Hepatoma-derived Cell Line Research Articles

Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin

AbstractHemojuvelin (HJV) is a coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. However, the precise combinations of BMP ligands and receptors used by HJV remain unknown. HJV has also been demonstrated to bind to neogenin, but it is not known whether this interaction has a role in regulating hepcidin expression. In the present study, we show that BMP-2, BMP-4, and BMP-6 are endogenous ligands for HJV in hepatoma-derived cell lines, and that all 3 of these ligands are expressed in human liver. We demonstrate in vitro that HJV selectively uses the BMP type II receptors ActRIIA and BMPRII, but not ActRIIB, and HJV enhances utilization of ActRIIA by BMP-2 and BMP-4. Interestingly, ActRIIA is the predominant BMP type II receptor expressed in human liver. While HJV can use all 3 BMP type I receptors (ALK2, ALK3, and ALK6) in vitro, only ALK2 and ALK3 are detected in human liver. Finally, we show that HJV-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or by inhibition of endogenous neogenin expression. Thus, HJV-mediated BMP signaling and hepcidin regulation occur via a distinct subset of BMP ligands and BMP receptors, independently of neogenin.

Replication of hepatitis C virus genome

The studies on the mechanism of HCV replication proliferated after the development of cell culture based-subgenomic HCV replicon system and genome-length HCV RNA replication system. Furthermore, these RNA replication systems have been improved to be suitable systems for the screening of anti-HCV reagents by the introduction of reporter genes such as luciferase. Genetic analysis of HCV RNAs obtained in long-term cell culture of HCV replicon or genome-length HCV RNA-harboring cells revealed that genetic mutations in HCV RNAs accumulated in a time-dependent manner. The genetic diversity of HCVs was also enlarged in a time-dependent manner. The appearance of adaptive mutation in HCV replicon or genome-length HCV RNA is one of characteristic features of HCV RNA replication system. Although human hepatoma-derived HuH-7 cell line was mainly used for HCV RNA replication systems, a specific combination of adaptive mutations led to develop the HCV RNA replication systems using a new human hepatoma cell line other than HuH-7.

Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg- siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2.

Interferon’s New Weapon

MicroRNAs (miRNAs) are small noncoding RNAs that inhibit the expression of target genes by promoting mRNA degradation or inhibiting mRNA translation. In plants and invertebrates, miRNAs are involved in the innate immune response to viruses, but it was not known whether they performed a similar function in mammals. Type I interferons such as interferon-β (IFN-β) are antiviral cytokines that are crucial to host defense against virus infection, and Pedersen et al . investigated whether miRNAs might play a role in this response. The authors used microarrays to identify about 30 miRNAs with altered abundance in an IFN-β-treated compared with untreated human hepatoma-derived cell line (Huh7). The sequences of eight of these miRNAs showed complementarities with the hepatitis C virus (HCV) RNA genome. The abundance of miR-122, a liver-specific miRNA that is essential for HCV replication, was decreased in IFN-β-treated Huh7 cells compared with untreated cells. The authors transfected a modified Huh7 cell line that contained an autonomously replicating HCV genome with synthetic miRNA mimics of the IFN-β-induced HCV-specific miRNAs and found that HCV replication (as assessed by quantitative measurement of HCV RNA) was reduced by five of these miRNAs compared with that in control miRNA-transfected cells. Transfection of HCV-containing Huh7 cells with a mixture of inhibitors of these same five miRNAs attenuated the ability of IFN-β to inhibit HCV replication. The addition of synthetic miR-122 further interfered with IFN-β-mediated inhibition of HCV replication. Together, these data show that miRNAs contribute to the antiviral effects of IFN-β, highlighting a new component to host defense mechanisms against viruses. I. M. Pedersen, G. Cheng, S. Wieland, S. Volinia, C. M. Croce, F. V. Chisari, M. David, Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 449 , 919-922 (2007). [PubMed]

Hepatoma cells inhibit the differentiation and maturation of dendritic cells and increase the production of regulatory T cells

Tumor cells may escape from the immune responses because of defective differentiation of dendritic cells (DC). Recent studies have found an increased number of regulatory T cells (Treg) in both peripheral blood and tissues from patients with hepatocellular carcinoma. In the present study, we used tumor culture supernatants (TSN) from hepatoma-derived cell lines to investigate whether TSN interfere with the differentiation of human monocyte-derived DC and/or their ability to increase Treg. The results showed that exposure to TSN significantly inhibited the differentiation of monocytes into DC with retained CD14 molecule and reduced expression of CD1a. These TSN-exposed immature DC also produced significant amount of immunosuppressive cytokine IL-10 and displayed an increased expression of co-stimulatory molecules. Upon stimulation with LPS, however, the TSN-exposed DC failed to undergo full maturation, with a blockage of the upregulation of co-stimulatory molecules on their surface and a switch to an IL-10 highIL-12 lowTNF-α low phenotype. Moreover, exposure of DC to TSN selectively inhibited their capacity to stimulate the proliferation of allogeneic CD8 + T cells, but promoted the generation of CD4 +CD25 hiFoxp3 + Treg cells. These findings, together with previous clinical studies showing that CD4 +CD25 hi Treg cells are concentrated within hepatocellular carcinoma tissue, suggest that the local tumor microenvironment may favor the induction of Treg cells through inhibiting the differentiation and maturation of DC.

Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function.

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells

Based on the reduced expression of ethanol-oxidizing enzymes in human hepatocellular carcinoma (HepG2) cells, we analyzed the role of nonoxidative metabolites in ethanol-induced apoptosis in HepG2 cells. For this purpose, an analysis of volatile metabolites of ethanol using ion-mobility spectrometry and gas chromatography-mass spectrometry was performed. HepG2 cells exposed to 1 mmol/L ethanol exhibited significant synthesis of undecan-2-one compared to untreated cells. Undecan-2-one is a fatty acid ethyl ester metabolite synthesized through a nonoxidative pathway. Undecan-2-one had a dose-dependent cytotoxic effect on HepG2 cells as shown by release of lactate dehydrogenase (LDH). The most notable finding of this study was the potentiation of ethanol-induced apoptosis demonstrated by an increased apoptotic rate induced by undecan-2-one in ethanol-treated HepG2 cells. The data presented in this study contribute to the better understanding of the molecular mechanisms of ethanol exposure at low concentration in HepG2 cells, a human hepatocellular carcinoma-derived cell line.

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.

Time‐ and concentration‐dependent effects of resveratrol in HL‐60 and HepG2 cells

Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 microm in both HL-60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 microm for HL-60 cells and 60 microm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.

Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E 2), testosterone (T), insulin, thyroxin (T 4) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms. Insulin, T 4 and high concentrations of E 2 decreased the secretion of CBG by HepG2 cells ( p < 0.05). Ethanol, the solvent used for E 2, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13–14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T 4 and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E 2 resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.

Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1

Variable expression of human arylamine N-acetyltransferase 1 (NAT1) due to genetic polymorphism, gene regulation or environmental influences is associated with individual susceptibility to various cancers. Recent studies of NAT1 transcription showed that most mRNAs originate at a promoter, P1, located 11.8 kb upstream of the single open reading frame (ORF) exon. We have now characterized an alternative NAT1 promoter lying 51.5 kb upstream of the NAT1 ORF. In the present study, analysis of human RNAs representing 27 tissue types by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR showed the upstream 51.5 kb promoter, designated P3, to be most active in specific tissues, including kidney, liver, lung, and trachea. All NAT1 P3 mRNAs included 5'-untranslated region (5'-UTR) internal exons of 61 and 175 nucleotides in addition to the 79 nucleotide 5'-UTR exon present in P1 mRNA. CAP-dependent amplification of 5'-P3 mRNA termini defined an 84 bp transcription start region in which most start sites are centrally clustered. The hepatoma-derived HepG2 cell line expressed a high level of P3 mRNA with the same spliced structure and start site pattern as found in normal tissues. A 435-bp minimal promoter was defined by transfection of HepG2 with luciferase expression constructs containing genomic fragments from the P3 start region. These findings imply a fundamental role for P3 in NAT1 regulation and define additional regions for genetic polymorphisms associated with enhanced cancer risk.

Abnormal activation of the synuclein-gamma gene in hepatocellular carcinomas by epigenetic alteration

Liver cancer is the fifth most common neoplastic disease and the fourth leading cause of cancer-related death. Identification of the key molecular targets involved in hepatocarcinogenesis has significant therapeutic implications. In this study, by conducting immunohistochemistry, we show that the neuronal protein, synuclein-gamma (SNCG), is abnormally expressed in a high percentage of liver cancer (46/70, 65.7%). The expression of SNCG in liver cancer exhibits a clear stage-specific pattern of low expression in stage I (1/19, 5.3%) and high expression in stages III to IV (44/50, 88%). Importantly, all patients with metastatic diseases expressed SNCG in their primary tumors (15/15, 100%). Consistent with the IHC results, RT-PCR assays demonstrate that SNCG mRNA is highly expressed in the tumor tissue of advanced hepatocellular carcinomas. Analyses of the methylation status of the CpG island of the SNCG gene by methylation-specific PCR confirmed that all tumor samples contained the demethylated gene. To determine whether demethylation of SNCG is an early event of genetic abnormality in the process of hepatocarcinogenesis, we examined the methylation status of SNCG in 70 non-malignant cirrhotic liver samples and showed that 64.3% cirrhotic liver samples contained the partially or completely demethylated gene. We further show that SNCG expression in liver cancer is not restricted to HBV- and HCV-infected tumors, implying the involvement of other hepatocarcinogenic risk factors in SNCG gene reactivation. Utilizing human hepatoma-derived cell line HepG2 as an in vitro model, we demonstrate that hepatic carcinogens aflatoxin B1 and N-nitrosodimethylamine (DMN) are strong inducers of SNCG expression. Collectively, these new findings suggest that SNCG protein expression in primary tumors is a strong indicator of distant metastasis and demethylation of SNCG CpG island is an early sign of genetic abnormality in liver cirrhosis preceding hepatocarcinogenesis. Our studies also suggest that inducing demethylation of SNCG by hepatocarcinogens may represent one underlying mechanism for the aberrant expression of SNCG in HCC.

Inflammatory Cytokine IL-1β Upregulates Hepcidin Expression in Human Hepatoma Cell Lines.

The anemia of chronic disease (ACD) is commonly observed in patients with inflammatory disorders, malignancies, and chronic infections. ACD is characterized by low serum iron concentration and elevated serum ferritin concentration. A number of previous findings suggest that impaired mobilization of iron from the reticuloendotherial system (RES), such as macrophages, and an inhibition of iron absorption from intestine contribute to ACD. However, the precise mechanisms for generating the conditions of ACD have long been unclear despite extensive investigations until hepcidin was recently found. Hepcidin was found as one of antimicrobial peptides, but hepcidin is now thought to be a key regulator in iron metabolism. Hepcidin inhibits iron absorption from enterocytes and iron efflux from RES, which have suggested that hepcidin could account for the pathogenesis of ACD. Some cytokines have also been shown to modulate iron metabolism in the condition of inflammation. Interleukin-6 (IL-6), one of inflammatory cytokines, has been reported to induce hepcidin production not only in vitro but also in vivo. However, it is not clear whether other cytokines have such effect or not. We, therefore, investigated the possibility that other inflammatory cytokines might have a regulatory effect on hepcidin expression. Because hepcidin is produced mainly by hepatocytes, we used human hepatoma-derived cell line HuH-7 cells and hepatoblastoma-derived cell line HepG2 cells. Cells are incubated with or without inflammatory cytokines, such as IL-1β, IL-6, tumor necrosis factor α (TNFα), and then total RNA was extracted. Quantitative RT-PCR was then performed, revealing that IL-1β upregulate hepcidin mRNA expression as well as IL-6, although TNFα down regulates hepcidin expression in both cell lines. We next investigated the dose-response effect of IL-1β and IL-6 on hepcidin mRNA expression. Strong induction of hepcidin mRNA by IL-1β was observed when cells were incubated with low concentrations of IL-1β (0.2 ng/ml), although much higher concentrations of IL-6 were needed for hepcidin mRNA upregulation. It was likely that the concentrations of IL-1β that needed for hepcidin upregulation were more physiological than those of IL-6. Since it has been reported that IL-1β induce IL-6 production in hepatocytes, there was a possibility that the effect of IL-1β on hepcidin mRNA expression was not direct but come from IL-6 induced by IL-1β. However, the manners of IL-6 mRNA induction and hepcidin mRNA induction by IL-1β stimulation were quite different when cells were incubated with IL-1β at the concentrations ranging from 0.1 to 10 ng/ml. Moreover, antibody against IL-6 did not inhibit the induction of hepcidin mRNA by IL-1β stimulation. Therefore, it is likely that the effect of IL-1β on hepcidin mRNA expression is independent from that of IL-6. We conclude that inflammatory cytokine IL-1β can induce hepcidin expression and might be a key cytokine in the condition of ACD as well as IL-6, and IL-1β might be more important than IL-6 in physiological situations.

In Vitro and Ex Vivo Permissivity of Hepatocytes for Leishmania donovani

Using models of ex vivo infection of murine, rat, and human primary hepatocytes by Leishmania donovani, we showed that hepatocytes are permissive for Leishmania at a low level. We then modeled the in vitro infection of a human hepatoma-derived cell line to examine the parasite's capability to proliferate and to cause direct damage to hepatocytes. Results showed that L. donovani can infect hepatocytes, but do not massively proliferate. This slight infection under our experimental conditions resulted in limited damage to hepatocytes. These results bring into question a possible role for hepatocytes as a parasite reservoir during latent infection.

First-line bevacizumab (Avastin) with 5-fluorouracil/leucovorin prolongs progression-free survival in metastatic colorectal cancer (mCRC) patients who are not optimal candidates for irinotecan therapy. ASCO 2004: Abstract No. (3516)

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells.

Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes

Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappaB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.

Cyanidin-3-O-β-glucopyranoside, a natural free-radical scavenger against aflatoxin B1- and ochratoxin A-induced cell damage in a human hepatoma cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2)

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B1 (AFB1). Induction of oxidative stress also plays an important role in the toxicity of another mycotoxin, ochratoxin A (OTA). In the present study, the protective effect of cyanidin-3-O-beta-glucopyranoside (C-3-G; an anthocyanin contained in oranges, blackberries, strawberries and cranberries) against AFB1- and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2). The ability of C-3-G to reduce the production of reactive oxygen species (ROS), the inhibition of protein and DNA synthesis and the apoptosis caused by the two mycotoxins was also investigated in both cell lines. Our experiments proved the significant cytoprotective effect of C-3-G in vitro against OTA- and AFB1-induced cell damage. In particular, 24 h of pretreatment with 50 microm-C-3-G inhibited the cytotoxicity of 10 microm-AFB1 (by 35 %) and of 10 microm-OTA (by 25 %) in Hep G2 cells (P < 0.001) and of 10 microm-AFB1 (by 10 %, P < 0.01) and of 10 microm-OTA (by 14 %, P < 0.05) in CaCo-2 cells. Moreover, 50 microm-C-3-G attenuated ROS production induced by the two toxins in both cell lines (P < 0.05). Inhibition of DNA and protein synthesis induced by the mycotoxins was counteracted by pretreatment with the antioxidant at 50 microm. Similarly, apoptotic cell death was prevented as demonstrated by a reduction of DNA fragmentation and inhibition of caspase-3 activation. The in vitro free-radical scavenging capacity of the anthocyanin was tested with the Briggs-Rauscher oscillating reaction. This system works at pH approximately 2. The results showed good scavenging power, in accordance with the observed inhibition of ROS production.

A novel technique to specifically analyze the secretome of cells and tissues

The secretome of cells and tissues may reflect a broad variety of pathological conditions and thus represents a rich source of biomarkers. The identity of secreted proteins, usually isolated from cell supernatants or body fluids, is hardly accessible by direct proteome analysis, because these proteins are often masked by high amounts of proteins actually not secreted by the investigated cells. Here, we present a novel method for the specific detection of proteins secreted by human tissue specimen as well as cultured cells and chose liver as a model. The method is based on the metabolic labelling of proteins synthesized during a limited incubation period. Then, the cell supernatant is filtered, precipitated, and subjected to two-dimensional gel electrophoresis. Whereas fluorography detected a large number of proteins derived from residual plasma and dead cells, the autoradiographs selectively displayed genuinely secreted proteins. We demonstrate the feasibility of this approach by means of the secretomes of the hepatocellular carcinoma-derived cell line HepG2 and human liver slices. The selective identification of cell- and tissue-specific protein secretion profiles may help to identify novel sets of biomarkers for wide clinical applications.

Alternative Usages of Multiple Promoters of the Acetyl-CoA Carboxylase β Gene Are Related to Differential Transcriptional Regulation in Human and Rodent Tissues

Acetyl-CoA carboxylase beta (ACCbeta) is a critical enzyme in the regulation of fatty acid oxidation and is dominantly expressed in the skeletal muscle, heart, and liver. It has been established that two promoters, P-I and P-II, control the transcription of the ACCbeta gene. However, the precise mechanism involved in controlling tissue-specific gene expression of ACCbeta is largely unknown yet. In this study we revealed that promoter P-I, active in the skeletal muscle and heart but not in the liver, could be activated by myogenic regulatory factors and retinoid X receptors in a synergistic manner. Moreover, P-I was also activated markedly by the cardiac-specific transcription factors, Csx/Nkx2.5 and GATA4. These results suggest that the proper stimulation of P-I by these tissue-specific transcription factors is important for the expression of ACCbeta according to the tissue types. In addition, CpG sites around human exon 1a transcribed by P-I are half-methylated in muscle but completely methylated in the liver, where P-I is absolutely inactive. In humans, the skeletal muscle uses P-II as well as P-I, whereas only P-I is active in rat skeletal muscle. The proximal myogenic regulatory factor-binding sites in human P-II, which are not conserved in rat P-II, might contribute to this difference in P-II usage between human and rat skeletal muscle. Hepatoma-derived cell lines primarily use another novel promoter located about 3 kilobases upstream of P-I, designated as P-O. This study is the first to explain the mechanisms underlying the differential regulation of ACCbeta gene expression between tissues in living organisms.

Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon.

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a beta-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.