Mahlavu Cells Research Articles

Silencing of Glucose-Regulated Protein 78 (GRP78) Enhances Cell Migration Through the Upregulation of Vimentin in Hepatocellular Carcinoma Cells

Glucose-regulated protein 78 (GRP78) plays an important role in embryonic development and cancer progression. However, there is little information regarding the regulation of GRP78 in hepatocellular carcinoma (HCC) metastasis. We used RNA silencing and cDNA expression vectors to manipulate target gene expression in HCC cells. The transwell migration assay and xCelligence biosensor system were applied to determine the proliferatory and migratory ability of the HCC cells. In this study, we found that GRP78 silencing enhanced cell migration in both HepJ5 and Mahlavu cells. Overexpressed GRP78 in skHep1 cells suppressed the migratory ability. In the insight mechanism dissection for GRP78-mediated cancer migration, we found that downregulation of GRP78 caused the increase of vimentin expression on HCC cells. Suppressed vimentin expression also decreased the migratory ability on HCC, indicating that vimentin expression levels modulated the cell migratory ability. We found that silencing GRP78 in HCC cells may enhance cell migration through the increase of vimentin expression.

Survivin-Mediated Cancer Cell Migration Through GRP78 and Epithelial-Mesenchymal Transition (EMT) Marker Expression in Mahlavu Cells

Survivin has multiple functions during the progression of cancer. However, the role of survivin in the progression and metastasis of hepatocellular carcinoma (HCC) remains unknown. Survivin expression in HCC cells (Mahlavu and Hep3B) was assessed using reverse transcription real-time PCR and Western blot analyses. In addition, survivin expression in HCC cells was manipulated using small interfering RNA (siRNA) or overexpression and proliferation and transwell migration assays were performed to monitor the effect of manipulated survivin expression on the growth rate and migratory ability of the transfected cells. Among the HCC cell lines tested, we found high endogenous expression of survivin mRNA and protein in Mahlavu cells. After silencing survivin expression in Mahlavu cells, there was a dramatic decrease in the cell growth rate and an increase in the metastatic potential of the cells. Overexpression of survivin in Hep3B cells suppressed the ability of the cell to migrate. The mechanism of enhanced cell migration caused by decreased survivin expression is mediated through the downregulation of glucose-regulated protein 78 (GRP78) and the upregulation of the epithelial-mesenchymal transition (EMT) marker, vimentin. Survivin may mediate metastasis in HCC. The knockdown of survivin expression may enhance cancer metastasis through the downregulation of GRP78 and upregulation of vimentin expression.

Abstract A171: Cytotoxicity of novel substituted purine derivatives against liver cancer cells

Abstract Cytotoxic purine and purine nucleoside analogs (PNA) are widely used in cancer treatment (1). In this study, three newly synthesized substituted purine derivatives, AUM18, AUM23 and AUM32* were evaluated for their cytotoxicities by Sulforhodamine B (SRB) assay initially on Hct116 (colon), T47D (breast), and Huh7 (liver) human carcinoma cell lines (2). The IC50 values for the cell lines analyzed were ∼2.0µM for AUM18, ∼0.5µM for AUM23 and ∼5nM for AUM32. The growth inhibitory effects that we observed with these PNAs were comparable to those of the known anticancer agents Camptothecin (IC50 ∼ 10 nM) and 5-Fluorouracil (IC50 ∼10µM). Subsequently, the cytotoxicity of each molecule was tested on the HCC cell line panel composed of Huh7, HepG2, Hep3B, Mahlavu, PLC, SkHep1, Focus, SNU-182 and SNU-475 cells. The IC50 values were again in micromolar varying between 0.1 to 10 µM range. However, we did not observe significant cytotoxic effect on SNU-182 cells with AUM18 PNA and on SNU-475 with AUM32. The differential inhibition that we observe, can be considered as an indication of the specificity of these inhibitors against their target. They might interfere with the activity of certain kinases rather than acting as a multi-kinase inhibitor. Furthermore, we evaluated the protein kinase activity inhibition potential of these novel purine analogs. Therefore, the kinase assay based on the detection of the amount of ATP in the reaction mixture through bioluminescence was performed with Huh7 and Mahlavu cells. Cells incubated with these novel kinase inhibitor candidates and Staurosporine (STS), a multi-kinase inhibitor used as positive control (4). The relative light units that we observed with novel PNAs were comparable to that of STS. Based on our observations, the newly synthesized PNAs, AUM18, AUM23 and AUM32 can be identified as protein kinase inhibitor candidates which must be further analyzed at the molecular level. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A171.

Generation and Modulation of Hepatocellular Carcinoma Circulating Cells: A New Experimental Model

To establish a new experimental model of human hepatocellular carcinoma by orthotopic implantation of tumoral cells with its subsequent removal, to generate and modulate circulating tumoral cells. Three human hepatoma cell lines (HepG2, PLC/PRF, and Mahlavu) were orthotopically implanted under the Glisson's capsule of the left lateral lobe of the liver in a total of 56 non-obese diabetic/severe combined immunodeficiency mice. Tumor removal was performed 30 d after injection, and a laparotomy without tumor removal was done in control mice. Generation of circulating cells was monitored by flow cytometry using fluorescein isothiocyanate-conjugated anti-HLA antibody. In 26 mice implanted with Mahlavu cells, 20 developed a unique tumor allowing a resection (77%), which was technically feasible in 80% of cases. The overall perioperative mortality was 30% (3/10) after resection; no mortality was observed in the control group. The circulating tumoral cells decreased dramatically after resection of the tumor as compared with control mice. This new model is feasible and may be an interesting useful tool to study the hepatocellular carcinoma metastatic process and is consistent with the human clinical practice.

6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant Mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism.

Mahlavu cells, poorly differentiated and p53 mutants of a human hepatoma subline, are known to be highly refractory to a number of chemotherapeutic agents and radiotherapy due to their high expressions of multidrug resistance gene-1 (MDR-1) and Bcl-2 proteins. Thus, it is desirable to search for an alternative strategy for effective eradication of this type of cancer cells. We present evidence here for the first time that 6-shogaol (6-SG), an alkanone isolated from the rhizomes of ginger, can effectively induce apoptotic cell death of Mahlavu cells via an oxidative stress-mediated caspase-dependent mechanism. The cascade of events in 6-SG-induced apoptosis of these cells involved an initial overproduction of reactive oxygen species (ROS) followed by a severe depletion of intracellular glutathione (GSH) contents. Both events consequently entailed a significant drop in mitochondrial transmembrane potential (DeltaPsim), which ultimately activated the activities of caspases 3/7 resulting in the DNA fragmentation. Interestingly, we also found that N-acetylcysteine (NAC), an antioxidant and a precursor of GSH biosynthesis, could offer a near complete protection of apoptotic cell death exerted by 6-SG. Similarly, exogenously added GSH could also provide protection with an equal efficacy. However, it was paradoxical that both Boc-Asp(OMe)-fmk (a broad caspases inhibitor) and cyclosporin A (an mitochondrial permeability transition opening inhibitor) could only partially protect these cells from 6-SG-induced apoptosis. Taking these data into consideration, it is obvious that GSH depletion is the major contributing factor in arbitrating 6-SG-induced apoptosis of Mahlavu cells. In conclusion, we provide here a novel modality that can help to eradicate a p53 mutant of human hepatoma cells by using a natural consistent isolated form of ginger. These data also provide evidence to reaffirm the notion that consumption of certain foodstuffs can be beneficial to health because some of the constituents contained in them may be anticarcinogenic.

Expression of T‐cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma

Overexpression of T-cadherin (T-cad) transcripts occurs in approximately 50% of human hepatocellular carcinomas (HCCs). To elucidate T-cad functions in HCC, we examined T-cad protein expression in normal and tumoral human livers and hepatoma cell lines and investigated its influence on invasive potential of HCC using RNA interference silencing of T-cad expression in Mahlavu cells. Whereas T-cad expression was restricted to endothelial cells (EC) from large blood vessels in normal livers, it was up-regulated in sinusoidal EC from 8/15 invasive HCCs. Importantly, in three of them (38%) T-cad was detected in tumor cells within regions in which E-cadherin expression was absent. Among six hepatoma cell lines, only Mahlavu expressed T-cad but not E-cadherin. T-cad exhibited a globally punctuate distribution in quiescent Mahlavu and additionally it concentrated at the leading edge of migrating cells. Matrigel invasion assay revealed that Mahlavu possess a high invasive potential that was significantly inhibited by T-cad silencing. Wound healing and random motility assays demonstrated that inhibition of T-cad expression in Mahlavu significantly reduced their motility. We propose that T-cad expression in tumor cells might occur by cadherin-switching during epithelial-mesenchymal transition and may represent an additional mechanism contributing to HCC metastasis.

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

Actinodaphnine induces apoptosis through increased nitric oxide, reactive oxygen species and down-regulation of NF-κB signaling in human hepatoma Mahlavu cells

Actinodaphnine, extracted from Cinnamomum insularimontanum (Lauraceae), possesses cytotoxicity in some cancers, but the mechanism by which actinodaphnine induces apoptosis in human hepatoma cells remains poorly understood. In this study, we investigated the mechanisms of apoptosis induced by actinodaphnine in human hepatoma Mahlavu cells. Treatment with actinodaphnine dose-dependently induced apoptosis in Mahlavu cells that correlated with increased intracellular nitric oxide (NO) and reactive oxygen species (ROS), disruptive mitochondrial transmembrane potential (Δ Ψ m), and activation of caspase 3/7. Our data also demonstrated that actinodaphnine down-regulated activity of nuclear factor κB (NF-κB). The apoptotic response to actinodaphnine was markedly decreased in Mahlavu cells pretreated with dexsamethasone, a NO inhibitor, N-acetylcysteine (NAC), an antioxidant, and Boc-Asp(OMe)-fmk, a broad caspases inhibitor. These results suggested that actinodaphnine-induced apoptosis is initially mediated through the NO and/or ROS increase and caspases-dependent pathway. In conclusion, our results indicate that an increase of ROS and/or NO is the initial essential event that results in the decrease of Δ Ψ m and the activation of caspases that commits the cells to the apoptotic pathway in actinodaphnine-treated hepatoma Mahlavu cells.

Aberrant caspase-activated DNase (CAD) transcripts in human hepatoma cells.

The gene of caspase-activated DNase (CAD), the key enzyme for nucleosome cleavage during apoptosis, is mapped at chromosome 1p36, a region usually associated with hemizygous deletions in human cancers, particularly in hepatoma (HCC). It is tempting to speculate that CAD plays a tumour-suppressive role in hepatocarcinogenesis. To address this, we examined the CAD transcripts in six human HCC cell lines, one liver tissue from a non-HCC subject, and peripheral blood leukocytes (PBL) from three healthy individuals. Alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were isolated in most of the examined samples including HCC cells and normal controls. However, relatively abundant alternatively spliced CAD transcripts with fusion of exon 2 to exon 6 or 7, in which the corresponding domain directing CAD interaction with ICAD was preserved, were found only in poorly differentiated Mahlavu and SK-Hep1 cells. Interestingly, an abnormal CAD transcript with its exon 3 replaced by a truncated transposable Alu repeat was isolated in Hep3B cells, indicative of the implication of an Alu-mediated genomic mutation. Moreover, mis-sense mutations in the CAD genes were identified in all six HCC cell lines. Upon UV-induced apoptosis, DNA fragmentation efficiency was found to be intact, partially reduced and remarkably reduced in Huh7 and J328, Hep3B and HepG2, and Mahlavu cells, respectively. That mutations and aberrantly spliced transcripts for the CAD gene are frequently present in human HCC cells, especially in poorly differentiated HCC cells, suggests a significant role of CAD in human hepatocarcinogenesis.

Dysregulation of glycogen synthase kinase-3β signaling in hepatocellular carcinoma cells

It has been reported that upstream components of the insulin-like growth factor (IGF) signaling axis could be overexpressed during hepatocarcinogenesis in humans and rodents. However, the signal transduction pathways activated downstream have been poorly studied. Here, we examined whether glycogen synthase kinase-3β (GSK-3β) could be a target in human hepatoma cell lines and transgenic ASV mice with hepatic expression of the SV40 large T antigen. In HuH7, Mahlavu, and Hep3B cells, basal levels of GSK-3β Ser9 phosphorylation were strongly elevated, indicating that GSK-3β was inhibited. GSK-3β phosphorylation was insensitive to exogenous IGFs and was blocked with an IGF-1 receptor–neutralizing antibody in Mahlavu and Hep3B cells. By using LY294002 and ML-9, which act as phosphatidylinositol 3-kinase (PI3-K) and Akt inhibitors, respectively, we showed that GSK-3β phosphorylation required PI3-K activation in both cell lines whereas downstream Akt activation was required only in Mahlavu cells. However, in the 2 cell lines, GSK-3β Ser9 phosphorylation was controlled by protein kinase C (PKC)ζ because it was blocked by an inhibitory PKCζ peptide. The blockage of GSK-3β phosphorylation markedly inhibited glycogen synthesis and decreased β-catenin expression. In addition, the overexpression of a constitutively active GSK-3β reduced AP-1–mediated gene transcription in Hep3B cells. Finally, we observed that reexpression of IGF-2 in tumoral livers from ASV mice was associated with a marked phosphorylation of GSK-3β. In conclusion, our results identify GSK-3β as a molecular target of the constitutive activation of the IGF axis in in vitro and in vivo models of hepatocarcinogenesis. Persistent phosphorylation of GSK-3β could be critical for regulation of glycogen metabolism and cell growth in hepatoma cells. (H EPATOLOGY 2002;36:1528-1536.)

Dysregulation of glycogen synthase kinase-3β signaling in hepatocellular carcinoma cells

It has been reported that upstream components of the insulin-like growth factor (IGF) signaling axis could be overexpressed during hepatocarcinogenesis in humans and rodents. However, the signal transduction pathways activated downstream have been poorly studied. Here, we examined whether glycogen synthase kinase-3β (GSK-3β) could be a target in human hepatoma cell lines and transgenic ASV mice with hepatic expression of the SV40 large T antigen. In HuH7, Mahlavu, and Hep3B cells, basal levels of GSK-3βSer9 phosphorylation were strongly elevated, indicating that GSK-3β was inhibited. GSK-3β phosphorylation was insensitive to exogenous IGFs and was blocked with an IGF-1 receptorneutralizing antibody in Mahlavu and Hep3B cells. By using LY294002 and ML-9, which act as phosphatidylinositol 3-kinase (PI3-K) and Akt inhibitors, respectively, we showed that GSK-3β phosphorylation required P13-K activation in both cell lines whereas downstream Akt activation was required only in Mahlavu cells. However, in the 2 cell lines, GSK-3βSer9 phosphorylation was controlled by protein kinase C (PKC)ξ because it was blocked by an inhibitory PKCξ peptide. The blockage of GSK-3β phosphorylation markedly inhibited glycogen synthesis and decreased β-catenin expression. In addition, the overexpression of a constitutively active GSK-3β reduced AP-1-mediated gene transcription in Hep3B cells. Finally, we observed that reexpression of IGF-2 in tumoral livers from ASV mice was associated with a marked phosphorylation of GSK-3β. In conclusion, our results identify GSK-3β as a molecular target of the constitutive activation of the IGF axis in in vitro and in vivo models of hepatocarcinogenesis. Persistent phosphorylation of GSK-3β could be critical for regulation of glycogen metabolism and cell growth in hepatoma cells. (Hepatology2002;36:1528-1536).

In vitro toxicity of 7 H-dibenzo[ c,g]carbazole in human liver cell lines

7 H-Dibenzo[ c,g]carbazole (DBC) is a model N-heterocyclic aromatic compound (NHA) which is both a hepatotoxin and hepatocarcinogen in rodents. The focus of this investigation was to determine whether human liver cell lines display differential sensitivities to DBC-induced toxicity. Treatment of cell lines with increasing DBC concentrations produced apoptosis only in HepG2 cells. Although DBC inhibited the clonogenic growth of all cell lines at high concentrations, only the survival of HepG2 cells was reduced at lower concentrations. DBC inhibited DNA synthesis in two (HepG2, HLF) of the three cell lines at lower concentrations and was effective only at a high concentration in Mahlavu cells. Differences in DBC uptake were not observed in any of the cell lines, suggesting that bioavailability was not a limiting factor. DBC–DNA adducts were not detected in HLF or Mahlavu cells at either low or high concentrations of DBC. Consistent with the DNA adduct data, RP-HPLC analysis indicated that DBC was metabolized to a lesser degree in the HLF and Mahlavu cells. These results suggest that human liver cell lines differ markedly in the ability to metabolize DBC to toxic species and that DBC-induced apoptosis is only observed in cells that produce detectable metabolites and DBC–DNA adducts.

Altered regulation of Src tyrosine kinase by transforming growth factor beta1 in a human hepatoma cell line.

Transforming growth factor betas (TGF-betas) are the potent growth inhibitors for various cell types. Certain transformed cells, however, show poor response to TGF-beta-induced growth inhibition, which contributes to their uncontrolled proliferation. Recently, we have reported that TGF-beta1 induces degradation of activated Src tyrosine kinase in rat fibroblasts. To elucidate the alteration in TGF-beta signaling pathway in tumor cells that cannot respond to the cytokine, we compared the effects of TGF-beta1 on Src kinase in two human hepatoma cell lines, TGF-beta1-insensitive Mahlavu cells and TGF-beta1-sensitive HepG2 cells. TGF-beta1 decreased Src kinase activity in HepG2 cells, but increased cellular Src levels and Src kinase activity in Mahlavu cells. Co-incubation of Mahlavu cells with TGF-beta1 and 12-O-tetradecanoyl phorbol 13-acetate (TPA) decreased Src protein levels and Src kinase activity, inducing TGF-beta1 sensitivity. TGF-beta1 induced tyrosine dephosphorylation of Ras guanosine triphosphatase-activating protein (Ras-GAP) and Ras inactivation in HepG2 cells, but induced Ras-GAP phosphorylation and Ras activation in Mahlavu cells. The Src kinase inhibitor abolished the increase of Src kinase activity in TGF-beta1-treated Mahlavu cells, and induced TGF-beta1 sensitivity. These findings suggest that regulation of Src kinase by TGF-beta1 is altered in Mahlavu cells. The altered regulation of Src may contribute to TGF-beta1 insensitivity in this cell line, at least in part through activation of Ras.

Regulation of heparin-binding EGF-like growth factor expression by phorbol ester in a human hepatoma-derived cell line

Heparin-binding EGF-like growth factor (HB-EGF) is a recently identified potent mitogen for smooth muscle cells and fibroblasts. HB-EGF has been shown to be an EGF receptor ligand, and also to stimulate epithelial cell growth. A human hepatoma-derived cell line, Mahlavu, was analyzed for the production of HB-EGF mRNA and active HB-EGF protein. It was found that the cell line synthesized very low or undetectable basal level of HB-EGF mRNA. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a rapid and transient rise in HB-EGF mRNA level. HB-EGF in Mahlavu cells appears to be regulated by a protein kinase C (PKC) pathway, since PKC inhibitors, H7, staurosporin, and calphostin C, abrogated the induction of HB-EGF mRNA by TPA. Unlike vascular smooth muscle cells, induction of HB-EGF gene transcription by TPA was blocked completely by incubation with cycloheximide, suggesting that protein synthesis may be a prerequisite for HB-EGF gene transcription in Mahlavu cells. Mahlavu cells were also found to release a bioactive HB-EGF-like protein into conditioned medium which stimulates DNA synthesis in EP170.7 cells. This activity was neutralized by an anti-HB-EGF antibody. These results indicate that HB-EGF gene transcription is regulated via a PKC pathway, resulting in secretion of active HB-EGF into the culture medium of hepatoma-derived Mahlavu cells.

Differential in situ hybridization for determination of mutational specific expression of the p53 gene in human hepatoma cell lines

The codon 249 mutation specific expression of the p53 gene was determined in 7 human hepatocellular carcinoma (HCC) cell lines. Two 20-base oligomers complementary to bases 872-891 of human p53 cDNA with a single nucleotide difference in the third position of codon 249 were end-labelled with biotin-conjugated dATP using terminal deoxynucleotidyltransferase (TdT). The hybridized oligomer was visually detected in situ using streptavidin-alkaline phosphatase (AP) conjugate and AP substrate. Expression of the codon 249 mutant p53 was steady in PLC/PRF/5 and Mahlavu cells (derived from African patients), while Huh4, Huh6, Huh7 and HCC-M cells (derived from Japanese patients) expressed only the codon 249 wild-type p53. The transcripts of the p53 gene were undetectable in Hep3B cells (derived from an American patient). Hybridizations of the codon 249 specific oligomers were specific to the p53 transcripts, since the cells that expressed p53 gene homogeneously were stained in the cytoplasm only by differential hybridization with a codon 249 specific oligomer; moreover, hybridization with a labelled oligomer non-complementary to the p53 cDNA showed nuclear stainings. Thus, detection of the codon 249 mutant p53 mRNA by differential in situ hybridization is a specific method for studying the mutation-specific expression of the p53 gene in liver cancers at the cellular level, while simultaneously visualizing the cell morphology. The results also support the notion that the p53 gene codon 249 mutation may have etiological implications involving HCC from various geographic areas.

Extralysosomal degradation of high-density lipoproteins in a human hepatoma cell line, Mahlavu

Lipoprotein metabolism has been investigated in a novel human hepatoma cell line, Mahlavu, which has been reported to possess the characteristics of hepatocytes. Low-density lipoprotein (LDL) was degraded by Mahlavu cells. LDL taken up by the cells suppressed intracellular cholesterol biosynthesis and promoted cholesterol esterification in a manner similar to that of HepG2 cells. High-density lipoprotein (HDL) was also degraded by Mahlavu cells, whereas it was not degraded by fibroblasts. We compared the mode of intracellular metabolism of HDL to that of LDL. In contrast to the LDL receptor pathway, the degradation of HDL was not inhibited by 100 μM chloroquine added to the medium, indicating that the degradation may not occur in lysosomes. Cholesterol taken up as HDL-cholesterol by the Mahlavu cells had no effect on the intracellular biosynthesis of cholesterol nor on cholesterol esterification. The conditioned media in which Mahlavu cells had been cultured did not promote the degradation of HDL, suggesting that HDL is degraded intracellularly. These data suggest that HDL is taken up and degraded by the liver cells in contrast to extrahepatic peripheral cells such as fibroblasts and macrophages in which the degradation of HDL does not occur. The results indicate that HDL-associated cholesterol may be processed via a pathway different from that of LDL metabolism and that the degradation of HDL occurs extralysosomally.

Interactions between Platelets and Human Hepatoma Cell Lines: The Influence of Endothelial Cells

The properties of human hepatoma cell lines in relation to their ability to aggregate isolated human platelets were investigated because of the possible relevance to tumour metastasis. Mahlavu, HepG2 and SK55 cells were all able to aggregate platelets irreversibly, but required the presence of small amounts of plasma, which could not be replaced by fibrinogen or other common plasma proteins. They also required the presence of Ca(2+). The plasma factor was non-dialysable and heat-labile. In the case of Mahlavu cells and SK55 cells the aggregatory activity was released from the cells as a non-dialysable material of high molecular weight. HepG2 cells were required intact and did not release materials which induced platelet aggregation. In all cases the aggregation was inhibited by hirudin. However, the requirements for plasma factors suggested that the material was not thrombin itself. The aggregatory property of all the cells was inhibited by prostacyclin and in the case of Mahlavu and HepG2 cells, to a lesser extent by nitric oxide. Addition of very small numbers of bovine aortic endothelial cells (but not human umbilical cord cells) inhibited the aggregation induced by Mahlavu and HepG2 cells, but not SK55 cells. If the endothelial cells were pre-treated with aspirin, this inhibitory property was abolished, indicating that a cyclo-oxygenase product was the principal agent.

Effects of phorbol ester on cell growth inhibition by transforming growth factor β 1 in human hepatoma cell lines

The effects of phorbol ester on cell growth inhibition by transforming growth factor β 1 (TGF-β 1) in human hepatoma cell lines, Mahlavu and PLC/PRF/5, were investigated. TGF-β 1 (2.5 to 10 pM) alone could not inhibit the growth of Mahlavu cells, whereas in the presence of 12-O-tetradecanol phorbol 13-acetate (TPA) at 1 ng/ml, TGF-β 1 could suppress their growth in a dose-dependent manner. The growth of PLC/PRF/5 cells could be inhibited by addition of TGF-β 1 (2.5 to 10 pM) alone in a dose-dependent manner, and this action was not affected by TPA (1 ng/ml). The TGF-β 1 inhibition induced by TPA in Mahlavu cells could not be cancelled by addition of protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) (10 μM) or staurosporin (1 nM). Thus, TPA could induce TGF-β 1 inhibition of cell growth in Mahlavu cells which did not respond to TGF-β 1 alone, and activation of protein kinase C does not seem to be behind this TPA action.

Modulation of c‐myc Expression by Transforming Growth Factor β1 in Human Hepatoma Cell Lines

The effects of transforming growth factor β1 (TGF‐β1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum‐free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF‐β1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c‐myc mRNA was suppressed by the addition of TGF‐β1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF‐β1 might regulate cell growth, in part, by modulating c‐myc expression, although there is no direct proof that c‐myc expression is really relevant to DNA synthesis mediated by TGF‐β1.

Abnormal structure and expression of p53 gene in human hepatocellular carcinoma.

There is little information regarding the molecular mechanisms of hepatocarcinogenesis. We studied the p53 gene at the DNA, RNA, and protein level in seven human hepatocellular carcinoma (HCC)-derived cell lines; six of seven showed p53 abnormalities. By Southern blotting, the p53 gene was found to be partially deleted in Hep 3B and rearranged in SK-HEP-1 cells. Transcripts of the p53 gene were undetectable in Hep 3B as well as in FOCUS cells that had no apparent deletion or rearrangement of the p53 gene. Immunoprecipitation after [35S]methionine labeling of HCC cells demonstrated that p53 protein was absent in Hep 3B and FOCUS and reduced in concentration in PLC/PRF/5 cells. p53 synthesized by Mahlavu cells showed a slower migration on SDS/polyacrylamide gels suggesting it was an abnormal protein. In Huh7 cells, p53 protein had a prolonged half-life leading to its accumulation in the nuclei; increased levels of p53 protein were also found by immunoblotting. The p53 gene and its expression appeared to be unaltered in the hepatoblastoma-derived Hep G2 cell line. We found that the loss of p53 expression did not occur as a late in vitro event in the FOCUS cell line because p53 protein was also nondetectable at an early passage. We conclude that the loss of p53 expression or the presence of abnormal forms of the protein are frequently associated with HCC cell lines. These observations suggest that alterations in p53 may be important events in the transformation of hepatocytes to the malignant phenotype.