series XXII). Production of human serum proteins was detected by immunoelectrophoretic analyses of concentrated serum-free hybrid culture supernatants. Human alpha 1-antitrypsin (AAT) was secreted by a subset of hybrids but not by the parental cells. The activated human AAT phenotype segregated concordantly with human chromosome 14 in 18 primarily HAT-selected and five azaguanine back-selected series XXII hybrids. All other chromosomes were excluded as playing a role in AAT expression. Therefore, the AAT gene (PI) is assigned to chromosome 14. This quasi-constitutive expression of a liver-specific function was not observed for the other serum proteins studied, nor was it seen in the skin fibroblast-derived hybrids (series XIX) although AAT was produced by some of them.     

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.     

Adenosine deaminase from deoxycoformycin-sensitive and -resistant rat hepatoma cells. Purification and characterization

Deoxycoformycin-resistant rat hepatoma cells exhibit up to 300-fold increase in adenosine deaminase activity compared to the sensitive parental cells. In order to determine the basis of the increased enzyme activity in deoxycoformycin-resistant cells, adenosine deaminase was purified from rat liver and deoxycoformycin-sensitive and -resistant cells. Physical, kinetic, and immunological properties of the purified enzymes were compared. Purified adenosine deaminase from all sources was found to be a monomer with an Mr approximately 45,000. In addition, the purified enzymes had a similar isozyme pattern in nondenaturing polyacrylamide gels. Km values for adenosine and Ki values for deoxycoformycin did not differ among the purified enzymes. By double diffusion analysis and quantitative immunoprecipitation, the purified enzymes were found to be immunologically indistinguishable. These data indicate that deoxycoformycin-resistant rat hepatoma cells produce increased amounts of adenosine deaminase protein which results in increased enzymatic activity.     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

Studies on the variations in generation times of rat hepatoma cells in culture

The proliferation kinetics of a cultured hepatoma cell line, HTC, was studied by time-lapse photography and autoradiography. Variability of G1 phase, S phase and intermitotic times of sister cells were compared with data on the variability of G1 phase, G2 phase, S phase and intermitotic times of unrelated cells. Evidence is presented that the variation in times spent in intermitosis by a pair of sisters is determined by the variation of G2 phase durations, while the variation in intermitotic times of unrelated cells is mainly determined by the variation of G1 phase times.     

Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate

Several human cell lines were studied for the production of gelatinases. Diploid fibroblasts, the melanoma cell line Bowes, the MG-63 osteosarcoma cell line and the human hepatoma cell line Malavu all constitutively produced a 67 kDa gelatinase. Gelatinolytic enzymes were quantified by a sensitive zymographic substrate conversion assay. Upon induction with phorbol 12-myristate 13-acetate (PMA), the human hepatoma cell line secreted considerable amounts of an 85 kDa gelatinase activity. The induction process was time- and dose-dependent. It represented a true increase in production per individual cell and was associated by a marked change of the cell morphology. The effect of various proteinase inhibitors and the maximal activity of the enzyme near neutral pH demonstrate that it is a neutral metalloproteinase. Characterization studies showed the 85 kDa gelatinase to be transformed to lower molecular weight, active forms by treatment with p-aminophenylmercuric acetate (APMA) or trypsin.