Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4‐II‐E cells overexpressing RC stably. H4‐II‐E cells were transfected with RC/pCXN2 vector and the multiple neomycin‐resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2‐transfected cells used in this study was 19.7‐fold as compared with that of the parental wild type H4‐II‐E cells. Wild type H4‐II‐E cells, pCXN2 vector‐transfected cells (mock type), and RC/pCXN2‐transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4‐II‐E cells was significantly suppressed in transfectants with culture for 12–48 h. The presence of anti‐RC monoclonal antibody (10–50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti‐RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 μM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4‐II‐E overexpressing RC stably. J. Cell. Biochem. 84: 143–149, 2002. © 2001 Wiley‐Liss, Inc.     

Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL—7404

Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells.In the cells of JX-1,a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research,3:75,1993),an enhanced rate(9.5%) of spontaneous apoptosis was detected by flow cytometry,whereas the rates of spontaneous apoptosis in JX-0 cells,a sub-clone of BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 cells were almost equal and about 1.7%.Serum-starvation for 72h increased the rate of apoptosis of JX-lcells up to 33.7%,while JX-0 and BEL-7404 cells,under the same condition,produced less than 5% of apoptotic cells.Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies often occurred in JX-1 cells,especially during serumstarvation.These results,combined with the data of DNA fragmentation Elisa test,suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca^2 level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0 cells indicated that antisense egfr might interrupt the EGF/EGFR sigaling pathway resulting in the decreass of intracellular Ca^2 pool content as well as the responsiveness of these cells to the extracellular signals.These findings suggest that antisense EGFR either directly or indirectly regulates Ca^2 storage in endoplasmic reticulum,thereby enhances apoptosis in the human hepatoma cells.     

Induction of sister chromatid exchanges by benzidine in rat and human hepatoma cell lines and inhibition by indomethacin

The genotoxic activity of benzidine was studied in two cell lines derived from rat (H4) and human (HepG2) hepatomas which have been shown to be capable of activating certain promutagens. The responses were compared to results in two lung-derived fibroblast lines (IMR-90 and V79) which appear to have little or no metabolizing capability. Benzidine was found to induce sister chromatid exchanges in the two liver-derived cell lines in a dose-dependent fashion but failed to induce sister chromatid exchanges in the fibroblast lines. Since one proposed pathway for benzidine activation involves prostaglandin-mediated metabolism, we tested the effect of pretreatment with indomethacin, an inhibitor of this metabolic pathway. Indomethacin was highly effective in inhibiting benzidine-induced sister chromatid exchanges in both H4 and HepG2 cells. These results suggest that some DNA damage may occur in the livers of fast acetylating species such as the rat without prior N-acetylation and that some amount of DNA damage may occur in the livers of slow acetylating species, even when the liver is not the target organ for carcinogenesis.Abbreviations RI replication index - SCE sister chromatid exchanges     

Effect of sodium butyrate on the hepatoma cell cycle: Possible use for cell synchronization

Summary Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse cinematography and [³H]thymidine incorporation studies. Evidence is presented that sodiunm butyrate affected the cell cycle at a specific point immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used for HTC synchronization. This work was supported by l'Institut Nationale de la Santé et de la Recherche Médicale and la Centre Nationale de la Recherche Scientifique (L. T. and J. K.).     

Heme-biosynthetic enzyme activities and porphyrin accumulation in normal liver and hepatoma cell lines of rat

The activities of four heme-biosynthetic enzymes, -aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilogen (PBG) dearninase, and ferrochelatase, were studied in five epithelial cell lines of normal rat liver origin (Re, REC-10, RLC-24, M, Culb-TC) and five cell lines derived from Yoshida ascites hepatoma (JTC-1, JTC-2, JTC-15, JTC-16, JTC-24). The JTC series of hepatoma-derived cell lines exhibited decreased ALA synthase activity and increased ALA dehydratase activity, although the activities of all four enzymes and the Km values for their respective substrates varied widely from one cell line to another, a finding suggesting that specific regulatory mechanisms for porphyrin metabolism might operate in each cell type. M cells, which were transformed by 4-dimethylaminoazobenzene in vitro, gave the most abnormal Km values of heme-biosynthetic enzymes among all the cell lines studies, and were found to accumu2ate hematoporphyrin derivative (HpD).Abbreviations ALA o-aminolevulinic acid - DAB 4-dimethyl aminoazobenzene - HpD hematoporphyrin derivative - 4NQO 4-nitroquinoline 1-oxide - PBG porphobilinogen     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

Characteristics and aldehyde dehydrogenase activity of four rat hepatoma cell lines produced by diethylnitrosamine-phenobarbital treatment

Summary Recent studies in our laboratory have shown that five established rat hepatoma cell lines provide a wide spectrum of tumor-associated aldehyde dehydrogenase (ALDH) activity representative of the range of activities of this enzyme seen in primary rat hepatocellular carcinomas. Four newly established rat hepatoma cell lines, RLT-2M, RLT-3C, RLT-9F, and RLT-5G, were derived from a primary hepatocellular carcinoma. The primary tumor was induced by a single injection of diethylnitrosamine (15 μM/g body weight) to a 1-d-old female S-D rat followed at weaning by chronic phenobarbital treatment. RLT-2M was established from outgrowths of minced tumor pieces. RLT-3C, RLT-9F, and RLT-5G were cloned from RLT-2M by the serial endpoint dilution. All four lines have been maintained in culture for over 100 passages. The ALDH phenotype in both the primary tumor and the four new cell lines was determined by total activity assay, gel electrophoresis, and histochemistry. By total activity assay, the primary tumor did not possess significant tumor-ALDH activity. In contrast, the four new cell lines expressed tumor-ALDH activity. However, they differed in their basal ALDH activities and in ALDH inducibility by 3-methycholanthrene, benzo(a)pyrene, and phenobarbital. Additionally, significant decreases in tumor-ALDH activity occurred when cells from each line were passaged in vivo. The four lines have been characterized by light and electron microscopic morphology, tumorigenicity, chromosome number, doubling time, and colony formation efficiency, in soft agar. This work was submitted by K.-H. L. in partial fulfillment of the requirements for the Doctor of Philosophy degree in The Graduate School of The University of Alabama. This work was supported by grant CA-21103 from the National Cancer Institute, Bethesda, MD.     

Immuno-overlay: A method for identification of hepatoma cell colonies that secrete albumin

Summary A screening technique was developed for the identification of clones of hepatoma cells that secrete albumin. The technique employs the overlay of a 1% agarose solution containing antiserum to albumin onto clones of hepatoma cells. A distinct immunoprecipitation complex is formed in the immuno-overlay that corresponds directly to the position of each secreting clone. Clones deficient in albumin secretion do not form an immunoprecipitate. Thus comparison of the immuno-overlay and the cell colonies results in identification of variant clones as well as those capable of secretion. Biochemical characterization of the region of agarose overlay from secreting and nonsecreting clones demonstrates the specificity of the method and its potential for selection of colonies that are secreting other hepatic or cellular proteins. This study was supported by Grant GM 22372 from the Public Health Service. G. J. D. is a recipient of an Established Investigatorship from the American Heart Association.     

Thapsigargin increases apoptotic cell death in human hepatoma BEL—7404 cells

Effects of thapsigargin,an inhibitor of Ca^2 -ATPase in surface of endoplasmic reticulum,on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron microscopy.Propidium iodide staining and flow cytometry revealed that in the serum-free condition,thapsigargin increased the rate of apoptosis of BEL-7404 cells in a dose-dependent manner.Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment.Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation,apoptotic bodies existed in TG-treated cells,supporting that thapsigargin is a potent activator of apoptosis in the cells.     

Expression of calcium-binding protein regucalcin mRNA in hepatoma cells

Whether the gene expression of hepatic Ca²⁺-binding protein regucalcin is altered in hepatomas was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). Rat hepatoma was induced by continuous feeding of basal diet containing 0.06% 3-methyl-4-dimethylaminoazobenzene (3-Me-DAB). After 35 weeks feeding, rats were sacrificed, and the non-tumorous and tumorous tissues of the livers were removed. In individual rats, the regucalcin mRNA levels in the tumorous tissues were generally decreased in comparison with that of the non-tumorous tissues of the chemical-fed rats, although the chemical administration might decrease the mRNA expression in normal rat liver, suggesting that the chemical administration causes a suppresive effect on the mRNA expression. When the genomic DNA extracted from the liver tumorous tissues was digested with restriction enzymes (EcoRI, BamHI and HindIII) and analyzed by Southern blotting, no rear-ranged band was found in the regucalcin gene from the hepatoma. Interestingly, in the transplantable Morris hepatoma cells, the regucalcin mRNA was markedly expressed, while the albumin mRNA was expressed only slightly. The present study demonstrates that regucalcin mRNA is clearly expressed in the transformed cells (Morris hepatoma cells).     

Use of an established human hepatoma cell line with endogenous bioactivation for gene mutation studies

Genetic toxicology assays that rely on S9 microsomal mixes are subject to artifacts related to the generation of mutagenic metabolites by acidic pHs, variation in individual isolations of microsomes and the failure of subcellular fractions to faithfully produce metabolites generated in intact cells. We have developed a gene mutation assay utilizing the human hepatoma cell line HepG2, which has been shown to metabolize a broad spectrum of promutagens. Optimal conditions for assaying the induction of 6-thioguanine-resistant mutants in this cell line include: 1) growth of colonies for three weeks on lethally irradiated feeder layers of 10⁶ thioguanine-resistant HepG2 cells (average plating efficiency = 60–80%); 2) a thioguanine concentration in selection dishes of 10^–4M with a maximum seeding density of 2.5 × 10⁵ cells per 100 mm culture dish; and 3) a minimum expression time of 6 days. In addition to ultraviolet light C (254 nm), a cytochrome P₄₅₀ (cyclophosphamide)-dependent and a cytochrome P₄₄₈ (aflatoxin B₁)-dependent promutagen were shown to induce cytotoxicity and mutations in this test system. The present studies, therefore, suggest that the HepG2 cell line may be useful for a variety of assays in genetic toxicology.Abbreviations HAT hypoxanthine, aminopterin, thymidine - TG 6-thioguanine     

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.     

Effect of protease inhibitors on protein degradation in rat hepatoma cells I. Effect on general protein degradation

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Metabolism of 4-hydroxynonenal by the rat hepatoma cell line MH1C1

The metabolism of the toxic lipid peroxidation product 4-hydroxynonenal was investigated in the well-differentiated rat heptoma cell line MH1C1. When exposed to 0.1 mM 4-hydroxynonenal (HNE), MH1C1 cells consumed it in a time-dependent manner. There was a linear relationship between the amount of aldehyde consumed and cell number in the range 0.5 - 4 X 10(6) cells ml-1. This process was unaffected by pyrazole, suggesting that alcohol dehydrogenase is not involved. The whole homogenate of MH1C1 cells consumed added HNE at a rate similar to that in intact cells. Fractionation of the homogenate showed that the highest HNE-metabolizing activity is in the cytosol. The dialysed cytosol had almost no capacity to metabolize HNE, but this was restored by supplementation with NAD, NADH, NADP and NADPH. The metabolism of HNE in MH1C1 cells is thus different from that in hepatocytes, which were shown to utilize cytosolic alcohol dehydrogenase for this process. Both reductive and oxidative pathways could be implicated in the metabolic activity of MH1C1 cells towards HNE as well as binding by glutathione.     

Hydrogen peroxide induces apoptosis in human hepatoma cells and alters cell redox status

Direct exposure of human hepatoma cell line SMMC-7721 to hydrogen peroxide (H2O2) can induce apoptosis. Apoptosis induced by H2O2 was inhibited by cycloheximide, actinomycin D, 3-aminobenzamide, EGTA or Zn2+. H2O2 can increase the level of intracellular Ca2+, downregulate GSH levels, slightly induce lipid peroxidation, and lead to change in the ratio of reduced ion components to oxidized ion components of cells. Analysis of flow cytometry indicates that H2O2 decreases the level of Bcl-2. The data indicate that H2O2-induced apoptosis requires new mRNA and protein syntheses; H2O2 can activate Ca2+/Mg2+-dependent endonuclease leading to internucleosomal DNA fragmentation and activation of poly (ADP-ribose) polymerase interfering with the energy metabolism of the cell. The H2O2 downregulation of GSH may be more important for apoptosis than H2O2 induction of lipid peroxidation, and the H2O2 induced changes in redox status of the cell may be among the original events which lead up to other biochemical changes.     

Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619-626). After culture for 72 h, cells were further cultured for 24-72 h in medium without FBS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The number of wild-type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (0.1 or 1 ng/ml) or thapsigargin (10(-7) or 10(-6) M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF-alpha (10 ng/ml) or thapsigargin (10(-5) M) caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity in wild-type cells was significantly increased by culture with TNF-alpha (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10(-5) M) caused a significant increase in Ca(2+)/calmodulin-dependent NO synthase activity in wild-type cells or transfectants. TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-nitro-L-arginine (10(-4) M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with thapsigargin (10(-6) M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin-induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF-alpha or thapsigargin.     

Lipid peroxidation and activities of tyrosine aminotransferase and glutamine synthetase in hepatoma and glioma cells grown in bovine colostrum-supplemented medium

Summary The growth stimulating properties of bovine serum and colostrum were compared in rat hepatoma (HTC) and glioma (C6) cell cultures. A colostrum concentration of 2% was optimal for HTc cells, which then reached a terminal density 40% of that in serum-supplemented medium. The corresponding figures for C6 cells were 10 and 81%, respectively. After 4 d in culture, levels of lipid hydroperoxides were measured and compared. Highest levels of lipid hydroperoxides were found in HTC and C6 cells grown in unsupplemented medium. HTC and C6 cells grown in serum supplemented medium contained levels of 52 and 64%, respectively, of that in unsupplemented medium. The corresponding levels for cells grown in presence of colostrum were 40% for HTC and 44% for C6 cells. To obtain information on any functional alterations in the cells due to the presence of colostrum the induction of tyrosine aminotransferase (EC 2.6.1.5) and glutamine synthetase (EC 6.3.1.2), by dexamethasone was studied. Although colostrum seemed to increase, the basal activities of the enzymes, no significant effects on the degree of induction could be detected. This work was supported by grants from The Wenner-Gren Foundation Arla Ekonomisk F?rening and the Nordic Society Against Painful Experiments on Animals (grant 82-07-15).