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1. Introduction Uremia is characterized by high levels of uremic toxins in the blood and immune dysfunction that provides a suitable microenvironment for malignant transformation and cancer progression (XiaoHong et [...], The purpose of this study was to explore the differences between exhausted [CD8.sup.+] T cells in hepatocellular carcinoma (HCC) patients with and without uremia. We enrolled 45 uremic patients who were recently diagnosed with HCC into the HCC + uremia cohort and similar patients with HCC but without uremia into the HCC-only cohort. Lymphocytes were obtained from the two cohorts, and exhausted [CD8.sup.+] T cells, comprising [PD-1.sup.+][CD8.sup.+], [TIM-3.sup.+][CD8.sup.+], and [LAG-3.sup.+][CD8.sup.+] T cells, were sorted and expanded in vitro. After expansion, the proportions of [PD-1.sup.+][CD8.sup.+], [TIM-3.sup.+][CD8.sup.+], and [LAG-3.sup.+][CD8.sup.+] T cells were significantly higher in the HCC-only cohort than in the HCC + uremia cohort. [CD8.sup.+] T cells expressing PD-1, TIM-3, or LAG-3 showed increased tumor reactivity and release of interferon-[gamma] in vitro; however, these cells demonstrated weaker anti-tumor activity in HCC + uremia patients than in HCC-only patients. Among the expanded lymphocytes, only the decreased proportion of [PD-1.sup.+][CD8.sup.+] T cells significantly correlated with the HCC + uremia cohort (odds ratio of 2.731, p = 0.009). We concluded that peripheral [CD8.sup.+] T cells expressing PD-1, TIM-3, or LAG-3 from the HCC + uremia cohort were dysfunctional in vitro. Among these populations, [PD-1.sup.+][CD8.sup.+] T cells were most evident in HCC patients with uremia. Key words: PD-1, TIM-3, LAG-3, hepatocellular carcinoma, uremia. Cette etude avait pour but d'explorer les differences entre differents lymphocytes T [CD8.sup.+] epuises chez des patients presentant un carcinome hepatocellulaire (CHC) avec et sans uremie. Nous avons inscrit a l'etude 45 patients presentant une uremie ayant recemment recu un diagnostic de CHC dans la cohorte CHC + uremie. Nous avons aussi inscrit a l'etude des patients similaires avec un CHC, mais sans uremie, la cohorte CHC seulement. Nous avons obtenu des lymphocytes des deux cohortes, et crible et fait croitre in vitro des lymphocytes T [CD8.sup.+] epuises, comprenant des lymphocytes T [PD-1.sup.+][CD8.sup.+], [TIM-3.sup.+][CD8.sup.+] et [LAG-3.sup.+][CD8.sup.+]. A la suite de la croissance, les proportions de lymphocytes T [PD-1.sup.+][CD8.sup.+], [TIM-3.sup.+][CD8.sup.+] et [LAG-3.sup.+][CD8.sup.+] etaient nettement plus elevees dans la cohorte CHC seulement que dans la cohorte CHC + uremie. Les lymphocytes T [CD8.sup.+] exprimant PD-1, TIM-3 ou LAG-3 ont presente une augmentation de la reactivite tumorale et entraine la liberation d'interferon y in vitro; toutefois, ces cellules ont presente une activite antitumorale plus faible chez les patients presentant un CHC avec uremie que dans la cohorte CHC sans uremie. Parmi les lymphocytes obtenus apres la croissance, uniquement une faible proportion des lymphocytes T [PD-1.sup.+][CD8.sup.+] correspondait a la cohorte CHC + uremie (rapport de cotes de 2,731, p = 0,009). En conclusion, les lymphocytes T [CD8.sup.+] peripheriques exprimant PD-1, TIM-3 ou LAG-3 obtenus a partir de la cohorte CHC + uremie etaient dysfonctionnels in vitro. Dans ces populations, les lymphocytes T [PD-1.sup.+][CD8.sup.+] etaient plus evidentes chez les patients presentant un CHC avec une uremie. [Traduit par la Redaction] Mots-cles : PD-1, TIM-3, LAG-3, carcinome hepatocellulaire, uremie.

Background: Polyphyllin I (PPI), a bioactive phytochemical isolated from the rhizoma of Paris polyphyllin, exerts preclinical anticancer efficacy in various cancer models. However, the effects of PPI on regulatory human [...]

ARTICLE INFO Keywords: Saponin Trillium tschonoskii Hepatocellular carcinoma Chemotherapy Multidrug resistance ABSTRACT Combating with multidrug resistance (MDR) is a major part of hepatocellular carcinoma (HCC) chemotherapy. Steroidal saponin from Trillium [...]

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of [beta]-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1[alpha]. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of [beta]-catenin in a subset of human hepatocellular carcinomas. Inactivation of METtransgenes led to regression of hepatocellular carcinomas despite the persistence of activated [beta]-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated [beta]-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway. [beta]-catenin | hepatocyte nuclear factor 1[alpha] | liver cancer | MET | mouse | hepatocellular carcinoma

We previously reported the in vitro and in vivo induction of thioredoxin reductase (TR) by sulforaphane (SF) purified from broccoli. The present study was designed to determine whether this induction is mediated by putative antioxidant response elements (ARE) found in the promoter. Luciferase reporter constructs were built using the TR promoter sequence. Sulforaphane, tert-butylhydroquinone and [beta]-napthoflavone, as well as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), increased luciferase activity in HepG2 cells transfected with the reporter construct (P < 0.0001). Quinone reductase (QR) is an enzyme with a well-characterized ARE, and QR reporter constructs built as positive controls showed similar patterns of induction. Mutation of the core sequence of a putative ARE in the TR promoter drastically decreased inducibility by SF, but mutations in nonconsensus areas of the ARE and outside of the ARE did not affect inducibility. Results from electrophoretic mobility shift assay analysis corroborated mutated reporter gene findings. Induction by TPA was not affected by mutation of the putative ARE. Se plus SF, and SF alone were equally effective for induction of TR reporter luciferase activity (P < 0.0001); Se alone had no effect. Se and SF independently increased TR activity (P < 0.0001) and when combined, increased TR activity synergistically (P = 0.036). These data suggest that TR is transcriptionally regulated by electrophilic compounds via an ARE in the 5' region of the gene, and that this mechanism is unrelated to the established Se-dependent induction of selenoproteins. KEY WORDS: * thioredoxin reductase * antioxidant response element * selenium * sulforaphane

Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter [B.sup.O] (AT[B.sup.O]), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by AT[B.sup.O]. The 2.9-kb AT[B.sup.O] mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. AT[B.sup.O] mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of AT[B.sup.O]-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels. amino acid transporter [B.sup.O]; ASCT2; SN1; SN2; hepatocellular carcinoma; transport; system A

Research demonstrates a proteasome-mediated endoplasm reticulum-associated degradation pathway for apolipoprotein B100 in the rat hepatoma cells. Results indicate that the chymotrypsin-like activity of the proteasome degrades apoB100 to the extent of about 50% with the remaining degraded by the endoplasm reticulum-associated nonproteasomal activity.

The objective of this study was to determine the effects of cis-9,trans-11 and trans-10,cis-12 CLA on the fatty acid desaturation in a human hepatoma cell line, HepG2. Therefore, experiments were conducted in which HepG2 cells were incubated with various concentrations of those fatty acids and the concentrations of fatty acids in various lipid fractions of HepG2 cells were determined. In the presence of linoleic acid as substrate, cells treated with 25 [micro]mol/L of trans-10,cis-12 CLA had lower ratios of dihomo-[gamma]-linoleic acid to linoleic acid and of arachidonic acid to linoleic acid in phospholipids than control cells; with [alpha]-linolenic acid as substrate, they had a lower ratio of eicosapentaenoic acid to [alpha]-linolenic acid in phospholipids than control cells. Cells treated with cis-9,trans-11 CLA did not differ in these ratios from control cells. Cells treated with trans-10,cis-12 CLA had also a markedly lower ratio of mononunsaturated fatty acids (MUFA) to saturated fatty acids (SFA) in lipids than control cells; cells treated with cis-9,trans-11 CLA had a slightly lower MUFA:SFA ratio than control cells. These findings suggest that trans-10,cis-12 CLA suppresses [DELTA]9-, [DELTA]6- and [DELTA]5-desaturation in HepG2 cells; cis-9,trans-11 CLA slightly reduces [DELTA]9-desaturation but does not inhibit [DELTA]6- and [DELTA]5-desaturation. Moreover, HepG2 cells treated with 100 [micro]mol/L of trans-10,cis-12 CLA released larger amounts of 6-keto-prostaglandin F[sub.1[alpha]] and prostaglandin F[sub.2[alpha]] than control cells. Treatment of cells with cis-9,trans-11 CLA did not alter the release of these eicosanoids compared with control cells. In conclusion, this study suggests that trans-10,cis-12 CLA has significant effects on the metabolism of essential fatty acids in HepG2 cells, whereas cis-9, trans-11 CLA does not have any effect in this respect. KEY WORDS: * human hepatoma cells * conjugated linoleic acid * fatty acids * desaturation * eicosanoids

Research demonstrates that hepatic-derived cells accumulate 9-cis-retinol in the biosynthetic pathway of 9-cis-retinoic acid and estrification of 9-cis-retinol. Data further reveal that free form of all trans-retinol is used in the biosynthesis of 9-cis-retinol and eventually 9-cis-retinoic acid.

A study was conducted to analyze the influence of extracellular ATP and other nucleotides on the cytosolic free Ca2+ concentration in single primary human hepatocytes and Hep G2 and HuH-7 hepatoma cells. Pieces of liver tissues were prepared from macroscopically normal areas of lobectomies carried out for unilocular metastases. Results suggested that extracellular nucleotides from different sources may influence the regulation of human liver cell functions.

The mechanisms of uptake of non-transferrin-bound iron by human hepatoma cells (HuH7) were studied by applying 59Fe-citrate and (14C)citrate. The quantity of iron associated with the cells increased linearly with time, while the citrate uptake attained a plateau after 45 min, resulting in a cellular accumulation of iron over citrate. The cells had high-affinity membrane binding sites for citrate with maximum binding of 118 +/- 17 pmol citrate/mg protein and dissociation constant of 21 +/- 2 microM.

In this study, we tested the hypothesis that dietary linoleic acid intake controls the arterial blood plasma linoleic acid concentration and the rates of tumor growth and linoleic acid metabolism in vivo. Seven groups of young male Buffalo rats (11-21 rats/group) were given free access to semipurified diets containing different amounts of corn and/or olive oils. Four other groups (7-11 rats/group) were 30% energy-restricted. Each experiment included periods for rat growth and plasma lipid stabilization (6 wk), measurement of mean daily arterial blood plasma fatty acid concentrations (3 wk), surgical implantation of a subcutaneous tissue-isolated hepatoma 7288CTC, tumor growth and harvest (2-4 wk). Linoleic + arachidonic acid (P = 0.007) and oleic acid (P = 0.002) concentrations in arterial blood plasma were increased as dietary intake of linoleic and oleic acids was increased, respectively. In rats given free access to food, tumor growth was directly dependent on the plasma concentrations of linoleic (P < 0.001) and arachidonic acids (P = 0.04). Tumor growth in energy-restricted rats was dependent only on the linoleic acid concentration (P = 0.008). Energy restriction itself caused a growth inhibition independent of plasma linoleic acid. The linoleic acid and total fatty acid concentrations of tumor triacylglycerols were directly dependent on the plasma linoleic acid concentration in rats given free access to food (P = 0.009). Hepatoma 7288CTC (both in vivo and during perfusion in situ) supported a dose-dependent conversion (P < 0.001) of plasma linoleic acid to the mitogen, 13-hydroxy-9, 11-octadecadienoic acid. We conclude that increased arterial blood plasma linoleic acid concentrations, caused by increased dietary intakes, specifically stimulate growth, lipid storage and linoleic acid metabolism in hepatoma 7288CTC in vivo. KEY WORDS: linoleic acid; hepatoma 7288CTC; tumor growth; tumor lipid content; 13-hydroxyoctadecadienoic acid; rats

The proto-oncogene c-myc induces hepatoma cell apoptosis under serum-free conditions in the presence of zinc without causing DNA-fragmentation. The amount of cell death is proportional to the zinc concentration, and independent of the level of c-myc expression. The addition of fetal calf serum or the overexpression of bcl-2 gene inhibits apoptosis. Cellular metallothionein mRNA levels and the rate of apoptosis due to copper or cadmium are independent of c-myc expression.

Farnesyl-pyrophosphate is required for the posttranslational modification of G proteins including p21 ras, prelamin A and lamin B, each of which plays an essential role in cell proliferation. As a consequence, competitive inhibitors of mevalonate synthesis, the rate-limiting substrate for the synthesis of the prenyl-pyrophosphates, arrest cultured cells at the G1/S interface of the cell cycle and initiate apoptotic cell death. Geraniol, an acyclic monoterpenoid alcohol, suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and concomitantly arrests the growth of cultured tumor cells. We evaluated the impact of dietary geraniol on the growth of two tumors. In the first study, geraniol (23 mmol/kg diet, 350 [[micro]mol]/d) was fed to male buffalo rats for 14 d before and for 42 d after the transplant of Morris 7777 hepatomas. Tumor growth was suppressed (P < 0.001). In the second study, the dose-dependent impact of geraniol on the growth of B16 melanomas was assessed. Dietary geraniol (0.65, 6.5 and 65 mmol/kg diet) was fed to female C57BL mice for 14 d before and for 21 d after tumor transplant. Tumor growth was suppressed (P < 0.02) by 6.5 and 65 mmol geraniol/kg diet. INDEXING KEY WORDS: * mevalonate biosynthesis * tumor growth * rats * mice * chemoprevention

A study was conducted on insulin stimulation effects on phosphotyrosine phosphatase (PTPase) activity in rat hepatoma cells. A 32P-labeled peptide that corresponds to the principal insulin receptor autophosphorylation site was used to measure PTP activity. Results indicate that 14% and 86% of PTPase activity occurred in the cystolic fraction and particulate fraction, respectively. When lectin chromatography was used to purify the particulate fraction, specific activity rose by 50%. Both fractions were active toward the tyrosine-phosphorylated insulin receptor in vitro.

Results show that the uptake of DNA frayed wires in the human hepatoma HepG2 cells is 2-3.5-fold greater than those exhibited by single-stranded DNA. Further, nuclear localization of DNA frayed wires is 10-13-fold greater than that of single-stranded DNA.

Roman, Richard M., Roderic L. Smith, Andrew P. Feranchak, Gerald H. Clayton, R. Brian Doctor, and J. Gregory Fitz. ClC-2 chloride channels contribute to HTC cell volume homeostasis. Am J Physiol Gastrointest Liver Physiol 280: G344-G353, 2001.--Membrane [Cl.sup.-] channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated [Cl.sup.-] currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying [Cl.sup.-] currents when non-ClC-2 currents were blocked by DIDS (100 [micro]M). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous [Cl.sup.-] currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane [Cl.sup.-] permeability and cell volume homeostasis. hepatocyte; purinergic receptors; liver

Decaens, C., and D. Cassio. Spatiotemporal expression of catenins, ZO-1, and occludin during early polarization of hepatic WIF-B9 cells. Am J Physiol Cell Physiol 49: C527-C539, 2001.--WIF-B9 is a suitable model for in vitro studies of hepatocyte polarity. To better understand polarity establishment, we have localized key proteins of the adhesion system, cytoskeleton, and tight junctions soon after plating, when most cells are isolated or in doublets. In isolated attached cells, only cytoskeletal proteins (tubulin, cytokeratins) displayed a precise localization. As soon as two cells formed a doublet, E-cadherin, [Alpha], [Beta], and [Gamma]-catenins, and p120 protein were present at the doublet contiguous membrane. Actin, ezrin, and zonula occludens-1 (ZO-1) colocalized at this membrane, but not in all doublets: ezrin was present only at contiguous membrane expressing ZO-1, and ZO-1 was present only at membrane expressing actin. In contrast, occludin was spread throughout the doublet cytoplasm. With time in culture, these proteins localized transiently, as in cells expressing simple epithelial polarity, and finally, as in hepatocytes. We conclude that during WIF-B9 early polarization, key proteins are settled according to a hierarchy, as has been shown for Madin-Darby canine kidney cells. Cytoplasmic complexes of E-cadherin-catenin were detected during the whole polarization process; they were more abundant in fully polarized cells. hepatoma hybrid WIF-B9; polarity establishment; cell-cell adhesion; cytoskeleton; tight junctions; zonula occludens-1

Soy phytoestrogens have been shown to increase plasma levels of HDL cholesterol and apolipoprotein (apo) A-I, its major protein component, in animal studies and in some human studies. The human hepatoma cell line Hep G2 was used to study the effect of the phytoestrogens genistein and daidzein on apo A-I secretion and gene expression in liver cells. Both genistein and daidzein increased apo A-I secretion in a dose-dependent fashion. Apo A-I concentration in the media of treated cells was increased approximately fivefold by 10/[micro]mol/L genistein (P [is less than] 0.001) and approximately onefold by 10/[micro]mol/L daidzein (P [is less than] 0.001) compared with control cells. The effect of genistein on apo A-I secretion was similar to that observed with 17-[Beta]-estradiol. Treatment of cells with genistein for 24 h increased the transcriptional activity of the apo A-I gene as measured by nuclear run-on assay. Transfection experiments with plasmids containing regulatory regions of the apo A-I gene cloned in front of the luciferase reporter gene indicated that the 5' region of the apo A-I gene contained between nucleotides -256 and -41 is responsible for the increased expression of this gene by genistein. J. Nutr. 130: 2489-2492, 2000. KEY WORDS: * phytoestrogens * genistein * daidzein * apolipoprotein A-I * gene expression