transforming-growth-factor-alpha and Carcinoma--Hepatocellular

Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumors worldwide. HCC is a complex process that is associated with several etiological factors, which in turn result in aberrant activation of different cellular and molecular pathways and the disruption of balance between activation and inactivation of protooncogenes and tumor suppressor genes, respectively. Since HCC most often occurs in the setting of a diseased or cirrhotic liver and most of the patients are diagnosed at the late stage of disease, prognosis is generally poor. At present, limited treatment options with marginal clinical benefits are available. Systemic therapy, particularly in the form of conventional cytotoxic drugs, are generally ineffective. In recent years, molecular-targeted therapies have been clinically used to treat various cancers, including liver cancer. This approach inhibits the growth of tumor cells by interfering with molecules that are involved in carcinogenesis, which makes it more selective and specific than cytotoxic chemotherapy. Many clinical trials have been carried out while using molecular targeted drugs in advanced HCC with many more in progress. The clinical trials in HCC to date have evaluated a single-targeted therapy alone, or two or more targeted therapies in parallel. The aim of this review is to provide insight of various molecular mechanisms, leading to HCC development and progression, and also the range of experimental therapeutics for patients with advanced HCC. The review will summarize different clinical trials data the successes and failures of these treatments, as well as the most effective and approved drugs designed against HCC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Incidence; Interleukin-6; Liver Neoplasms; NF-kappa B; Risk Factors; Signal Transduction; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Epidermal growth factor receptor system plays a central hepato-protective and pro-regenerative role in liver. Transforming growth factor-α (TGF-α) is an important autocrine growth regulator of hepatocytes that plays a role in development of hepatocellular carcinoma (HCC) among patients with chronic hepatitis C (CHC). This study was done on 40 core liver biopsies from patients with CHC, 20 liver specimens from HCC cases on top of CHC as well as five normal controls. All were immunohistochemically stained with epidermal growth factor receptor (EGFR) and TGF-α antibodies. Some selected HCC cases were submitted for FISH technique to detect EGFR gene alteration. By immunohistochemistry EGFR and TGF-α were overexpressed in HCC and cirrhotic cases compared to CHC cases without cirrhosis. Also, their expression was stronger in CHC cases with higher grades of activity and stages of fibrosis compared to lower ones. FISH positive results for EGFR were detected in 33.3% of the examined HCC cases. EGFR and TGF-α can be used as predictive markers for activity, fibrosis, and carcinogenesis in CHC patients. Overexpression of EGFR in HCC patients can be promising in selecting those who can get benefit from anti-EGFR target therapy.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Hepatocellular; ErbB Receptors; Female; Hepacivirus; Hepatitis C, Chronic; Hepatocytes; Humans; In Situ Hybridization, Fluorescence; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cetuximab; Clinical Trials, Phase II as Topic; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Ligands; Liver Neoplasms; Multicenter Studies as Topic; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Quinazolines; Rats; Signal Transduction; Transforming Growth Factor alpha

Hepatocarcinogenesis is a slow process during which genomic changes progressively alter the hepatocellular phenotype to produce cellular intermediates that evolve into hepatocellular carcinoma. During the long preneoplastic stage, in which the liver is often the site of chronic hepatitis, cirrhosis, or both, hepatocyte cycling is accelerated by upregulation of mitogenic pathways, in part through epigenetic mechanisms. This leads to the production of monoclonal populations of aberrant and dysplastic hepatocytes that have telomere erosion and telomerase re-expression, sometimes microsatellite instability, and occasionally structural aberrations in genes and chromosomes. Development of dysplastic hepatocytes in foci and nodules and emergence of hepatocellular carcinoma are associated with the accumulation of irreversible structural alterations in genes and chromosomes, but the genomic basis of the malignant phenotype is heterogeneous. The malignant hepatocyte phenotype may be produced by the disruption of a number of genes that function in different regulatory pathways, producing several molecular variants of hepatocellular carcinoma. New strategies should enable these variants to be characterized.

Topics: Carcinoma, Hepatocellular; Chromosome Aberrations; DNA Methylation; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Humans; Insulin-Like Growth Factor II; Liver Neoplasms; Precancerous Conditions; Transforming Growth Factor alpha

The experimental three-stage hepatocarcinogenesis protocol of initiation, promotion, and progression, coupled with the analytical technique of stereology, permits quantitative analysis of the carcinogenic process, including the derivation of biologically based risk assessment models. The aberrant expression of the placental isozyme of glutathione S-transferase (PGST) is an efficient marker for initiated, preneoplastic, and neoplastic hepatocytes. Putatively initiated cells and their clonal progeny can be identified, enumerated, and their growth characteristics determined on the basis of their aberrant expression of this protein. A lack of suitable markers has made the identification and quantitation of hepatocytes in the early stage of progression more difficult. One characteristic of cells in the stage of progression is the evolution of relatively autonomous growth. The alteration of growth factor signalling pathways may provide one mechanism for this observation. The expression of transforming growth factor alpha (TGF alpha) is seen in many malignancies. The initiation-promotion-progression protocol has been used to induce progression in the rat liver. The focal expression of TGF alpha was found to correlate with areas of progression in rats subjected to this protocol. The ability to identify and quantitate cells in the stage of progression should facilitate application of the Moolgavkar-Venzon-Knudson model for assessing human risk from carcinogens active at each of these three stages. Validation of this model will require determination of the number and growth characteristics of hepatocytes in the stage of progression.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Count; Cell Transformation, Neoplastic; Diethylnitrosamine; Disease Progression; Glutathione Transferase; Liver Neoplasms; Models, Biological; Neoplasm Staging; Photogrammetry; Rats; Risk Assessment; Transforming Growth Factor alpha

A series of changes in the genes that control hepatocyte growth, or interference with the protein products of these genes, appears to have an important role in the etiology of hepatocellular carcinoma (HCC). Mutations of the p53 tumor suppressor gene have been identified in 30-50% of HCC patients in some geographic areas. Abnormalities of the RB tumor suppressor gene have been found in 20-25% of HCCs, including 80-86% of HCCs with p53 mutations. Overexpression of transforming growth factor alpha (TGF-alpha), insulin-like growth factor II (IGF-II), and the oncogenes N-ras, c-myc, and c-fos have been found in high percentages of HCC patients. The cumulative effect of these changes may be more important than the order in which they occur. Some of these changes may explain the mechanism(s) by which the hepatitis B virus participates in the development of HCC.

Topics: Carcinoma, Hepatocellular; Genes, Tumor Suppressor; Hepatitis B; Humans; Insulin-Like Growth Factor II; Liver Neoplasms; Oncogenes; Transforming Growth Factor alpha; Virus Integration

Duloxetine, a selective reuptake inhibitor for serotonin and norepinephrine, is a medication widely used for major depression. Currently, duloxetine is also recommended for pain related to chemotherapy-induced peripheral neuropathy or cancer. Previously, we showed that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC)-derived HuH7 cells through the activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and AKT. In the present study, we investigate whether duloxetine affects cell migration and its mechanism. Duloxetine significantly enhanced the TGF-α-induced migration of HuH7 cells. Fluvoxamine and sertraline, specific inhibitors of serotonin reuptake, also upregulated the TGF-α-induced cell migration. On the contrary, reboxetine, a specific norepinephrine reuptake inhibitor, failed to affect cell migration. Duloxetine significantly amplified the TGF-α-stimulated phosphorylation of JNK, but not p38 MAPK and AKT. In addition, fluvoxamine and sertraline, but not reboxetine, enhanced the phosphorylation of JNK. SP600125, a JNK inhibitor, suppressed the enhancement by duloxetine, fluvoxamine, or sertraline of TGF-α-induced migration of HuH7 cells. Taken together, our results strongly suggest that duloxetine strengthens the TGF-α-induced activation of JNK via inhibition of serotonin reuptake in HCC cells, leading to the enhancement of cell migration.

Topics: Carcinoma, Hepatocellular; Duloxetine Hydrochloride; Fluvoxamine; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Norepinephrine; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Serotonin; Sertraline; Transforming Growth Factor alpha; Up-Regulation

Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time-of-day dependent changes of proliferation and DNA damage in HCC. Using transgenic c-myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ-H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev-erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ-H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time-of-day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Chlorides; Chronotherapy; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; Mice; Mice, Transgenic; Neoplasms, Experimental; Period Circadian Proteins; Photoperiod; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha; Tumor Cells, Cultured; Zinc Compounds

Incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are hormones secreted from small intestine accompanied with oral intake. We previously showed that transforming growth factor (TGF)-α stimulates the migration of hepatocellular carcinoma (HCC) cells via mitogen-activated protein (MAP) kinases, AKT and Rho-kinase. However, it remains to be elucidated whether incretins affect HCC cell functions. In the present study, therefore, we investigated whether incretins affect the migration of HCC cells using human HCC-derived HuH7 cells. GLP-1, but not GIP, reduced both TGF-α- and hepatocyte growth factor (HGF)-induced cell migration. IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, enhanced the suppressive effect of GLP-1. GLP-1 attenuated the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) by TGF-α and HGF. Our results strongly suggest that GLP-1 suppresses TGF-α- and HGF-induced migration of HCC cells through inhibiting the SAPK/JNK signaling pathway, and that the inhibition by GLP-1 is due to cAMP production.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cyclic AMP; Glucagon-Like Peptide 1; Hepatocyte Growth Factor; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Mitogen-Activated Protein Kinases; Phosphorylation; Transforming Growth Factor alpha

Flavonol, which is found abundantly in plants such as fruits and vegetables, belongs to the family of flavonoid, natural polyphenols. Quercetin, one of the flavonol, reportedly has anti-cancer effects and prevents the proliferation of various cancer cells, including hepatocellular carcinoma (HCC). However, the effects of quercetin on HCC cells migration have not yet been clarified. We have previously shown that the migration of human HCC-derived HuH7 cells induced by hepatocyte growth factor (HGF) or transforming growth factor-α (TGF-α) is mediated through p38 MAPK and AKT. In this study, we investigated whether quercetin affects the HGF- or TGF-α-induced migration of HuH7 cells. Quercetin significantly suppressed both HGF- and TGF-α-induced migration of HuH7 cells in a dose-dependent manner. In addition, myricetin, another flavonol, also showed significant inhibition of the cell migration. Each HGF- and TGF-α-induced autophosphorylation of receptors were not affected by quercetin or myricetin. Quercetin did not suppress HGF- or TGF-α-induced p38 MAPK phosphorylation. On the contrary, quercetin and myricetin inhibited the growth factors-induced phosphorylation of AKT. Our results strongly suggest that quercetin suppresses the growth factor-induced migration of HCC cells by inhibiting the signaling pathway of AKT but not p38 MAPK.

Topics: Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Flavonols; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Liver Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Quercetin; Signal Transduction; Transforming Growth Factor alpha

Small heat shock proteins (HSPs) regulate a variety of cell functions. Among them, HSP22 and HSP20 are recognized to be ubiquitously expressed in various tissues. With regard to hepatocellular carcinoma (HCC) cells, we previously reported that phosphorylated HSP20 plays a suppressive role in transforming growth factor (TGF)-α-induced cell migration and invasion. In the present study, we investigated whether or not HSP22 is implicated in HCC cell migration. We detected HSP22 protein expression both in human HCC tumor (189.9±68.4ng/mg protein) and the adjacent non-tumor liver tissues (167.9±94.6ng/mg protein). The cases of low-quantity HSP22 protein level group (88.3≧ng/mg protein, the optimum cut-off value of HSP22) were increased in tumor tissues compared with the adjacent non-tumor tissues. The migration of human HCC-derived HuH-7 cells stimulated by TGF-α or hepatocyte growth factor (HGF) was significantly enhanced by the knockdown of HSP22 expression. Down-regulation of HSP22 protein in the cells markedly strengthened the AKT phosphorylation induced by TGF-α or HGF. Inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathway, which suppressed the TGF-α-induced migration, significantly reduced the amplification by HSP22 knockdown. PI3K but not AKT was coimmunoprecipitated with HSP22 in HuH-7 cells. In addition, in human HCC tissues, a significantly lower HSP22 protein level in tumor tissues than in adjacent non-tumor tissues was observed more frequently in cases of moderately or poorly differentiated HCC than well-differentiated HCC. Taken together, our results strongly suggest that HSP22 represses HCC progression, especially HCC cell migration, by the down-regulation of the PI3K/AKT signaling pathway.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Female; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Liver Neoplasms; Male; Molecular Chaperones; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor alpha

The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytosol; DNA; Gene Deletion; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Liver Neoplasms; Mice; Oncogenes; Phosphatidylinositol 3-Kinases; Protein Stability; Protein Transport; Suppressor of Cytokine Signaling 1 Protein; Transforming Growth Factor alpha

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.

Topics: Anthracenes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; HSP27 Heat-Shock Proteins; Humans; Liver Neoplasms; MAP Kinase Kinase 4; Neoplasm Invasiveness; Neoplasm Proteins; Phosphorylation; Signal Transduction; Transforming Growth Factor alpha

Nanoparticle (NP)-based contrast agents that enable high resolution anatomic T1-weighted magnetic resonance imaging (MRI) offer the prospect of improving differential diagnosis of liver tumors such as hepatocellular carcinoma (HCC). In the present study, we investigated the possibility of employing novel non-toxic human serum albumin nanoparticles conjugated with Gd-DTPA and rhodamine 123 (Gd-Rho-HSA-NPs) for the detection of HCC by T1-weighted MRI. In addition, the influence of surface coating of the NPs with poloxamine 908, which alters the absorptive behavior of NPs and changes their distribution between the liver and tumor was examined. MRI of transgenic mice with endogenously formed HCCs following intravenous injection of Gd-Rho-HSA-NPs revealed a strong negative contrast of the tumors. Contrasting of the HCCs by NP-enhanced MRI required less Gd as compared to gadolinium-ethoxybenzyl-diethylenetriaminepentaacetic acid-enhanced MRI, which currently provides the most sensitive detection of HCC in patients. Immunohistochemical analyses revealed that the Gd-Rho-HSA-NPs were localized to macrophages, which were - similar to HCC in patients - fewer in number in HCC as compared to the liver tissue, which is in agreement with the negative contrasting of HCC in Gd-Rho-HSA-NP-enhanced MRI. Poloxamine-coated NPs showed lower accumulation in the tumor macrophages and caused a longer lasting enhancement of the MRI signal. These data indicate that Gd-Rho-HSA-NPs enable sensitive detection of HCC by T1-weighted MRI in mice with endogenous HCC through their uptake by macrophages. Poloxamine coating of the NPs delayed the tumor localization of the NPs.

Topics: Animals; Carcinoma, Hepatocellular; Cell Survival; Contrast Media; Ethylenediamines; Excipients; Gadolinium DTPA; Genes, myc; Humans; Liver; Liver Neoplasms; Macrophages, Peritoneal; Magnetic Resonance Imaging; Mice; Mice, Transgenic; Nanoparticles; Particle Size; Polyethylene Glycols; Rhodamine 123; Serum Albumin; Tissue Distribution; Transforming Growth Factor alpha

The multi-kinase inhibitor sorafenib is now used as standard therapy for advanced hepatocellular carcinoma (HCC). Predictive biomarkers of response to sorafenib are thus necessary. The purpose of this study was to assess the feasibility of using targeted DNA and RNA sequencing to elucidate candidate biomarkers of sorafenib response using fine-needle biopsy, formalin-fixed paraffin-embedded (FFPE) specimens in patients with HCC. Targeted DNA and RNA deep sequencing were feasible for the evaluation of fine-needle biopsy FFPE specimens obtained from 46 patients with HCC treated with sorafenib. Frequent mutations of suppressor genes, such as CTNNB1 (34.8%) and TP53 (26.1%), were detected in the HCC tumors. After excluding these suppressor genes, the average numbers of detected oncogene mutations differed significantly between the non-PD and PD groups (P = 0.0446). This result suggests that the oncogene mutational burden in the tumor might be associated with the clinical response to sorafenib. We have identified candidate gene expression (TGFa, PECAM1, and NRG1) in tumor for the prediction of sorafenib response and PFS by RNA sequencing. Our findings provide new insights into biomarkers for sorafenib therapy and allow us to discuss future therapeutic strategies.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; beta Catenin; Biomarkers; Biomarkers, Tumor; Biopsy, Fine-Needle; Carcinoma, Hepatocellular; Female; Formaldehyde; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Liver Neoplasms; Male; Middle Aged; Mutation; Neuregulin-1; Niacinamide; Paraffin Embedding; Phenylurea Compounds; Platelet Endothelial Cell Adhesion Molecule-1; Sequence Analysis, DNA; Sequence Analysis, RNA; Sorafenib; Transforming Growth Factor alpha; Treatment Outcome; Tumor Suppressor Protein p53

The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

Topics: Animals; Carcinoma, Hepatocellular; DNA Primers; Dog Diseases; Dogs; Electrophoresis, Agar Gel; Epidermal Growth Factor; ErbB Receptors; Focal Nodular Hyperplasia; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Proto-Oncogene Proteins c-sis; Real-Time Polymerase Chain Reaction; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha

Dietary isoprenic derivatives such as β-ionone (βI) are a promising class of chemopreventive agents. In this study, cellular aspects of βI protective activities during early hepatocarcinogenesis were evaluated. Male Wistar rats were submitted to "resistant hepatocyte" model and then received daily 16 mg/100 g body weight (b.w.) of βI (βI group) or only 0.25 mL/100 g b.w. of corn oil (vehicle, control group [CO]) during 4 wk, specifically during early promotion phase. Compared to controls, βI inhibited (P < 0.05) the development of persistent preneoplastic lesions (pPNL), considered to be potential hepatocellular carcinoma (HCC) progression sites, and increased remodeling PNL (rPNL) (P < 0.05) that tend to regress to a normal phenotype. Increased βI hepatic levels (P < 0.05), in the βI group, were associated with its chemopreventive actions. Compared to control rats, βI reduced the frequency of both pPNL and rPNL positive for tumor growth factor (TGF)-α (P < 0.05), reduced the frequency of pPNL stained for p65 (nuclear factor-kappaB; NF-κB) (P < 0.05), and reduced the frequency of pPNL positive for cytoplasmic p53 (P < 0.05). Our data demonstrated that βI targets TGF-α, NF-κB, and p53 in initial phases of hepatocarcinogenesis and specifically inhibits PNL with increased probability to progress to HCC. This isoprenoid may represent a chemopreventive agent of choice for HCC control.

Topics: Animals; Anticarcinogenic Agents; Carcinoma, Hepatocellular; Chemoprevention; Liver; Liver Neoplasms, Experimental; Male; NF-kappa B; Norisoprenoids; Rats; Rats, Wistar; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

This case-control study aimed to evaluate the diagnostic application of urinary transforming growth factor (TGF) α and serum α-fetoprotein (AFP) in hepatocellular carcinoma (HCC). TGFα and AFP were determined in 90 pairs of age- and gender-matched patients with cirrhotic HCC and patients with cirrhosis alone and 60 healthy controls. The results indicated that TGFα and AFP levels in patients with HCC were higher than in those with cirrhosis alone or healthy controls (each P = 0.0001). Multivariate analysis indicated that TGFα (odds ratio (OR) 1.03, 95% confidence interval (CI) 1.05-1.16) and AFP (OR 1.03, 95% CI 1.01-1.06) were closely associated, in a dose-related fashion, with the development of HCC. The optimal cutoff values, determined with the receiver operating characteristic (ROC) curves, were 29 μg/g creatinine for TGFα and 100 ng/ml for AFP, respectively. The areas under ROC curve (AUC) were 0.74 for TGFα and 0.78 for AFP, respectively. Both biomarkers showed the same sensitivity (52.2%), high specificity, high positive predictive value, and moderate positive likelihood ratio. Determination of both markers in parallel significantly increased the AUC (0.91) and diagnostic accuracy (92.2%), with a high sensitivity (86.7 %), specificity (97.8%), positive predictive value (PPV; 97.5%), and moderate positive likelihood ratio (PLR; 39.4). Among 31 cirrhotic HCC with AFP ≤ 20 ng/ml, the calculated AUC for TGFα was 0.79, with a sensitivity of 64.5%, specificity of 96.7%, PPV of 87.0%, and PLR of 19.5. In conclusion, urinary TGFα and serum AFP are complementary tumor markers for detection of HCC with low AFP production.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Male; Middle Aged; ROC Curve; Transforming Growth Factor alpha

Sorafenib, the first-line systemic drug for advanced hepatocellular carcinoma (HCC), has demonstrated limited benefits with very low response rates. Thus it is essential to investigate the underlying mechanisms for the resistance to sorafenib and seek potential strategy to enhance its efficacy. Hypoxic cells inside solid tumors are extremely resistant to therapies as their survival ability is increased due to the cellular adaptive response to hypoxia, which is controlled by hypoxia-inducible factor (HIF)-1 and HIF-2. Sorafenib inhibits HIF-1α synthesis, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways and providing a mechanism for more aggressive growth of tumors. The present study has demonstrated that upregulation of HIF-2α induced by sorafenib contributes to the resistance of hypoxic HCC cells by activating the transforming growth factor (TGF)-α/epidermal growth factor receptor (EGFR) pathway. Blocking the TGF-α/EGFR pathway by gefitinib, a specific EGFR inhibitor, reduced the activation of STAT (signal transducer and activator of transcription) 3, AKT and ERK (extracellular signal-regulated kinase), and synergized with sorafenib to inhibit proliferation and induce apoptosis of hypoxic HCC cells. Transfection of HIF-2α siRNA into HCC cells downregulated the expression of VEGF (vascular endothelial growth factor), cyclin D1, HIF-2α and TGF-α, and inhibited the activation of EGFR. HIF-2α siRNA inhibited the proliferation and promoted the apoptosis of HCC cells in vitro, and synergized with sorafenib to suppress the growth of HCC tumors in vivo. The results indicate that targeting HIF-2α-mediated activation of the TGF-α/EGFR pathway warrants further investigation as a potential strategy to enhance the efficacy of sorafenib for treating HCC.

Topics: Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Niacinamide; Phenylurea Compounds; Quinazolines; Signal Transduction; Sorafenib; Transforming Growth Factor alpha; Up-Regulation

Alterations in the Wnt signaling pathway are associated with diverse cancers, including hepatocellular carcinoma (HCC). The development of HCC is thought to be a multistage process in which multiple genetic alterations are necessary. Few studies have assessed the effect of aberrant Wnt signaling activity in association with other molecular alterations in HCC. Here we sought to determine whether co-overexpression of c-Myc or TGFα, 2 signaling molecules known to contribute to HCC development, enhanced the development of hepatic lesions associated with a stabilized β-catenin. The coexpression of mutant β-catenin with either c-Myc or TGFα within hepatocytes increased the severity of hepatic lesions compared with that associated with any of the transgenes expressed individually. The coexpression of mutant β-catenin with c-Myc or TGFα resulted in severe hepatomegaly necessitating the euthanasia of mice by an average of 156 and 128 d, respectively, after the cessation of doxycycline. The expression of mutant β-catenin alone resulted in mild to moderate hepatomegaly that prompted the euthanasia of mice by an average of 75 d after the cessation of doxycycline. Collectively, these findings indicate that coexpression of c-Myc or TGFα delays the onset of endstage hepatic disease yet enhances the severity of hepatic lesions due to mutant β-catenin.

Topics: Analysis of Variance; Animals; beta Catenin; Breeding; Carcinoma, Hepatocellular; Hepatomegaly; Immunohistochemistry; Kaplan-Meier Estimate; Liver; Liver Neoplasms; Mice; Mice, Transgenic; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha; Wnt Signaling Pathway

Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling and carcinogenesis. Overexpression of COX-2 in hepatocytes constitutes an ideal condition to evaluate the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in genetic hepatocarcinogenesis has been investigated in triple c-myc/transforming growth factor α (TGF-α) transgenic mice that express human COX-2 in hepatocytes on a B6CBAxCD1xB6DBA2 background. Analysis of the contribution of COX-2-dependent PGs to the development of hepatocarcinogenesis, evaluated in this model, suggested a minor role of COX-2-dependent prostaglandins to liver oncogenesis as indicated by liver histopathology, morphometric analysis and specific markers of tumor progression. This allows concluding that COX-2 is insufficient for modifying the hepatocarcinogenesis course mediated by c-myc/TGF-α.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cyclooxygenase 2; Disease Progression; Female; Gene Expression; Humans; Liver; Liver Neoplasms; Male; Mice; Mice, Transgenic; Proto-Oncogene Proteins c-myc; Signal Transduction; Transforming Growth Factor alpha

The hepatitis B virus (HBV) X gene, which encodes the hepatitis B virus x protein (HBx), is essential for viral infection and genome replication, virus-associated liver disease, and development of hepatocellular carcinoma. However, the exact role(s) of HBx remain controversial. In this study, we focus on studying the role of HBx in the regulation of cell cycle and apoptosis in normal liver and hepatoma cell lines. We established the Huh7-X and Chang-X cell lines that constitutively express HBx. There were differences between the two cell lines in terms of cell cycle regulation and expression of p27 and transforming growth factor-β. Expression of HBx proteins dramatically increases expression of Bcl-2 and reduces levels of cleaved PARP protein in Chang-X cells, and it inhibits apoptosis under unfavourable conditions, such as serum starvation, in both cell lines. Our findings provide clues about the intracellular roles of HBx and demonstrate that expression of this protein is important for multiple cellular processes, that is, cell cycle progression and apoptosis, in hepatoma cells and normal liver cell lines.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; DNA Replication; Gene Expression Regulation, Viral; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; Liver; Liver Neoplasms; Models, Biological; Protein Transport; Signal Transduction; Trans-Activators; Transforming Growth Factor alpha; Viral Regulatory and Accessory Proteins

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors. It is important to detect disease and recurrence at its earlier period. We aimed to evaluate the usefulness of TGF-alpha and VEGF in diagnosis of HCC patients. Thirty patients with liver cirrhosis, 30 patients with confirmed HCC and 20 healthy volunteers were subjected to abdominal ultrasonography, and alpha-fetoprotein, TGF-alpha and VEGF were assessed. Serum level of AFP was significantly higher in HCC than cirrhotic patients and controls and in cirrhosis patients than controls. The level of TGF-alpha was significantly increased in HCC and cirrhosis groups than in control group with no difference between cirrhosis and HCC groups. Serum VEGF was higher in HCC than in cirrhosis group and in both groups than in control group. Sensitivity and specificity of makers in diagnosis of HCC were 63%, 90% respectively for AFP using a cutoff value of 19.96 ng/ml; 60% and 92% for VEGF at cut off 268 and 73% and 84 % for TGF-alpha using a cutoff value of 13.95 pg/ml. VEGF may be useful serum marker for detection of HCC in addition to traditional markers.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A

Genetically engineered mouse models, such as double transgenic c-myc/TGFα mice, with specific pathway abnormalities might be more successful at predicting the clinical response of hepatocellular carcinoma (HCC) treatment. But a major drawback of the tumour models is the difficulty of visualizing endogenously formed tumours. The optimal imaging procedure should be brief and minimally invasive. Magnetic resonance imaging (MRI) satisfies these criteria and gadoxetate acid-enhanced MRI improves the detection of HCC. Fat content is stated to be an additional tool to help assess tumour responses, for example, in cases of radiofrequency ablation. Therefore the aim of this study was to investigate if gadoxetate acid-enhanced MRI could be used to detect HCC in c-myc/TGFα transgenic mice by determining the relation between the signal intensity of HCC and normal liver parenchyma and the corresponding fat content as a diagnostic marker of HCC. In our study, 20 HCC in c-myc/TGFα transgenic male mice aged 20-34 weeks were analyzed. On gadoxetate acid-enhanced MRI, the signal intensity was 752.4 for liver parenchyma and 924.5 for HCC. The contrast to noise ratio was 20.4, the percentage enhancement was 267.1% for normal liver parenchyma and 353.9% for HCC. The fat content was 11.2% for liver parenchyma and 16.2% for HCC. There was a correlation between fat content and signal intensity with r = 0.7791. All parameters were statistically significant with P < 0.05. Our data indicate that gadoxetate acid contrast enhancement allows sensitive detection of HCC in c-myc/TGFα transgenic mice and determination of the fat content seems to be an additional useful parameter for HCC.

Topics: Animals; Carcinoma, Hepatocellular; Contrast Media; Fats; Gadolinium DTPA; Image Enhancement; Liver; Liver Neoplasms; Magnetic Resonance Imaging; Male; Mice; Mice, Transgenic; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. In Egypt, the disease is usually detected in an advanced stage at which no treatment may be effective including surgery. Early detection of the disease is thus an important goal allowing the patient to be treated before the enlargement of the tumor or its metastasis to distant organs. Tumor markers are serological agents which serum level may be useful in predicting the presence of the tumor at early stages. Alpha fetoprotein (AFP) which is the golden marker for HCC is of low sensitivity, therefore, additional markers such as alpha-L-fucosidase (AFU), transforming growth factors alpha and beta (TGF-α and TGF-β) and interleukin-8 (IL-8) are suggested to be simultaneously evaluated in order to enhance the detection of HCC. A total of 96 patients with different liver diseases such as HCC, hepatitis C virus (HCV), hepatitis B virus (HBV) and cirrhotic patients are included in this study. Sixteen healthy volunteers are used as a control group. In patients with HCC each of AFP, AFU, TGF-α and TGF-β recorded significantly higher levels than the other patient groups and controls. HCC patients recorded significantly lower level of IL-8 compared to the other patient groups but significantly higher than the control. For AFP, AFU, TGF-α, TGF-β and IL-8, at the optimal cut-off values (obtained from the receiver operating characteristic (ROC) curves), the calculated sensitivities are 46%, 72.97%, 67.56%, 54.05% and 83.8%, respectively. The simultaneous evaluation using all of the suggested markers resulted in increasing the sensitivity up to 100%. It thus recommended that, if patients with cirrhosis, as high risk patients, are subjected to regular examination using these markers in addition to AFP, HCC may be detected by 100% sensitivity in an early stage and as a consequence an effective treatment can be achieved.

Topics: Adult; Aged; alpha-Fetoproteins; alpha-L-Fucosidase; Biomarkers, Tumor; Carcinoma, Hepatocellular; Case-Control Studies; Early Detection of Cancer; Egypt; Female; Fibrosis; Hepatitis, Viral, Human; Humans; Interleukin-8; Liver Neoplasms; Male; Middle Aged; ROC Curve; Transforming Growth Factor alpha; Transforming Growth Factor beta

A comprehensive understanding of molecular mechanisms driving cancer onset and progression should provide a basis for improving early diagnosis, biomarker discovery and treatment options. A key value of genetically engineered mice for modeling human cancer is the possibility to analyze the entire process of tumor development. Here, we applied functional genomics approach to study step-by-step development of hepatocellular carcinoma (HCC) in the c-Myc/Tgfα transgenic mouse model of aggressive human liver cancer. We report that coexpression of c-Myc and Tgfα induces progressive and cumulative transcriptional alterations in the course of liver oncogenesis. Functional analysis of deregulated genes at the early stage of HCC disease supports a model of active hepatocyte proliferation on the background of chronic oxidative stress generated by a general metabolic disorder. In addition, early and persistent deregulation of numerous immune-related genes suggested that disruption of immune microenvironment may contribute to oncogenic process in this model of accelerated liver carcinogenesis. In particularly, by flow cytometry analysis, we found loss of the major histocompatibility complex class I expression in dysplastic hepatocytes followed by upregulation of numerous activating ligands for natural killer (NK) cells concomitant with a drastic decrease in hepatic NK cell frequency. In conclusion, our study provides a comprehensive characterization of sequential molecular changes during a stepwise progression of preneoplastic lesions toward HCC and highlights a critical role of metabolic disorders and innate immunity at the early stages of liver cancer.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Disease Models, Animal; Disease Progression; Flow Cytometry; Gene Expression Profiling; Genomics; Hepatocytes; Humans; Killer Cells, Natural; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor alpha

Our previous animal study had demonstrated that partial liver irradiation (IR) could stimulate regeneration in the protected liver, which supported the measurements adopted in radiotherapy planning for hepatocellular carcinoma. The purpose of this present study is to investigate whether cirrhotic liver repopulation could be triggered by partial liver IR. The cirrhosis was induced by thioacetamide (TAA) in rats. After cirrhosis establishment, TAA was withdrawn. In Experiment 1, only right-half liver was irradiated with single doses of 5 Gy, 10 Gy and 15 Gy, respectively. In Experiment 2, right-half liver was irradiated to 15 Gy, and the left-half to 2.5 Gy, 5 Gy and 7.5 Gy, respectively. The regeneration endpoints, including liver index (LI); mitotic index (MI); liver proliferation index (LPI); PCNA-labeling index (PCNA-LI); serum HGF, VEGF, TGF-α and IL-6, were evaluated on 0 day, 30-day, 60-day, 90-day, 120-day and 150-day after IR. Serum and in situ TGF-β1 were also measured. In both experimental groups, the IR injuries were sublethal, inducing no more than 9% animal deaths. Upon TAA withdrawal, hepatic regeneration decelerated in the controls. In Experiment 1 except for LI, all other regeneration parameters were significantly higher than those in controls for both right-half and left-half livers. In Experiment 2 all regeneration parameters were also higher compared with those in controls for both half livers. Serum HGF and VEGF were increased compared with that of controls. Both unirradiated and low dose-irradiated cirrhotic liver were able to regenerate triggered by sublethal partial liver IR and higher doses and IR to both halves liver triggered a more enhanced regeneration.

Topics: Animals; Biomarkers; Carcinoma, Hepatocellular; Dose-Response Relationship, Radiation; Hepatocyte Growth Factor; Humans; Interleukin-6; Liver Cirrhosis, Experimental; Liver Neoplasms; Liver Regeneration; Male; Rats; Rats, Wistar; Transforming Growth Factor alpha; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Liver carcinogenesis is associated with multiple genetic changes in affected cells, including alterations in the Hras signalling pathway.. To define the biological contributions of Hras to mouse hepatocarcinogenesis, we quantified in vivo interactions between mutant Hras and other genetic alterations frequently associated with liver cancer, including overexpression of the transcription factor c-myc and the epidermal growth factor receptor ligand transforming growth factor alpha (TGFα).. To accomplish this aim, we initiated expression of an activated Hras in hepatocytes of adult mice with or without simultaneous overexpression of either c-myc or TGFα. Potential interactions also were assessed through the use of the comparative hepatocyte growth assay, a hepatocyte transplantation assay that measures effects of altered gene expression on hepatocyte growth in vivo.. Hras expression caused diffuse liver enlargement (hepatomegaly), and this phenotype was not changed by coexpression of c-myc or TGFα. Using the transplant system, we found that expression of mutant Hras alone was sufficient to induce hepatocyte focus growth in a quiescent liver. Paradoxically, adding expression of TGFα or c-myc reversed this Hras effect. Finally, the frequencies of transplant foci with the preneoplastic feature of extreme growth potential and of liver neoplasms were increased for Hras and both combinations when compared with control hepatocytes, but did not differ among oncogene-expressing groups.. Hras-associated hepatocyte growth deregulation is not complemented by activation of c-myc or TGFα growth signalling pathways in mouse liver. This finding emphasizes the tissue-specific character of molecular growth regulation.

Topics: Adenoma, Liver Cell; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Genotype; Hepatocytes; Hepatomegaly; Liver Neoplasms; Mice; Mice, Transgenic; Mutation; Phenotype; Proto-Oncogene Proteins c-myc; ras Proteins; Signal Transduction; Time Factors; Transforming Growth Factor alpha

Previous studies showed that several genetic polymorphisms might influence the clinical outcome of chronic hepatitis B virus (HBV) infection, including HBV clearance or development of hepatocellular carcinoma (HCC). The aim of this study was to determine whether polymorphisms of the transforming growth factor-alpha (TGF-alpha) gene are associated with clinical outcome of HBV infection. A total of 1096 Korean subjects having either present or past evidence of HBV infection were prospectively enrolled between January 2001 and August 2003. Among 16 genetic variants in TGFA gene, nine variants were genotyped using TaqMan assay and the genetic association with HBV clearance and HCC occurrence was analysed. Statistical analyses revealed that TGFA+103461T>C, TGFA+106151C>G and TGFA-ht2 were marginally associated with clearance of HBV infection. However, only TGFA-ht2 retained significance after multiple correction (OR = 0.39, P(corr) = 0.007 in recessive model). Although no variants were significant after multiple correction, TGFA+88344G>A and TGFA+103461T>C were weakly associated in recessive model in the analysis of HCC occurrence. In addition, Cox relative hazards model also revealed that TGFA+88344G>A was associated with onset age of HCC occurrence in subjects (RH = 1.46, P(corr) = 0.04). TGF-alpha polymorphisms might be an important factor in immunity, progression of inflammatory process and carcinogenesis, which explains the variable outcome of HBV infection at least in part. Further biological evidence is warranted in the future to support these suggestive associations.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Genotype; Hepatitis B virus; Hepatitis B, Chronic; Humans; Korea; Liver Neoplasms; Male; Middle Aged; Models, Statistical; Polymorphism, Genetic; Prospective Studies; Transforming Growth Factor alpha; Treatment Outcome

Hepatocellular carcinoma (HCC) results from the cumulative effects of deregulated tumor suppressor genes and oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and their downstream signaling pathway components. The overexpression of transforming growth factor alpha (TGF-alpha) and the inhibition of TGF-beta signaling are especially common in human liver cancer. Thus, we assessed whether TGF-alpha overexpression and TGF-beta signaling inactivation cooperate in hepatocarcinogenesis using an in vivo mouse model, MT1/TGFa;AlbCre/Tgfbr2(flx/flx) mice ("TGFa;Tgfbr2(hepko)"), which overexpresses TGF-alpha and lacks a TGF-beta receptor in the liver. TGF-beta signaling inactivation did not alter the frequency or number of cancers in mice with overexpression of TGF-alpha. However, the tumors in the TGFa;Tgfbr2(hepko) mice displayed increased proliferation and increased cdk2, cyclin E and cyclin A expression as well as decreased Cdkn1a/p21 expression compared to normal liver and compared to the cancers arising in the TGF-alpha overexpressing mice with intact TGF-beta receptors. Increased phosphorylated ERK1/2 expression was also present in the tumors from the TGFa;Tgfbr2(hepko) mice and correlated with downregulated Raf kinase inhibitor protein expression, which is a common molecular event in human HCC. Thus, TGF-beta signaling inactivation appears to cooperate with TGF-alpha in vivo to promote the formation of liver cancer that recapitulates molecular features of human HCC.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Integrases; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta

Mice lacking hepatocyte IKKbeta (Ikkbeta(Delta hep)) are defective in TNFalpha-activation of hepatocellular transcription factor NF-kappaB, and highly susceptible to hepatotoxicity. Following diethylnitrosamine (DEN) exposure, Ikkbeta(Delta hep) mice develop more hepatocellular carcinoma (HCC) than control mice due partly to enhanced DEN-induced hepatocyte death. Here we show that Ikkbeta(Delta hep) hepatocytes display growth advantages over normal hepatocytes consisting of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G(0)-->G(1) transitions), and enhanced recovery efficiency, cyclin D1 expression and cell proliferation after plating. Ex vivo deletion of Ikkbeta also accelerates hepatocyte growth. Ikkbeta(Delta hep) hepatocyte proliferative responses show heightened sensitivity to TGFalpha and TNFalpha, and heightened expression of fibronectin, collagens I/III, nidogen, beta-actin and integrin beta1 mRNAs. These findings suggest that altered mitogen signaling and expression of extracellular matrix and its associated components underlie growth advantages. Increased HCC development in Ikkbeta(Delta hep) mice may also be caused by growth advantages of surviving Ikkbeta-deleted hepatocytes.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; G1 Phase; Gene Deletion; Gene Targeting; Hepatocytes; I-kappa B Kinase; Liver Neoplasms; Liver Regeneration; Mice; Mice, Knockout; Proliferating Cell Nuclear Antigen; Resting Phase, Cell Cycle; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Global transcriptome analysis has been successfully applied to characterize various human tumors, including hepatocellular carcinomas. This novel technology can facilitate early diagnosis, as well as prognostic and therapeutic diversification of cancer patients. To enhance access to the genomic information buried in archived pathology samples, we assessed RT-PCR amplification rates in paraffin-embedded tissues preserved in three different fixatives. Reliable amplification could be achieved from all paraffin-embedded specimens, when the amplicon size did not exceed 225 bp. A longer amplicon size resulted in rapid decrease of yield and reproducibility. In addition, formalin provided superior morphology and better reactivity with claudin-4 and -7 immunohistochemistry. Amplification of the initial sample is often required before transcriptome analysis of clinical specimens could be performed. We introduced a random nonamer primed T3 polymerase reaction into the conventional linear RNA amplification protocol. The modified T3T7 method generated a sense strand product ideal for synthesizing indirectly labeled cDNA templates. Microarray analysis of amplified frozen and laser-microdissected Myc and Myc/TGFalpha mouse liver tumors confirmed good reproducibility (r=0.9) of the reaction and conservation of original transcriptional patterns (r=0.78). Finally, we tested the utility of expression profiling for the classification of human HCC samples. By comparing expression data from HGF-treated c-Met conditional knock-out and control primary mouse hepatocytes, we identified 690 HGF/c-Met target genes. Functional analysis of the significant gene set implicated c-Met as key regulator of hepatocyte motility and oxidative homeostasis. Cross comparison of the c-Met-induced transcription signature with human HCC expression profiles revealed a group of tumors (27%) with potentially activated c-Met signaling (MET+). These tumors were characterized by higher vascular invasion rate, increased microvessel density, and shortened survival. A prediction model based on 111 cross-species conserved c-Met signature genes was able to diversify HCC patients into good and bad prognostic groups with 83-95% accuracy. Our results therefore demonstrate that careful experimental design and state-of-the-art laboratory methods could open the way for global expression profiling of archived and limited availability pathologic samples. Comparative functional genomics based analysis of the cancer tra

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Claudin-4; Claudins; Early Detection of Cancer; Genomics; Humans; Immunohistochemistry; Liver Neoplasms; Membrane Proteins; Mice; Microarray Analysis; Microdissection; Prognosis; Proteomics; Proto-Oncogene Proteins c-met; Proto-Oncogene Proteins c-myc; Reproducibility of Results; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transforming Growth Factor alpha

Chronic ethanol intake is a significant risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). The effects of ethanol on extracellular signal-regulated kinase (ERK) activation, transforming growth factor alpha (TGF-alpha), and HCC growth were examined in this study.. HepG2, SKHep, Hep3B human HCC cells, or normal human hepatocytes were treated with ethanol (0-100 mM), exogenous TGF-alpha, TGF-alpha neutralization antibody or the MEK inhibitor U0126. TGF-alpha levels were quantified by ELISA. Growth was determined by trypan blue-excluded cell counts. Cell cycle phase distribution was determined by flow cytometry. Protein expression was determined by Western blot.. Ethanol treatment (10-40 mM) increased ERK activation in HepG2 and SKHep HCC cells but not in Hep3B or human hepatocyte cells. Growth increased in HepG2 (174 +/- 29%, P < 0.05) and SKHep (149 +/- 12%, P < 0.05) cells in response to ethanol treatment. Correspondingly, ethanol increased S phase distribution in these cells. U0126 suppressed ethanol-induced growth increases. Ethanol treatment for 24 h also raised TGF-alpha levels in HepG2 cells (118%-198%) and SKHep cells (112%-177%). Exogenous administration of recombinant TGF-alpha mimicked the ethanol-induced growth in HepG2 and SKHep cells; TGF-alpha neutralization antibody effectively abrogated this effect. The TGF-a neutralization antibody also prevented ERK activation by ethanol in HepG2 cells.. These data demonstrate that clinically relevant doses of ethanol stimulate ERK-dependent proliferation of HCC cells. Ethanol up-regulates TGF-alpha levels in HCC cells and enhances growth through cell cycles changes, which appear to be mediated through TGF-alpha-MEK-ERK signaling. Ethanol-MEK signaling in normal hepatocytes is absent, suggesting that ethanol promotion of HCC growth may in part depend upon the acquisition of cancer-specific signaling by hepatocytes.

Topics: Antibodies; Butadienes; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Central Nervous System Depressants; Enzyme Inhibitors; Ethanol; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Kinase 1; MAP Kinase Signaling System; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Transforming Growth Factor alpha

Hepatocellular carcinoma (HCC) causes 600,000 mortalities per year worldwide. Previous studies from our lab provide evidence for altered mitogen-activated protein kinase and extracellular signal-regulated kinase kinase (MEK) signaling in HCC pathogenesis. We hypothesized that pharmacologic targeting of MEK may prevent HCC. Transforming growth factor-alpha-transgenic mice (CD1-MT42) exposed to diethylnitrosamine were randomized to 20 (trial I) or 35 (trial II) weeks of MEK inhibitor PD0325901 (1, 10 mg/kg) or control via orogastric gavage. Ten HCC (44%) formed in trial I controls versus 0 in treatment arms (p<0.05). Fourteen HCC (50%) formed in trial II controls versus 1 (9%) in treatment arms (p<0.05). Mean HCC volume was 578 mm3 in control versus 46 mm3 in the single tumor formed in trial II. In trial I, foci of altered hepatocytes (FAH) formed in 78% of control versus 40% and 0% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). In trial II, incidence of FAH was 80% in control versus 20% and 50% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). Hepatocyte expression of phosphorylated extracellular signal-regulated kinase dose-dependently decreased in trial I but remained the same in trial II. Control and treated HCC demonstrated similar proliferation rates, but apoptosis appeared increased with treatment. MEK targeting is effective HCC chemoprevention, perhaps by lowering the apoptotic threshold.

Topics: Alkylating Agents; Animals; Apoptosis; Benzamides; Carcinoma, Hepatocellular; Cell Proliferation; Chemoprevention; Diethylnitrosamine; Diphenylamine; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Follow-Up Studies; Hepatocytes; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms, Experimental; Mice; Mice, Transgenic; Transforming Growth Factor alpha

The purpose of this study is to investigate if the EGFR-Stat3 signal pathway contributes to the carcinogenesis of hepatoma in rats. Hepatoma was induced in rats by 3'Me-DAB as a model. EGFR, TGFalpha, Stat3, p-Stat3 in different stages of carcinogenesis were detected by immunohistochemistry and Western blot. In situ hybridization was applied to investigate the expression of Stat3 mRNA. The expressions of signal molecules were assessed by KS400 Image Analysis system. The data were statistically evaluated. EGFR, TGFalpha, Stat3 were highly expressed in the stages of liver necrosis and repairment. All hepatocellular carcinoma cases revealed elevated expression of EGFR, TGFalpha. Elevation of Stat3 mRNA and protein levels were identified, increase of activation of Stat3 was also observed. In HCC, there was positive correlation between p-Stat3 level and the expression of TGFalpha and PCNA. Increased expression of Bcl-2 (P < 0.05) coincided with elevated level of p-Stat3. Therefore, the EGFR-Stat3 signal pathway was related to the development of hepatoma in rats. TGFalpha-EGFR autocrine ring formation may lead to the activation of Stat3 and in turn, promote proliferation and regulate the transcription of genes regulating cell apoptosis and cell cycle.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Transformation, Neoplastic; Disease Models, Animal; ErbB Receptors; Immunohistochemistry; In Situ Hybridization; Liver Cirrhosis; Liver Neoplasms, Experimental; Male; Models, Biological; p-Dimethylaminoazobenzene; Phosphorylation; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor alpha

ErbB receptor proteins are transmembrane tyrosine kinase receptors; when they are activated by interaction with ligands, they generate diverse cellular responses, especially during lesion development and progression to cancer. In this study the expression of ErbB receptors and TGF-alpha were investigated using an experimental cirrhosis rat model giving rise to hepatocellular neoplasms, similar to human liver diseases.. Fifty three male rats received intraperitoneal injection of diethylnitrosamine (DEN, 50 mg/kg), weekly for 18 weeks. Until the eighth week, two rats were sacrificed every two weeks and from the tenth to the eighteenth week, five rats were sacrificed weekly. Grossly, dyschromatic and dysmorphic nodules were counted and categorized into three groups: N1/N2/N3: 3 mm < or = x < 5 mm/5 mm < or = x < 10 mm/x > or = 10 mm in diameter. All nodules were examined, histologically. Antibodies for GSTp, TGF-alpha, EGF-R, ErbB2, ErbB3 and ErbB4 were used for immunohistochemistry.. The onset of cirrhoses was noted from the twelfth week. Preneoplastic foci, hepatocellular adenomas (HCA) and hepatocellular carcinomas (HCC) were noted from the second, eleventh and fifteenth week, respectively. The nodules (N1/N2/N3: 397/258/64) included regenerating nodule; RN (N1/N2/N3: 72.3%/15.9%/0%), HCA (N1/N2/N3: 27.2%/82.2%/7.6%) and HCC (N1/N2/N3: 0.5%/ 1.9%/92.4%). EGF-R was expressed in 12.5% of RN, 64.7% HCA and 75.2% HCC. TGF-alpha was expressed in 92.4% of RN, 91.3% HCA and 93.2% HCC. Sixty eight percent of TGF-alpha expressing nodules showed concurrent EGF-R expression. ErbB2 was expressed in 83.6% of RN, 72.9% HCA and 88.7% HCC. ErbB4 was expressed in 95.2% of RN, 86.3% HCA and 62.5% HCC.. Increased expression of EGF-R and decreased expression of ErbB4, might be related with tumor progression during DEN-induced hepatocarcinogenesis.

Topics: Adenoma, Liver Cell; Animals; Carcinoma, Hepatocellular; Diethylnitrosamine; ErbB Receptors; Glutathione Transferase; Immunohistochemistry; Liver Neoplasms, Experimental; Male; Rats; Rats, Wistar; Receptor, ErbB-2; Receptor, ErbB-4; Transforming Growth Factor alpha

Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c-Myc and c-Myc/Tgf-alpha transgenic mice and human HCCs. S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and liver-specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c-Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patient's survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c-Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c-Myc/Tgf-alpha lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c-Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c-Myc/Tgf-alpha HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c-Myc/Tgf-alpha tumors, may develop in the absence of alterations in DNA methylation.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; DNA Methylation; DNA, Neoplasm; Genes, myc; Genomic Instability; Humans; Immunoblotting; Liver; Liver Neoplasms; Methionine; Mice; Mice, Transgenic; Prognosis; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Transforming Growth Factor alpha

Intrahepatic transplantation of ovarian fragments in ovariectomized rats results in morphological abnormalities. The liver acini draining blood from ovarian grafts show alterations resembling chemically induced amphophilic hepatocellular preneoplasias. We investigated the long-term development of these estrogen-induced foci of altered hepatocytes (FAH). We divided 451 Lewis rats into one main group (MG) and 11 (7 female, 4 male) control groups and observed them for up to 30 months. MG animals were ovariectomized and received ovarian transplants into the right liver part. Different combinations of castration, transplantation of ovarian or testicular fragments, and administration of antiestrogenic toremifene were used in controls. In the MG, transplants showed signs of gonadotropic stimulation, and estrogen levels were strongly increased in the downstream liver acini. After 6 and 12 months, FAH developed in hepatocytes downstream of the transplants. After 18 months, 27% of the MG animals showed transformation of FAH into hepatocellular adenomas; this figure increased to 42% after 24 months (8/19), significantly outnumbering four spontaneous adenomas that developed between 18 and 30 months in 258 control animals. Hepatocellular carcinoma (HCC) appeared only in the MG. At 24 and 30 months, 18 HCCs developed; thus, 78% of MG animals showed at least one carcinoma. Administration of toremifene in ovariectomized and transplanted animals completely prevented hepatocarcinogenesis. Testicular grafts showed no influence on liver tissue. In conclusion, initially adaptive but preneoplastic alterations in hepatocytes downstream of intrahepatically transplanted ovarian fragments may transform into HCC, indicating a strong hepatocarcinogenic potential of high local levels of endogenous estrogens in the rat liver.

Topics: Adenoma, Liver Cell; Animals; Aorta; Carcinoma, Hepatocellular; Estradiol; Estrogen Antagonists; Estrogens; Female; Gonadal Steroid Hormones; Gonadotropins; Hepatic Veins; Hepatocytes; Immunohistochemistry; Liver; Liver Neoplasms; Male; Ovariectomy; Ovary; Precancerous Conditions; Rats; Rats, Inbred Lew; Testis; Toremifene; Transforming Growth Factor alpha; Transplantation, Heterotopic

Hepatitis C virus (HCV) infection is a major cause of human hepatocellular carcinoma (HCC). The precise mechanism of hepatocarcinogenesis in humans by HCV is currently unclear. It was recently shown, however, that transgenic mice with the HCV core gene often develop HCC, suggesting tumorigenic activity of the HCV core protein. Further, the HCV core protein expressed in HepG2 cells transfected with the core gene was shown to stimulate proliferation of transfectants through activation of nuclear factor-kappaB (NF-kappaB). The downstream target molecule(s) of NF-kappaB activated by the HCV core protein to evoke cell proliferation is not yet identified. Transforming growth factor (TGF) alpha, which is often overexpressed in various tumour tissues such as HCC, has been shown to stimulate hepatocyte proliferation through activation of the mitogen-activated protein kinase or extracellular signal-related protein kinase (MAPK/ERK) cascade.. To explore the possibility that TGFalpha might be a target molecule for NF-kappaB activated by the HCV core, and that TGFalpha participates in the growth promotion of the core transfectants in an autocrine manner, activating the MAPK/ERK pathway.. A HCV core expression vector was transfected into human hepatoma Huh-7, HepG2 and Hep3B cells. NF-kappaB activity was examined by an electrophoretic mobility shift assay. TGFalpha transcription was assessed by a luciferase reporter assay. TGFalpha protein was determined by immunoblot and ELISA. MAPK/ERK activity was examined by an in vitro kinase assay. Cell proliferation was assessed by a water-soluble tetrazolium salt-1 assay.. In the HCV core transfectants, NF-kappaB bound to the kappaB site in the TGFalpha proximal promoter region, resulting in an increase in TGFalpha transcription. Immunoblot as well as ELISA showed increased TGFalpha expression in the HCV core transfectants. SN50, a specific inhibitory peptide for NF-kappaB, cancelled HCV core-induced TGFalpha expression. HCV core protein increased cell proliferation as well as ERK activity of the HCV core transfectants as compared with the mock transfectants. The growth-promoting activity and activation of ERK by the HCV core protein were negated by treatment with anti-TGFalpha antibodies.. These results suggest that the HCV core protein promotes proliferation of human hepatoma cells by activation of the MAPK/ERK pathway through up regulation of TGFalpha transcription via activation of NF-kappaB. Our finding provides a new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Enzyme Activation; Hepacivirus; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinases; Neoplasm Proteins; NF-kappa B; Peptides; Transcription, Genetic; Transfection; Transforming Growth Factor alpha; Viral Core Proteins

Hepatocellular carcinoma (HCC) is becoming one of the common malignant tumors worldwide and is characterized by high vascularity. Angiogenesis (formation of new microvessels) is critical for growth and progression of various human solid tumors. Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family, and its signal action is mediated through EGF receptors (EGFR). In this study, to understand the role of BTC in relation to EGFR in HCC, we examined localization, expression, and involvement in angiogenesis of BTC and EGFR. The results revealed that expression of BTC, EGFR, and tumor growth factor-alpha messenger RNA in HCC was increased by 80%, 60%, and 40%, respectively, as compared with those in the nontumorous tissues. Increased expression of BTC protein was observed in 31 (61%) of 51 HCC specimens, and the level of tumor growth factor-alpha protein was higher in 17 (33%) of 51 HCC specimens than in nonmalignant hepatocytes. Betacellulin was predominantly expressed in HCC cells, whereas EGFR was observed in sinusoidal endothelial cells of HCC in 25 tumors (49%). Betacellulin was secreted in all 4 examined HCC cell lines. The HCC specimens showing positive EGFR expression in tumor endothelial cells had a significantly higher microvessel density than those without EGFR expression (P < .005). A strong correlation was found between BTC expression in cancer cells and EGFR expression in tumor endothelial cells (P < .001). These findings suggest that overexpression of BTC by HCC cells and EGFR by tumor endothelial cells enhance vascularity in a paracrine manner.

Topics: Adult; Aged; Betacellulin; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; ErbB Receptors; Female; Fluorescent Antibody Technique, Direct; Fluorescent Antibody Technique, Indirect; Gene Expression; Hepatocytes; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; RNA, Messenger; Transforming Growth Factor alpha

Epidermal growth factor receptor (EGFR) binds transforming growth factor alpha (TGF-alpha) which is mitogenic for hepatocytes. Diverse lines of evidence suggest that activation of the TGF-alpha /EGFR pathway contributes to hepatocellular carcinoma (HCC) formation. Herein, we developed an experimental model of cirrhosis giving rise to HCC and tested the antitumoral effect of gefitinib, a selective EGFR tyrosine kinase inhibitor, in this model. Rats received weekly intraperitoneal injections of diethylnitrosamine (DEN) followed by a 2-week wash-out period that caused cirrhosis in 14 weeks and multifocal HCC in 18 weeks. Hepatocyte proliferation was increased in diseased tissue at 14 weeks compared with control liver and at even higher levels in HCC nodules compared with surrounding diseased tissues at 18 weeks. Increased proliferation was paralleled by upregulation of TGF-alpha messenger RNA expression. A group of DEN-treated rats received daily intraperitoneal injections of gefitinib between weeks 12 and 18. In rats treated with gefitinib, the number of HCC nodules was significantly lower than in untreated rats (18.1 +/- 2.4 vs. 3.7 +/- 0.45; P < .05), while EGFR was activated to a lesser extent in the diseased and tumoral tissues of these animals compared with untreated rats. HCC nodules from both untreated and gefitinib-treated animals displayed insulin-like growth factor 2 overexpression that contributed to tumor formation in treated animals. In conclusion, the blockade of EGFR activity by gefitinib has an antitumoral effect on the development of HCC in DEN-exposed rats, suggesting that it may provide benefit for the chemoprevention of HCC.

Topics: Alkylating Agents; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Diethylnitrosamine; ErbB Receptors; Gefitinib; Liver Cirrhosis; Liver Neoplasms; Male; Quinazolines; Rats; Rats, Wistar; Signal Transduction; Transforming Growth Factor alpha

Nuclear factor-kappaB (NF-kappaB) plays an important role during liver neoplastic development through transcriptional regulation of prosurvival genes, which then counteract the death-inducing signals elicited by the host immune response. The c-Myc proto-oncogene is frequently deregulated in liver tumors. Furthermore, enforced expression of c-Myc in the liver promotes the development of hepatocellular carcinomas, a process that is accelerated by coexpression with transforming growth factor-alpha (TGF-alpha). TGF-alpha/c-Myc-derived hepatocellular carcinomas display reduced apoptotic levels compared with those of single c-Myc transgenic hepatocellular carcinomas, suggesting that TGF-alpha provides a survival advantage to c-Myc-transformed hepatocytes. Given that TGF-alpha/c-Myc hepatocellular carcinomas display constitutive NF-kappaB activity, here, we have tested the hypothesis that enforced expression of TGF-alpha results in constitutive NF-kappaB activation and enhanced cell survival using TGF-alpha/c-Myc-derived hepatocellular carcinoma cell lines. We show that TGF-alpha induces NF-kappaB through the phosphatidylinositol 3-kinase/Akt axis in these bitransgenic hepatocellular carcinomas. Furthermore, we found that adenovirus-mediated inhibition of NF-kappaB activity impairs the ability of TGF-alpha/c-Myc-derived tumor cells to grow in an anchorage-independent fashion due to sensitization to c-Myc-induced apoptosis. Lastly, we show that NF-kappaB inhibits c-Myc-induced activation of caspase-9 and caspase-3 through up-regulation of the antiapoptotic target genes Bcl-X(L) and X-linked inhibitor of apoptosis (XIAP). Overall, these results underscore a crucial role of NF-kappaB in disabling apoptotic pathways initiated by oncogenic transformation.

Topics: Animals; Apoptosis; Blotting, Northern; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Liver Neoplasms; Mice; Mice, Transgenic; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Transcription Factor RelA; Transforming Growth Factor alpha; Up-Regulation

To evaluate the expression of transforming growth factor-alpha (TGF-alpha) and hepatitis B surface antigen (HBsAg) in human hepatocellular carcinoma (HCC) tissues and its significance.. Seventy specimens of HCC tissues were detected by immunohistochemical method. Five specimens of normal human liver tissues were used as control.. The TGF-alpha positive expression rates in HCC and its surrounding tissues were 74.3%(52/70) and 88.1%(52/59), respectively. TGF-alpha positive granules were mainly in the cytoplasm and fewer existed on the karyotheca. The TGF-alpha positive expressing rate in well differentiated HCC was significantly higher than that in moderately and poorly differentiated HCC (P<0.05). The TGF-alpha positive expression also was observed in intrahepatic bile ducts (part of those were hyperplastic ducts). The HBsAg positive expression rates in HCC and its surrounding tissues were 21.4%(15/70) and 79.7%(47/59), respectively. HBsAg positive granules were in the cytoplasm, inclusion and on the karyotheca. There was a prominent positive correlation between TGF-alpha and HBsAg expression in HCC surrounding tissues (P<0.05, gamma=0.34). TGF-alpha was usually existed with HBsAg in regenerated and/or dysplastic liver cells. In the five normal liver tissues, TGF-alpha and HBsAg were not detectable in hepatocytes and bile ducts.. Hepatitis B virus infection is closely related with hepatocarcinogenesis. The overexpression of TGF-alpha in the liver seems to be associated with the regeneration of hepatocytes injured by HBsAg. The continued expression of TGF-alpha might lead to dysplasia of liver cells and development of HCC. Furthermore, TGF-alpha might play a role in morphogenesis and regeneration of intrahepatic bile ducts.

Topics: Carcinoma, Hepatocellular; Hepatitis B Surface Antigens; Humans; Immunohistochemistry; Liver Neoplasms; Staining and Labeling; Transforming Growth Factor alpha

A change in the balance between proliferation and apoptosis in the course of hepatocellular carcinoma (HCC) development and progression has been suspected. We wanted to identify related genes whose mRNA levels could provide markers of severity and prognosis after resection. The extent of cell apoptosis, proliferation, and differentiation was measured with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling assay, and the Ki-67 index was determined in paired tumor and cirrhotic tissue samples from patients who had undergone HCC resection after diagnosis of hepatitis C-related or alcoholism-related cirrhosis. These patients included two groups with highly versus poorly differentiated tumor cells, and the latter was split into two subgroups of those with versus without early recurrence. The mRNA levels for various apoptosis-related or proliferation-related genes and those for the growth factor/receptor systems were measured by quantitative reverse transcriptase-polymerase chain reaction in paired tumor and cirrhotic liver samples from every patient, and some of the corresponding proteins were detected by immunohistochemistry. In all instances, protein expression was highly heterogeneous within groups and similar between groups. In contrast, some differences in mRNA level between tumor and cirrhotic tissues were quite informative. Low levels of hepatocyte growth factor and transforming growth factor alpha mRNAs were found concomitantly in highly differentiated tumors, whereas overexpression of mRNAs for the cognate receptors c-met and epidermal growth factor receptor were found in poorly differentiated tumors and primarily in patients with early tumor recurrence. These results argue for growth factor-dependent HCC development and provide novel and combined prognosis markers after HCC surgery.

Topics: Aged; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; ErbB Receptors; Female; Gene Expression Regulation; Hepatitis C; Hepatocyte Growth Factor; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Oncogenes; Prognosis; Proto-Oncogene Proteins c-met; Transforming Growth Factor alpha

The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; CSK Tyrosine-Protein Kinase; Endosomal Sorting Complexes Required for Transport; Endosomes; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Genes, src; HeLa Cells; Humans; Liver Neoplasms; Mice; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Proto-Oncogene Proteins c-yes; Recombinant Fusion Proteins; Signal Transduction; src-Family Kinases; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

The activin-follistatin system is a potent growth regulatory system of liver tissue homeostasis. Activin A inhibits hepatocellular DNA synthesis and induces cell death. Follistatin binds activin and sequesters it from the signaling pathway. Consistently, follistatin has been reported to act as an inducer of DNA synthesis in the liver. Using RNase protection analysis, we studied the expression of follistatin in rat and mouse liver tumors as a possible mechanism to overcome activin growth control. Approximately 40% of the tumors (nine of 24 each), most of them hepatocellular carcinomas, displayed increased levels of follistatin mRNA when compared to tumor-surrounding liver tissue. The degree of overexpression was highly variable but independent of the carcinogen treatment that animals had received. It was also independent from the histological stage of malignancy and further found in rat liver adenomas. Follistatin expression was also observed in cell lines derived from human hepatocellular carcinomas. Overexpression of follistatin may represent a unique strategy of hepatic tumors to overcome the inhibitory action of a growth factor, activin, by decreasing its local bioavailability.

Topics: Activins; Adenoma; Alternative Splicing; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Division; Diethylnitrosamine; Follistatin; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred Strains; Nafenopin; Phenobarbital; Rats; Reference Values; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

The HBx protein is known as a transactivator and potential oncogene, and TGF-alpha as a potent mitogen in hepatocellular carcinoma. By assays of serial deletion of the promoter of TGF-alpha gene and the cotransfection of HBx and AP-2 expression vectors, we observed that the HBx significantly activated the promoter activity through AP-2 sites located in the proximal region of the TGF-alpha promoter (-136 to -30). This effect was also observed in the heterologous promoter assay system containing AP-2 sites. The mutation analyses of three AP-2 sites in the promoter revealed that all three AP-2 sites contributed to the activation of the TGF-a gene in the presence of HBx. Accordingly, the mRNA level of TGF-alpha was significantly elevated in the HBx-expressing cell, HepG2-HBx and the HBV-producing cell, HepG2-K8. These results suggest that the HBx protein could increase the mitogenic effect of TGF-alpha by the transactivation of the gene through AP-2 binding sites and consequently, these interactions may accelerate the process of hepatocarcinogenesis.

Topics: Carcinoma, Hepatocellular; DNA-Binding Proteins; Humans; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Response Elements; RNA, Messenger; Sequence Deletion; Trans-Activators; Transcription Factor AP-2; Transcription Factors; Transcriptional Activation; Transforming Growth Factor alpha; Viral Regulatory and Accessory Proteins

A 45-year-old woman with chronic hepatitis B underwent partial hepatectomy for hepatocellular carcinoma (HCC). However, the HCC recurred 2 months after surgery and rapid progression of the disease resulted in her death. Immunohistochemistry showed that transforming growth factor-alpha (TGFalpha) was barely expressed in the liver specimens obtained at hepatic resection, whereas autopsy specimens were strongly stained with anti-TGFalpha antibody in the cytoplasm of both non-tumourous and tumourous liver cells. A higher level of Ki67 expression, a proliferating marker, was observed in the recurrent HCC, similar to that of TGFalpha. Thus, we speculate that the partial hepatectomy increased the level of TGFalpha leading to recurrence and progression of HCC through an autocrine/paracrine mechanism.

Topics: Carcinoma, Hepatocellular; Fatal Outcome; Female; Hepatectomy; Hepatitis B, Chronic; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Middle Aged; Neoplasm Recurrence, Local; Transforming Growth Factor alpha

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Topics: Animals; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-met; Quinazolines; Rats; Receptor Cross-Talk; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.

Topics: Acyl-CoA Oxidase; Animals; Apoptosis; Carcinoma, Hepatocellular; Carnitine O-Acetyltransferase; Cell Division; Cell Nucleus; DNA; DNA Replication; Hepatoblastoma; Liver; Liver Neoplasms; Oxidoreductases; Peroxisome Proliferators; Peroxisomes; Rats; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Sex difference has been shown to play a major role in susceptibility to the development of hepatocellular carcinoma in humans and rodents. In order to clarify the necessity of androgens in hepatic tumorigenesis in transgenic mice overexpressing transforming growth factor (TGF) alpha (MT42), androgen supplement after castration and the LH-RH analogue, leuprolerin acetate, were tested in an experimental model in MT42.. Male MT42 mice were castrated and supplemented with dihydrotestosterone (DHT) every three months up to 15 months and hepatic tumorigenesis was observed. Leuprolerin acetate was administered to both male and female MT42 mice once a month from 2 months after birth to 15 months to observe the effect on hepatic tumorigenesis. Northern hybridization was performed to detect messenger RNA (mRNA) of TGFalpha expression and the rate of proliferative cell nuclear antigen (PCNA) staining compared with the castrated and non-treated mice.. Castration tended to decrease both body and liver weight in MT42 mice which was then restored by DHT. Untreated MT42 males developed 11 liver tumors in 6 mice. Hormonal treatment including castration and DHT supplementation did not change the expression of TGFalpha-mRNA. Castrated transgenic mice developed 2 liver tumors in 2 out of 6 mice and DHT supplementation after castration restored the number of liver tumors to 9 in 5 of 6 mice. PCNA labelling indexes of liver tumors and adjacent non-tumorous-liver were 7.1% (p<0.05): 0.6% in untreated MT42, 3.2%: 0.2% in castrated MT42 and 10.1% (p<0.05): 0.5% in MT42 with castration and DHT supplementation (significant difference compared with castrated mice). Leuprolerin acetate-treated MT42 males developed one liver tumor in 6 mice compared to MT42 administered with saline as a vehicle control in which group 7 liver tumors in 6 male MT42 were observed. Tumors in castrated-MT42 and leuprolerin treated-MT42 were smaller than those in control MT42 mice.. TGFalpha related hepatocarcinogenesis and hepatocyte proliferation are increased by androgenic stimulation. Suppression of androgens may be useful for the treatment of TGFalpha related liver tumors.

Topics: Animals; Blotting, Northern; Body Weight; Carcinoma, Hepatocellular; Cell Division; Dihydrotestosterone; Disease Models, Animal; Female; Gene Expression; Hormone Replacement Therapy; Leuprolide; Liver; Liver Neoplasms; Male; Mice; Mice, Transgenic; Orchiectomy; Organ Size; Proliferating Cell Nuclear Antigen; RNA, Messenger; Sex Characteristics; Testosterone; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGFalpha) is an important autocrine growth factor of hepatocytes. The authors evaluated the roles of TGFalpha in chronic viral hepatitis (CVH) and hepatocellular carcinoma (HCC).. The authors measured the amounts of TGFalpha mRNA in liver tissues from 18 patients with HCC, 31 patients with CVH, and 7 normal controls. " Hot-start" reverse transcription-polymerase chain reaction (RT-PCR) using oligo-dT and specific primers detected TGFalpha mRNA in total cellular RNA extracted from liver tissues. The levels of TGFalpha mRNA were determined by the end point titers of serial, two-fold dilutions of cDNA. The amounts of hepatitis B virus RNA (HBV-RNA) in livers of patients with chronic hepatitis B also were measured by Northern blot hybridization.. TGFalpha mRNA levels were extremely higher in patients with HCC compared with patients with CVH and normal controls, and the levels in patients with CVH also were elevated compared with normal controls. The levels of TGFalpha mRNA were overexpressed in the underlying livers of patients with HCC compared with patients with CVH, although they were lower than those found in HCC tissues. The levels of TGFalpha mRNA were higher in samples from patients with chronic hepatitis B than in samples from patients with chronic hepatitis C. The levels of TGFalpha mRNA were not correlated with serum alanine aminotransferase or HBV-RNA levels in liver tissues in patients with chronic hepatitis B. However, the expression of TGFalpha mRNA tended to be higher in the livers of patients with raised serum alpha-fetoprotein levels.. The overexpression of TGFalpha mRNA in the liver seems to be associated with the regeneration of hepatocytes rather than hepatic necrosis or viral replication. Also, it may be related closely to the development or progression of HCC, especially in the livers of patients with chronic hepatitis B.

Topics: Adult; Carcinoma, Hepatocellular; Chronic Disease; Disease Progression; Female; Hepatitis, Viral, Human; Humans; Liver; Liver Neoplasms; Male; Middle Aged; RNA, Messenger; Statistics as Topic; Transforming Growth Factor alpha

In 6 HCC cell lines, clear expressions of EGFR and TGF-alpha were found in flow cytometry, while expressions of EGF, HB-EGF and AR were quite low. TGF-alpha secretion into culture supernatants became measurable when TPA 0.5 microM was added. TPA accelerated the proliferation of KYN-3 cells, and anti-TGF-alpha neutralizing antibody suppressed this proliferation in a dose-dependent manner. Addition of exogenous TGF-alpha, EGF, AR, or HB-EGF with heparin accelerated cell proliferation. In non-stimulated cultures, cell proliferation was suppressed by anti-EGFR neutralizing antibody, but not by the antibodies for EGF, TGF-alpha, AR and HB-EGF. HCC may possess a paracrine system regulated by these 4 ligands, and an autocrine system, under a certain condition, via TGF-alpha and EGFR.

Topics: Amphiregulin; Antibodies; Carcinoma, Hepatocellular; Cell Division; Cell Line; Culture Media; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Liver Neoplasms; Transforming Growth Factor alpha

Identification of specific and primary chromosomal alterations during the course of neoplastic development is an essential part of defining the genetic basis of cancer. We have developed a transgenic mouse model for liver neoplasia in which chromosomal lesions associated with both the initial stages of the neoplastic process and the acquisition of malignancy can be analyzed. Here we analyze chromosomal alterations in 11 hepatocellular carcinomas from the c-myc/TGF-alpha double-transgenic mice by fluorescent in situ hybridization with whole chromosome probes, single-copy genes, and 4'-6-diamidino-2-phenylindole (DAPI-) and G-banded chromosomes and report nonrandom cytogenetic alterations associated with the tumor development. All tumors were aneuploid and exhibited nonrandom structural and numerical alterations. A balanced translocation t(5:6)(G1;F2) was identified by two-color fluorescent in situ hybridization in all tumors, and, using a genomic probe, the c-myc transgene was localized near the breakpoint on derivative chromosome der 6. Partial or complete loss of chromosome 4 was observed in all tumors with nonrandom breakage in band C2. Deletions of chromosome 1 were observed in 80% of the tumors, with the most frequent deletion at the border of bands C4 and C5. An entire copy of chromosome 7 was lost in 80% of the tumors cells. Eighty-five percent of the tumor cells had lost one copy of chromosome 12, and the most common breakpoint on chromosome 12 occurred at band D3 (28%). A copy of chromosome 14 was lost in 72%, and band 14E1 was deleted in 32% of the tumor cells. The X chromosome was lost in the majority of the tumor cells. The most frequent deletion on the X chromosome involved band F1. We have previously shown that breakages of chromosomes 1, 6, 7, and 12 were observed before the appearance of morphologically distinct neoplastic liver lesions in this transgenic mouse model. Thus breakpoints on chromosome 4, 9, 14, and X appear to be later events in this model of liver neoplasia. This is the first study to demonstrate that specific sites of chromosomal breakage observed during a period of chromosomal instability in early stages of carcinogenesis are later involved in stable rearrangements in solid tumors. The identification of the 5;6 translocation in all of the tumors has a special significance, being the first balanced translocation reported in human and mouse hepatocellular carcinoma and having the breakpoint near a tumor susceptibility gene an

Topics: Age Factors; Animals; Carcinoma, Hepatocellular; Chromosome Breakage; Chromosome Deletion; Chromosome Mapping; Genes, myc; In Situ Hybridization, Fluorescence; Karyotyping; Liver Neoplasms; Mice; Mice, Transgenic; Transforming Growth Factor alpha; Translocation, Genetic

We have previously shown in transgenic mice that transforming growth factor (TGF)-alpha dramatically enhances c-myc-induced hepatocarcinogenesis by promoting proliferation and survival of hepatocellular carcinoma (HCC) cells. As transgenic livers display increased levels of mature TGF-beta1 from the early stages of hepatocarcinogenesis, we have now assessed whether impairment of TGF-beta1 signaling contributes to the deregulation of cell cycle progression and apoptosis observed during this process. Focal preneoplastic lesions lacking expression of TGF-beta receptor type II (TbetaRII) were detected in c-myc/TGF-alpha but not in c-myc livers. In c-myc/TGF-alpha mice, 40% (2/5) of adenomas and 90% (27/30) of HCCs showed down-regulation of TbetaRII expression in comparison with 11% (2/18) of adenomas and 47% (14/30) of HCCs in c-myc mice. Down-regulation of the TGF-beta1-inducible p15(INK4B) mRNA and reduced apoptotic rates in TbetaRII-negative HCCs further indicated the disruption of TGF-beta1 signaling. Furthermore, both TbetaRII-negative and -positive c-myc TGF-alpha HCCs, but not c-myc HCCs, were characterized by decreased levels of the cell cycle inhibitor p27. These results suggest 1) an inverse correlation of decreased p27 expression with the particularly strong expression of TGF-alpha in these lesions, consistent with the capacity of TGF-alpha signaling to post-transcriptionally regulate p27, and 2) the presence of alternative, downstream defects of TGF-beta1 signaling in c-myc/TGF-alpha HCCs that may impair the growth-inhibitory response to TGF-beta1. Thus, the accelerated neoplastic development in c-myc/TGF-alpha mice is associated with an early and frequent occurrence of TbetaRII-negative lesions and with reduced levels of p27 in HCC cells, indicating that disruption of TGF-beta1 responsiveness may play a crucial role in the enhancement of c-myc-induced hepatocarcinogenesis by TGF-alpha.

Topics: Animals; Blotting, Northern; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; Enzyme Inhibitors; Immunohistochemistry; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-myc; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Suppressor Proteins

Transforming growth factor-alpha (TGF-alpha) is a potent mitogen of normal and neoplastic hepatocytes. In addition, TGF-alpha has been reported to play a pivotal role in hepatocarcinogenesis. To evaluate the significance of TGF-alpha in chronic liver diseases and hepatocellular carcinoma, we examined serum TGF-alpha, and expression of TGF-alpha, epidermal growth factor receptor (EGFR) and proliferating cell nuclear antigen (PCNA) mRNA in liver tissues.. Thirty-five patients with chronic hepatitis (CH), 33 with liver cirrhosis (LC), 55 with hepatocellular carcinoma (HCC) and 53 normal controls (C) were enrolled in this study. Serum TGF-alpha levels were measured by an enzyme-linked immunosorbent assay. Expression of TGF-alpha, EGFR, PCNA and beta-actin mRNA in liver tissues were examined by reverse transcription polymerase chain reaction.. Serum TGF-alpha levels in C, CH, LC and HCC were 5.6+/-2.1, 33.2+/-8.3, 404.0+/-173.0 and 100.3+/-39.2 pg/ml, respectively. Serum TGF-alpha level in LC was higher than in other diseases (p<0.01, compared to CH, HCC and C, respectively). Serum TGF-alpha levels exhibited a significant positive correlation with total bilirubin, ICGR15 and Pugh score (p<0.01, p<0.01 and p<0.05, respectively), and increased in parallel with severity of disease according to Child classification. Although the ratios of TGF-alpha, EGFR and PCNA mRNA to beta-actin mRNA were not significantly different among the diseases, the TGF-alpha/beta-actin ratio correlated with EGFR/beta-actin and PCNA/beta-actin ratios (p<0.001 and p<0.0001, respectively), and EGFR/beta-actin ratio was related to PCNA/beta-actin ratio in all patients, especially with HCC.. The results of the present study suggest that serum TGF-alpha levels are closely related to severity of liver dysfunction, and that hepatic expression of TGF-alpha and EGFR correlates with proliferation of normal and neoplastic hepatocytes.

Topics: Actins; Adult; Aged; Biomarkers; Blotting, Southern; Carcinoma, Hepatocellular; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Female; Hepatitis, Chronic; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

A new human hepatocellular (HCC) cell line, HAK-3, was established from a resected HCC of a Japanese, female patient. HAK-3 retains morphologic features of the original HCC, and proliferates in a monolayered sheet (doubling time: 26 h). HAK-3 is a single aneuploid cell population with a DNA index of 2.42, the karyotype is human, chromosomes are 80-85 (mode: 83), and secretes fibronectin and tissue polypeptide antigen. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) dose-dependently accelerated the cell proliferation, while deletion-type hepatocyte growth factor (dHGF) tended to suppress the proliferation, and transforming growth factor (TGF)-alpha showed almost no influence. dHGF induced the decrease of cell adhesiveness, changed the cell morphology to spindle-shaped cells, increased cell movement, and showed chemotactic effects with the increase of its concentration gradient in cultures. HAK-3 would be useful in studies on the acceleration mechanisms of cancer cell proliferation by growth factors and of chemotaxis by dHGF.

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Movement; Cell Size; Chemotaxis; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Growth Substances; Hepatocyte Growth Factor; Humans; Karyotyping; Liver Neoplasms; Middle Aged; Ploidies; Transforming Growth Factor alpha; Tumor Cells, Cultured

To assess the expression of TGF alpha in hepatocellular carcinoma and its relationship with HBV infection.. Using in situ hybridization and streptavidin-peroxidase immunohistochemistry to detect TGF alpha mRNA, TGF alpha protein and HBV DNA in 53 cases of HCC and 14 cases of controls.. Positive rates of TGF alpha mRNA and TGF alpha protein in carcinoma tissue were 30.2% and 73.6% respectively, which were higher than in normal controls (P < 0.05). The expression of TGF alpha mRNA and TGF alpha protein was increased significantly in dysplastic liver cells (DLC) locating aside from the tumor tissue in comparing with those of non-dysplastic liver cells (NDLC) (P < 0.05). Positive rates of HBV DNA in carcinoma tissue and paratumorous liver tissue were 56.6% and 60.4% respectively, which were higher than in the normal controls (P < 0.001). Positive rate of HBV DNA in the nuclei of tumor tissues, tissue of the dysplastic areas and non-dysplastic areas was lowering down successively (P < 0.05). There was a significant relationship between expression of TGF alpha mRNA and TGF alpha protein in the carcinoma tissues and HBV DNA in the tumorous and its surrounding tissues respectively.. Expression of TGF alpha in carcinoma tissues was closely associated with the existence of HBV DNA and TGF alpha may participate in the early stage of liver carcinogenesis. The increased expression of HBV DNA in nuclei may serve as an important morphology marker in distinguishing carcinoma from DLC and NDLC.

Topics: Carcinoma, Hepatocellular; Cell Nucleus; DNA, Viral; Gene Expression Regulation, Neoplastic; Hepatitis B; Hepatitis B virus; Humans; Immunohistochemistry; In Situ Hybridization; Liver Neoplasms; RNA, Messenger; Transforming Growth Factor alpha

The Insulin like growth factor 2 (IGF2) gene is expressed in several types of tumors in humans and mice and has been implicated as an important growth factor in tumor progression. IGF2 expression in the TGF alpha transgenic mice was analysed in liver and tumors from animals which also contained one or two functional IGF2 alleles. In a two by two mating experiment using transgenic mice containing either a TGF alpha transgene or a IGF2 gene knockout, we have investigated whether IGF2 imprinting is reversed during hepatocarcinogenesis and the consequences of IGF2 expression for tumor growth. We observed that: (1) 100% of the hepatocellular carcinomas expressed IGF2 (2) the normally imprinted maternal allele is active in the tumors in which the paternal allele is knocked out and (3) all three of the murine IGF2 promoters upstream of the reactivated maternal alleles are transcriptionally active in tumors. We also observed that the total tumor burden of animals with two wild type IGF-2 alleles (paternal and maternal) was the same as the tumor burden in animals which contained only a single reactivated maternal allele. The 100% incidence of reactivation of the imprinted maternal allele suggests that IGF2 expression is selected during murine hepatocarcinogenesis and can substitute for the paternal allele when it is inactivated.

Topics: Alleles; Animals; Carcinoma, Hepatocellular; Genomic Imprinting; Insulin-Like Growth Factor II; Liver Neoplasms; Mice; Mice, Transgenic; Polymerase Chain Reaction; Promoter Regions, Genetic; Transforming Growth Factor alpha

We examined stage T1 to T4 hepatocellular carcinomas (HCC) to determine whether transforming growth factor alpha (TGFalpha) presence differed between early- and late-stage HCC and between tumors with low and high proliferative rates. Paraffin sections from 36 HCC were evaluated for TGFalpha and the proliferation markers Kiel 67 antigen (Ki67) or proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining. In 12 cases, double staining for TGFalpha and Ki67 was also performed. Eighty-one percent of tumors and 94% of adjacent liver sections contained TGFalpha. A trend toward inverse correlation was seen between the percentage of TGFalpha-positive tumor cells and the proliferative rate as determined by Ki67 staining. No clear correlation of TGFalpha to either tumor stage or percentage of PCNA-positive cells was seen. This study confirms the presence of TGFalpha in the majority of early- and late-stage HCC. Positivity within tumor tissue is consistent with autocrine or paracrine stimulation. A trend toward inverse correlation between TGFalpha-producing cells and the number of cycling cells suggests that rapidly proliferating tumors may consume this growth factor at an accelerated rate. Alternatively, other hepatic mitogens may have more functional significance in these latter tumors. Finally, the presence of TGFalpha in peritumoral hepatocytes suggests these cells as potential sources of paracrine stimulation for HCC.

Topics: Arteries; Carcinoma, Hepatocellular; Cell Count; Endothelium; Humans; Immunohistochemistry; Ki-67 Antigen; Liver; Liver Cirrhosis; Liver Neoplasms; Proliferating Cell Nuclear Antigen; Transforming Growth Factor alpha

Hepatitis B virus (HBV) causes acute and chronic liver injury, and integration of HBV DNA is considered to be an important pathogenic determinant for hepatocarcinogenesis and tumor development. Transforming growth factor alpha (TGF-alpha) drastically accelerates hepatocarcinogenesis when it is overexpressed in TGF-alpha transgenic mice (C. Jhappan et al., Cell, 61: 1137-1146, 1990). In HBV-infected patients, hepatocellular carcinoma (HCC) cells show elevated expression of TGF-alpha (C. C. Hsia et al., J. Med. Virol., 43: 216-221, 1994), the mechanism for which, however, has not been clarified yet. We here show that preS1, a part of the HBV large surface protein, carries a transcriptional transactivation domain and activates the transcription of the TGF-alpha gene by 2-fold in human HCC HuH6 cells. The responsive elements are restricted to the 315-bp segment of the proximal TGF-alpha promoter (-373 to -59). Furthermore, the expression of TGF-alpha was markedly increased in permanently preS1-producing HuH6 transformants. The crucial role for HBV preS1 in hepatocarcinogenesis and tumor development through transactivation of the TGF-alpha gene may give us new insight into the understanding of pathogenesis and therapy of viral hepatocarcinogenesis.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; DNA Primers; Gene Expression Regulation; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Mice; Polymerase Chain Reaction; Protein Precursors; Transcriptional Activation; Transforming Growth Factor alpha; Tumor Cells, Cultured; Viral Envelope Proteins

Surgical excision of liver tumors represents the only curative treatment for primary and metastatic liver malignancies. It has been suspected that hepatectomy may stimulate growth of microscopic tumors. To determine whether local or systemic factors after hepatectomy are responsible for enhancement of tumor growth, the effects of hepatectomy on the experimental growth of liver or pulmonary tumors were examined.. One hour after injection of 10(6) Morris hepatoma cells into either the portal or femoral vein, which produces isolated liver and lung tumors, respectively, animals were randomized to undergo 0%, 30%, or 70% partial hepatectomy (PH).. Animals that underwent portal injection of tumor had significantly increased liver tumor burden after PH (sham, 25 +/- 7 vs PH, 94 +/- 17; p < 0.01), whereas animals that underwent femoral injection had no change in lung tumor burden after PH. PH was associated with significantly increased levels of transforming growth factor-alpha, transforming growth factor-beta, and basic fibroblast growth factor in the liver but not in the lung.. Changes in liver cytokine-growth factor activation may contribute to enhanced tumor growth in the liver after hepatectomy.

Topics: Animals; Carcinoma, Hepatocellular; Fibroblast Growth Factor 2; Hepatectomy; Injections, Intravenous; Liver; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred BUF; Transforming Growth Factor alpha; Transforming Growth Factor beta

Little is known about the influence of amino acid imbalance by supplementation of branched-chain amino acids (BCAAs) on human hepatocellular carcinoma (HCC). We investigated the effect of various BCAA concentrations in culture medium on the growth and metabolism of two human HCC cell lines: Hep G2 and KYN-1. DNA and protein syntheses were studied by radiolabeled thymidine and leucine uptake, Amino acid concentrations in cell and medium were measured with an amino acid analyzer. Expression and excretion of transforming growth factor (TGF)-alpha and TGF-beta1 were studied by immunostaining and enzyme linked immunosorbent assay methods. The cell growth was suppressed in media with higher and lower BCAA concentrations than Dulbecco's modified Eagle's medium, and this was grossly correlated with the incorporation of [3H]thymidine into DNA and incorporation of [3H]leucine into intracellular protein. Pretreatment with 10,000 nmol/ml of BCAA did not suppress the cell growth in subsequent culture in the standard Dulbecco's modified Eagle's medium, indicating that the effect of BCAAs was not cytocidal but cytostatic and reversible. BCAA and aromatic amino acid concentrations in cells increased in parallel as the BCAA level in medium was increased, despite the fixed aromatic amino acid level. Intracellular expression and extracellular excretion of TGF-alpha were higher at BCAA concentrations of 100 and 1,000 nmol/ml than at 10 or 1,000 nmol/ml. The present finding that the in vitro growth of HCC can be suppressed by enriched BCAA levels in medium may indicate that amino acid-imbalanced therapy with enriched BCAAs is useful in the treatment of HCC.

Topics: Amino Acids; Amino Acids, Branched-Chain; Carcinoma, Hepatocellular; Cell Division; Culture Media; Culture Media, Conditioned; DNA; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Protein Biosynthesis; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

Topics: Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; DNA-Binding Proteins; Epithelial Cells; ErbB Receptors; Head and Neck Neoplasms; Humans; Kinetics; Liver Neoplasms; Mouth Mucosa; Recombinant Proteins; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transforming Growth Factor alpha; Tumor Cells, Cultured

There is strong evidence that selenium protects against certain human cancers, but the underlying mechanism is unknown. Glutathione peroxidase (GPX1) and thioredoxin reductase (TR), the most abundant antioxidant selenium-containing proteins in mammals, have been implicated in this protection. We analyzed the expression of TR and GPX1 in the following model cancer systems: (1) liver tumors in TGFalpha/c-myc transgenic mice; (2) human prostate cell lines from normal and cancer tissues; and (3) p53-induced apoptosis in a human colon cancer cell line. TR was induced while GPX1 was repressed in malignancies relative to controls in transgenic mice and prostate cell lines. In the colon cell line, p53 expression resulted in elevated GPX1, but repressed TR. The data indicate that TR and GPX1 are regulated in a contrasting manner in the cancer systems tested and reveal the p53-dependent regulation of selenoprotein expression. The data suggest that additional studies on selenoprotein regulation in different cancers are required to evaluate future implementation of selenium as a dietary supplement in individuals at risk for developing certain cancers.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line; Colonic Neoplasms; Enzyme Induction; Epithelial Cells; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, myc; Glutathione Peroxidase; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Mice, Transgenic; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Thioredoxin-Disulfide Reductase; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Protein p53

To make a better understanding of the molecular mechanisms involved in recurrence and metastasis of the hepatocellular carcinoma (HCC), some invasion related oncogenes, and growth factors have been investigated.. The studies were separately carried out, the results of which were summarized in this article with relation to tumor size and invasiveness of HCC.. The aberration rates of p53 and CDKN2 in HCC were 45.9% and 36.4% respectively, which were higher in invasive HCC compared with non-invasive HCC. H-ras expression was positive in 29.3% of HCC, which was associated with recurrence and extrahepatic metastasis of HCC. Intralesional injection of H-ras antisense gene markedly inhibited the tumor growth and metastasis of HCC in nude mice. The positive rates of transforming growth factor (TGF)-alpha, epidermal growth factor receptor (EGFR) and c-erbB-2 were 45.7%, 47.1% and 92.3% respectively. The expression of EGFR was closely related to TGF-alpha, which was related to HCC recurrence. But no obvious difference of TGF-alpha or c-erbB-2 expression was found between HCC with and without recurrence, or with and without extrahepatic metastasis. Expression of nm23/tissue inhibitor of metalloproteinase (TIMP)-2 was positively associated with the prognosis of HCC patients (Log-rank, P < 0.001). The alterative rates of above-mentioned genes and growth factors in small HCC were slightly lower than that in large ones, but no significant difference was shown except the p53 mutation.. The p53/CDKN2 mutation, over-expression of H-ras/EGFR, were associated with the invasiveness and recurrence of HCC. H-ras antisense gene might be of potential implication in the control of HCC recurrence and metastasis. Expression of nm23/TIMP-2 was closely related to the prognosis of HCC patients. Biological characteristics remained critical points to the prognosis even in small HCC.

Topics: Animals; Carcinoma, Hepatocellular; Genes, erbB-1; Genes, p16; Genes, p53; Genes, ras; Humans; Liver Neoplasms; Mice; Mice, Nude; Monomeric GTP-Binding Proteins; Mutation; Neoplasm Invasiveness; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Transcription Factors; Transforming Growth Factor alpha

A cell line derived from a Japanese man with hepatocellular carcinoma was established in culture and designated OCUH-16. The cell line has the morphological and chromosomal features of hepatocellular carcinoma cells and has a short doubling time (approximately 33 h). OCUH-16 cells were shown to express transforming growth factor-alpha (TGF-alpha) in addition to albumin, DNA polymerase-alpha, c-JUN, and the retinoblastoma gene product. Electron microscopy revealed TGF-alpha immunoreactivity associated with the cell membrane, but TGF-alpha was not detected in medium conditioned by OCUH-16 cells by enzyme-linked immunosorbent assay. Reverse transcription and polymerase chain reaction analysis revealed the presence of TGF-alpha messenger RNA in these cells. Culture of OCUH-16 cells in the presence of a neutralizing antibody to TGF-alpha inhibited cell proliferation and induced many cells to undergo apoptosis (programmed cell death). These observations suggest that endogenous TGF-alpha is necessary for OCUH-16 cell growth.

Topics: Antibodies, Monoclonal; Apoptosis; Binding, Competitive; Carcinoma, Hepatocellular; Cell Survival; Humans; Immunohistochemistry; Liver Neoplasms; Male; Transforming Growth Factor alpha; Tumor Cells, Cultured

We have previously demonstrated in short-term experiments that altered hepatocytes in liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-induced diabetic rats with mild persisting diabetes resemble those in preneoplastic foci of altered hepatocytes. We now present the results of long-term studies (up to 22 months) in this animal model. Glycogen-storing foci (which were the first parenchymal alteration observed some days after transplantation) persisted at least for 6 months, when the first mixed-cell foci and the first hepatocellular adenoma emerged. After 15 to 22 months, 86% of the animals exhibited at least one hepatocellular adenoma and four animals (19%) showed a hepatocellular carcinoma. The transplants were found in a close spatial relationship with the preneoplastic foci and the hepatocellular neoplasms. The mitotic indices, the 5-bromo-2'-desoxyuridine labeling indices and the apoptotic indices showed significant differences between the unaltered liver parenchyma, different types of preneoplastic foci, and hepatocellular neoplasms. The immunohistochemical expression of transforming growth factor-alpha increased during the stepwise development from glycogen-storing liver acini to hepatocellular carcinomas. Hepatocarcinogenesis in this new animal model is probably due to the hormonal and growth-stimulating effects of insulin secreted by the intraportally transplanted islets of Langerhans in diabetic rats.

Topics: Adenoma; Animals; Blood Glucose; Body Weight; Carcinoma, Hepatocellular; Case-Control Studies; Cause of Death; Cytoplasm; Diabetes Mellitus, Type 2; Endoplasmic Reticulum; Immunohistochemistry; Islets of Langerhans Transplantation; Liver Neoplasms, Experimental; Male; Microscopy, Electron; Neoplasm Staging; Precancerous Conditions; Rats; Rats, Inbred Lew; Streptozocin; Transforming Growth Factor alpha

Sensitive and reliable laboratory parameters are necessary to evaluate the degree of liver regeneration serially in patients after partial hepatectomy for liver cancer. We evaluated the serum levels of transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF), both of which are potent mitogens for hepatocytes, in 22 hepatectomized patients with liver cancer: 10 patients with hepatocellular carcinoma and 12 patients with metastatic liver tumors. Ten patients who underwent laparotomy for nonhepatic surgery were also studied as surgical controls. The serum TGF-alpha and HGF levels were measured by sandwich enzyme-linked immunosorbent assay techniques. Both the serum TGF-alpha and HGF levels increased after partial hepatectomy. However, there was no correlation between the levels of TGF-alpha and HGF. The maximal level of TGF-alpha achieved in each case correlated significantly with the resected liver volume and the increased volume of the remaining liver. Hepatocyte growth factor showed no such correlations. After nonhepatic surgery, the HGF level also increased significantly, while the TGF-alpha level did not. These results suggest that the serum TGF-alpha level varies depending on the regenerative stimulus to the liver, and that its increase corresponds with the degree of liver regeneration that occurs in patients after partial hepatectomy for liver cancer. In contrast, it is unlikely that the serum HGF level reflects liver regeneration. In conclusion, the serum TGF-alpha level can be used as a parameter for evaluating liver regeneration in patients who have undergone partial hepatectomy.

Topics: Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Female; Hepatectomy; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Liver Regeneration; Male; Middle Aged; Transforming Growth Factor alpha

Chronic infection with hepatitis B virus (HBV) can cause liver cancer in humans. Transgenic mice expressing the major envelope protein of HBV, HBV surface antigen (HBsAg), represent an experimental model for some of the histopathological effects of infection in humans, including prolonged hepatocellular injury, necrosis, hyperplasia, and an elevated incidence of liver tumors. The regenerative hyperplastic response to the chronic liver damage is thought to be a critical factor in the increased risk of cancer. However, little is known about the cellular factors that mediate regenerative proliferation. One candidate is the hepatocyte mitogen transforming growth factor alpha (TGF-alpha); in HBV-infected patients with liver cancer, TGF-alpha and HBsAg accumulate in the same hepatocytes. Transgenic mice overexpressing TGF-alpha demonstrate enhanced hepatocyte proliferation rates and develop hepatocellular carcinomas. In this study, we have analyzed the effect of TGF-alpha and HBsAg coexpression in the liver using a bitransgenic mouse model. We show that hepatocytes harboring both the TGF-alpha and HBsAg transgenes exhibited an increase in growth relative to hepatocytes with either transgene alone. Furthermore, bitransgenic males but not females had a dramatically accelerated appearance of hepatocellular carcinomas, compared to single transgenic TGF-alpha or HBsAg littermates. These results demonstrate synergistic activity between HBsAg and TGF-alpha in the liver, probably by first stimulating quiescent hepatocytes to enter G1 and by subsequently promoting their transit through the cell cycle, respectively. Moreover, our data support the contention that TGF-alpha participates in HBV-induced hepatocarcinogenesis in infected patients.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Female; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Male; Mice; Mice, Transgenic; Necrosis; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; RNA, Messenger; Sex Factors; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF-alpha) is thought to be involved in liver regeneration, cellular proliferation, and hepatocarcinogenesis. We have looked at the relationship between TGF-alpha and it's receptor, and have attempted to relate the expression of TGF-alpha and it's receptor to the differentiation of hepatocellular carcinoma (HCC) on serial sections of HCC. We examined immunohistochemically the expression of the TGF-alpha and of epidermal growth factor receptor (EGFR) proteins in the same area of 53 nodules (< 5 cm in diameter) of HCC obtained from patients. Immunoreactive proteins were visualized by using a biotin-streptoavidin system (LSAB Kil, Dako). TGF-alpha was strongly expressed in 29 of 53 (54.7%) nodules. Specimens strongly positive for TGF-alpha were found mainly in well-differentiated HCC, while specimens positive for EGFR were found mainly in poorly differentiated HCC (p < 0.05). In the tissues that stained weakly positive for TGF-alpha, the expression of EGFR differed significantly, according to the degree of HCC histologic differentiation (p < 0.05). These results led us to speculate that the expression of TGF-alpha and EGFR might be related to the pattern of histologic differentiation of HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Differentiation; ErbB Receptors; Female; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

The molecular role of hepatitis C virus (HCV) in liver disease has yet to be clarified. In this study, we analyzed the relationship of HCV replication with mRNA expression of growth factors and mutation of tumor suppressor gene, ie, transforming growth factor-beta 1 (TGF-beta 1), which promotes cirrhotic changes; TGF-alpha, insulin-like growth factor-II (IGF-II), which are both related to hepatocyte transformation; and tumor suppressor gene p53, which is associated with HCC progression. A semiquantitative RNA polymerase chain reaction (RNA-PCR) was used to analyze genetic expression in 31 cirrhotic liver specimens from patients with HCV. In order to detect HCV replication, the minus-strand RNA of HCV, which serves as a template for the synthesis of genomic plus-strand RNA, was examined. The expression of the growth factors was semiquantified by RNA-PCR, and the mutation of p53 was detected using PCR-single-strand conformation polymorphism. According to the semiquantitative analysis, HCV replication was not associated with the expression of TGF-beta 1 but was significantly so with the overexpression of TGF-alpha (r = 0.74) and IGF-II (r = 0.65) in the HCV-positive cirrhotic livers. No mutation of p53 was recognized in any of the samples. Our investigation thus suggested that the replication of HCV might mediate the coexpression of TGF-alpha and IGF-II and act as a possible initiating factor for hepatocarcinogenesis.

Topics: Actins; Carcinoma, Hepatocellular; Cocarcinogenesis; Genes, p53; Hepacivirus; Humans; Insulin-Like Growth Factor II; Liver; Liver Cirrhosis; Liver Neoplasms; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; RNA, Viral; Transforming Growth Factor alpha; Virus Replication

Synthetic 4,5-didehydro GGA (geranylgeranoic acid), a potent ligand both for cellular retinoic acid-binding protein and for nuclear retinoid receptors, induced apoptosis in human hepatoma-derived cell line HuH-7 but not in primary hepatocytes, although all-trans or 9-cis retinoic acid did not induce any growth inhibition. Either exogenous transforming growth factor-alpha (TGF alpha) or epidermal growth factor(EGF) prevented the cells from apoptosis in the presence of 4,5-didehydro GGA, but hepatocyte growth factor, insulin-like growth factor-II, insulin or triiodothyronine was essentially inactive. 4,5-Didehydro GGA down-regulated the cellular levels of TGF alpha mRNA as early as 30 min after the treatment. Either anti-TGF alpha or anti-EGF receptor monoclonal antibody induced apoptosis in HuH-7 cells without using the acid. Taken together, the present study strongly suggests that 4,5-didehydro GGA induced apoptosis in HuH-7 cells through the destruction of autocrine loop consisting of TGF alpha and EGF receptor, due to the down regulation of TGF alpha gene expression.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Insulin-Like Growth Factor II; Kinetics; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Transforming growth factor alpha (TGF alpha) is alleged to play a role in malignant progression as well as normal cell growth in an autocrine manner and its serum levels have been reported to increase during this progression. Most hepatocellular carcinomas (HCC) develop in cirrhotic livers in which hepatocyte necrosis and regeneration prevail. The significance of serum TGF alpha levels in the diagnosis of HCC complicating cirrhosis should be clarified.. One hundred twenty-four patients with cirrhosis were studied, 80 with HCC (HCC) patients and 44 without (LC) patients. There was no difference in clinical features between the two groups. One hundred eighty-two healthy adults were also studied as controls. Serum TGF alpha levels were measured by enzyme-linked immunosorbent diffusion assay (ELISA).. Serum TGF alpha levels were significantly higher in HCC patients than in healthy adults or LC patients (mean +/- SD: 45 +/- 40 vs. 21 +/- 15 or 25 +/- 19 pg/ml, respectively). In LC patients, serum TGF alpha levels were significantly correlated with serum albumin and total bilirubin levels (r = -0.44 and 0.32, respectively). When the cutoff level was defined as 25 pg/ml from receiver operating characteristic curve, sensitivity and specificity for the diagnosis of HCC in the presence of cirrhosis were 69% and 66%, respectively. Serum TGF alpha levels were decreased after successful treatment for HCC in 60% of the HCC patients. Serum TGF alpha levels showed no correlation with serum alpha-fetoprotein levels; the levels were greater than 25 pg/ml in 67% of the HCC patients whose serum alpha-fetoprotein levels were within 20 ng/ml.. Serum TGF alpha levels may provide useful information for the diagnosis of HCC developing in the presence of cirrhosis.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity; Transforming Growth Factor alpha

We investigated whether stress increases tumorigenesis in male transgenic mice that overexpress the gene encoding human transforming growth factor alpha (TGF alpha). At the age of 10-15 months, these mice begin to develop spontaneous hepatocellular carcinomas at high incidence. The male TGF alpha mice were housed with their siblings (non-stressful environment), housed in social isolation, or housed with aggressive non-siblings (stressful environment). Some animals in each group were exposed once a week to a second stressor (swim stress), beginning at the age of 7 months. Housing with aggressive non-siblings increased neoplastic growth in the male TGF alpha mice: the incidence and multiplicity of liver tumors, and tumor burden were higher in these animals than in the sibling-housed mice. Among the isolated TGF alpha mice, only the tumor burden was increased, when compared with the sibling-housed TGF alpha mice. Swim stress significantly increased the incidence of liver tumors and tumor burden in the sibling-housed TGF alpha mice. Plasma levels of 17 beta-estradiol (E2) that are elevated in the TGF alpha mice, were modestly but significantly higher in the non-sibling housed transgenic mice than in the sibling-housed. Natural killer (NK) cell activity, reduced in these mice, was not affected by housing environment. These data suggest that stress promotes the growth of hepatocellular tumors in the male TGF alpha mice. Whether estrogens are involved in mediating this association remains to be determined.

Topics: Aggression; Animals; Carcinoma, Hepatocellular; Estradiol; Housing, Animal; Killer Cells, Natural; Liver Neoplasms; Male; Mice; Mice, Transgenic; Stress, Physiological; Swimming; Transforming Growth Factor alpha

Expression of transforming growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR), in hepatocellular carcinomas (HCCs) and adjacent nontumorous livers from 25 Japanese patients were examined using immunoperoxidase staining of paraffin-embedded sections. TGF-alpha was detected in 24 of 25 (96%) HCCs and 23 of 24 (96%) available adjacent nontumorous livers. EGFR was detected in 16 of 25 (64%) HCCs and 17 of 24 (71%) adjacent nontumorous livers. TGF-alpha and EGFR were not detected by immunohistochemical staining in normal livers. Fifteen of 25 HCCs contained an apparent area of a second tumor (two of the 15 also contained a third tumor) that had a less-differentiated histological grade developing within or adjacent to the first tumor. In those cases, staining in the less-differentiated area of tumor was usually less intense than in the more highly differentiated area (80% of cases for TGF-alpha; 91% for EGFR). These data confirm that increased expression of TGF-alpha and EGFR occur frequently in human HCC. Furthermore, the detection of greater staining in more highly differentiated portions of the tumors suggests that increased expression of TGF-alpha and EGFR may be events of the early stages of human hepatocarcinogenesis.

Topics: Adult; Aged; Carcinoma, Hepatocellular; ErbB Receptors; Female; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using 125I-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Prothrombin; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Rats; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Vitamin K

In the present study we investigated the expression of transforming growth factor alpha and insulin-like growth factor II to explain the role of these growth factors in the development of hepatocellular carcinoma from chronic active hepatitis B and cirrhosis. The expression of transforming growth factor alpha and insulin-like growth factor II was tested in 38 tissue samples from patients with chronic active hepatitis B, 32 cirrhosis and 31 hepatocellular carcinoma, by immunohistochemical staining using monoclonal anti-transforming growth factor alpha and anti-insulin-like growth factor II. All patients were seropositive for HBsAg. Transforming growth factor alpha was expressed in 26 (68.4%) of 38 chronic active hepatitis B, 18 (56.3%) of 32 cirrhosis and 16 (51.6%) of 31 hepatocellular carcinoma tissue samples. Transforming growth factor alpha was found in the periportal hepatocytes of chronic active hepatitis B and in regenerating hepatocytes of cirrhotic nodules. In hepatocellular carcinoma tissues, transforming growth factor alpha-containing tumor cells were evenly distributed within the tumor tissues but focal distribution limited to a part of tumor tissues was also observed. The expression of insulin-like growth factor II was observed in 30 (93.8%) of 32 cirrhosis and all the 31 hepatocellular carcinoma tissue samples tested, but not in chronic active hepatitis B samples. Insulin-like growth factor II was expressed in most hepatocytes of regenerating nodules and in tumorous as well as non-tumorous hepatocytes of hepatocellular carcinoma tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Chronic Disease; Female; Hepatitis B; Hepatitis, Chronic; Humans; Immunohistochemistry; Insulin-Like Growth Factor II; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Radioimmunoassay; Transforming Growth Factor alpha

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cattle; Cell Division; DNA; Epidermal Growth Factor; Fetal Blood; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Insulin-Like Growth Factor II; Liver Neoplasms; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers; Transforming Growth Factor alpha; Tumor Cells, Cultured

Single and double immunohistochemical staining for transforming growth factor (TGF)-alpha and epidermal growth factor-receptor (EGF-R) was done in order to identify the localization of TGF-alpha and EGF-R in human hepatocellular carcinoma (HCC). Single immunohistochemical staining for TGF-alpha showed immunoreactivity in the cytoplasm of hepatoma cells in 22 of 30 cases of HCC. The localization of TGF-alpha was heterogeneous from HCC cells to HCC cells. In the surrounding regenerative nodules, the hepatocytes were mildly to moderately positive for TGF-alpha. The proliferating bile ductules and peripheral nerves were also immunopositive for TGF-alpha. Single immunohistochemical staining for EGF-R demonstrated a linear localization of EGF-R along the cell membrane of the HCC cells in 21 of the 30 cases of HCC. In the regenerative nodules, the hepatocytes also showed linear staining along the cell membrane. Double staining for TGF-alpha and EGF-R in 12 cases of HCC showed a concurrent localization of TGF-alpha and EGF-R in some hepatoma cells and isolated localization of the two substances of other HCC cells. These combinations either abruptly moved around or intermingled with each other. These immunohistochemical results thus support the theory of an autocrine, paracrine, and endocrine mechanism of TGF-alpha and EGF-R on the proliferation of human hepatocellular carcinoma.

Topics: Adolescent; Adult; Carcinoma, Hepatocellular; ErbB Receptors; Female; Humans; Immunohistochemistry; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha; Tumor Cells, Cultured

Hepatocyte growth factor (HGF) and transforming growth factor alpha (TGF alpha) are potent mitogens for mature hepatocytes in primary culture (Nakamura, et al. 1984, Mead et al., 1989). However, these cytokines have completely different effects on tumor cell growth (Jhappan et al., 1990, Shiota et al, 1992). To clarify dual effects of these cytokines in liver, we developed double transgenic mice of HGF and TGF alpha using albumin promoter-HGF cDNA transgenic mice (AlbHGF) and metallothionein promoter-TGF alpha transgenic mice (MthTGF alpha). Double transgenic mice were examined on DNA synthesis, c-myc mRNA and occurrence of hepatocelular carcinoma (HCC). Higher labeling indices using BrDU were observed, in sequence, in AlbHGF mice, AlbHGF/MthTGF alpha, MthTGF alpha and wild type mice. Hepatic expression of c-myc mRNA in AlbHGF mice was elevated, compared to that in AlbHGF/MthTGF alpha or MthTGF alpha mice. After long-term observation, MthTGF alpha mice developed HCC in 6/10 (60%), whereas AlbHGF/MthTGF alpha mice developed HCC in 3/9 (33%). These data suggest that HGF is the potent mitogen, stimulating DNA synthesis and up-regulating c-myc mRNA. In addition, HGF may excert some inhibitory effect on occurrence of HCC by TGF alpha.

Topics: Animals; Body Weight; Bromodeoxyuridine; Carcinoma, Hepatocellular; DNA; Hepatocyte Growth Factor; Mice; Mice, Transgenic; Organ Size; Proto-Oncogene Proteins c-myc; RNA, Messenger; Transforming Growth Factor alpha

Studies on the natural course of virus-associated hepatocellular carcinoma (HCC) in high risk areas, particularly hepatitis B virus (HBV), have shown a stage of persistent liver cell hyperplasia characterized by a low level elevation of serum alpha-fetoprotein (AFP). We have recently identified a population of epithelial cells with distinct structure and expression of cytokeratin and AFP in non-neoplastic liver tissues from humans with HBV-associated HCC. These cells were characterized by oval nuclei, scant pale cytoplasm, small cell size, and cross-reactivity to a monoclonal antibody against rat oval cells. These human epithelial cells, putative human oval-type cells, stained strongly positive for cytokeratin 19 and displayed considerable heterogeneity in AFP and albumin expression. These findings suggest that a cell population structurally and phenotypically similar to oval cells seen in the early stages of chemical hepatocarcinogenesis in the rat is also present in humans in regenerating liver lesions observed in HBV-associated HCC. Hepatitis B surface antigen (HBsAg) was detected in 69, 81, and 85% of oval-type cells, transitional cells, and hepatocytes, respectively, but not in bile ducts or ductular cells. Also, high levels of expression of transforming growth factor alpha (TGF-alpha) were frequently seen in oval-type and transitional cells expressing HBsAg. These data suggest the possibility that oval-type cells are a target cell population for HBV infection; in the presence of elevated TGF-alpha expression, these cells may constitute a progenitor population for human HCC.

Topics: Animals; Carcinoma, Hepatocellular; Hepatitis B; Hepatitis B Core Antigens; Hepatitis B Surface Antigens; Humans; Liver; Liver Neoplasms; Rats; Transforming Growth Factor alpha

Recent studies have identified epithelial cell populations in human livers that are similar to the "oval cells" and "transitional cells" seen in rat livers during the early stages of chemical carcinogenesis. It has been suggested that these cells might be precursors of hepatocytes and theoretically could be involved in hepatocarcinogenesis. The hepatitis B virus (HBV) also is believed to play a role in the etiology of hepatocellular carcinoma (HCC). Therefore, a study was conducted in nontumorous livers adjacent to HCCs obtained from 26 patients from China to determine whether HBV antigens could be identified in oval cells and transitional cells using an immunohistochemical technique. Hepatitis B surface antigen (HBsAg) was detected in the nontumorous livers of 22/26 (85%) patients. HBsAg was detected in oval cells in 18/26 (69%), in transitional cells in 21/26 (81%), and in mature hepatocytes in 22/26 (85%), but not in bile duct or ductule cells. Transforming growth factor-alpha (TGF-alpha) was expressed in oval cells, transitional cells, and bile duct cells in 24/26 (92%) patients, an in mature hepatocytes in 25/26 (96%). Coexpression of HBsAg and TGF-alpha was identified in the same cells in populations of oval cells and transitional cells of selected patients. Because of the possibility that oval cells could be a source of evolving HCC, these findings suggest that expression of TGF-alpha associated with HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. Thus, oval cells could be a site (or one of the sites) where HBV participates in the development of HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Epithelium; Female; Hepatitis B Core Antigens; Hepatitis B Surface Antigens; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations mus

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Transformation, Neoplastic; Culture Media, Serum-Free; DNA Replication; Epidermal Growth Factor; Insulin-Like Growth Factor II; Liver; Male; Mice; Mice, Nude; Mice, Transgenic; Niacinamide; Ploidies; RNA, Messenger; Time Factors; Transforming Growth Factor alpha

A well-differentiated trabecular hepatocellular carcinoma (HCC) and a well-differentiated tumor resembling HCC from each of two chimpanzees were found to have histochemical and immunohistochemical staining characteristics similar to those in human HCCs. Transforming growth factor alpha was overexpressed in both tumors. Oval cells, thought to be liver stem cell progeny with a possible role in hepatocarcinogenesis, were observed among nontumorous hepatocytes, particularly near the tumors. Hepatic tumors are rare in chimpanzees but their similarities to human HCC provides a useful research model.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Differentiation; Cell Nucleus; Histocytochemistry; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Male; Pan troglodytes; Primate Diseases; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

The expression of transforming growth factor-alpha (TGF-alpha) was studied in 33 surgically excised human hepatocellular carcinomas (HCCs), focal nodular hyperplasias (FNHs), and the surrounding liver tissue from patients who were life-long residents of Hungary. Monoclonal antibodies were used to localize TGF-alpha and hepatitis B surface antigen (HBsAg) using an avidin-biotin-peroxidase immunohistochemical method on paraffin-embedded sections. Transforming growth factor-alpha was detected in the tumor tissue in 13 of 16 patients with HCC (81%) and in 12 of 17 patients with FNH (71%). Three patients with HCC were actively infected with hepatitis B virus, indicated by the detection of HBsAg in the serum and in adjacent nontumorous liver. Transforming growth factor-alpha was detected in the same nontumorous hepatocytes as HBsAg, often in areas of the hepatocyte cytoplasm, with a "ground glass" appearance. Bile duct cells were stained for TGF-alpha in the surrounding nontumorous liver tissue and a more intensive immunostaining was observed in the proliferating bile ducts in the FNH cases, which suggests that TGF-alpha may participate in the growth regulation of bile ducts.

Topics: Adolescent; Adult; Aged; Carcinoma, Hepatocellular; Female; Hepatitis B; Hepatitis B Surface Antigens; Humans; Hungary; Hyperplasia; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGF-alpha) expression is associated with hepatocyte DNA replication both in vivo and in culture. Our previous work using TGF-alpha transgenic mice showed that constitutive overexpression of this growth factor in the liver causes hepatic tumors in 75 to 80% of the animals at 12 to 15 months of age. To understand the cellular events by which TGF-alpha overexpression leads to abnormal liver growth, we examined hepatocyte proliferative activity in young and old TGF-alpha transgenic mice and hepatocyte ploidy in normal, dysplastic, and neoplastic livers of these animals. At 4 weeks of age, transgenic mice had higher liver weights and liver weight/body weight ratios than non-transgenic mice of the same age and hepatocyte proliferative activity, measured by 3H-thymidine incorporation after 3- and 7-day infusion, proliferating cell nuclear antigen staining, and mitotic index determination, was 2 to 3 times higher than in controls. In both transgenic and non-transgenic mice hepatocyte proliferation declined with age but the decrease was much more pronounced in control animals, so that at 8 months of age, hepatocyte replication was 8 to 10 times higher in transgenic animals. Surprisingly, however, transgenic and non-transgenic mice at this age had similar liver weight/body weight ratios. Labeling studies done in 3-month-old animals revealed that hepatocyte turnover was much faster in transgenic than in control animals, suggesting that a homeostatic compensatory mechanism involving cell death tended to restore normal liver weight/body weight ratios in older transgenic mice. Ploidy analyses showed that at 4 weeks of age transgenic mice had a higher proportion of diploid and tetraploid hepatocytes and that the hepatocellular tumors which developed in TGF-alpha transgenic mice at 13 months of age contained a higher fraction of diploid hepatocytes than that present in adjacent tissue or in dysplastic livers. The results demonstrate that constitutive overexpression of TGF-alpha causes increased hepatocyte proliferation and liver enlargement in young animals and is associated with a delay in the establishment of hepatic polyploidy. These findings as well as the response of transgenic mice to partial hepatectomy show that constitutive overexpression of TGF-alpha initially caused increased but regulated hepatocyte proliferation which in older animals was compensated in part by a faster cell turnover. At 8 to 10 months of age, proliferative

Topics: Aging; Animals; Body Weight; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Liver; Liver Neoplasms; Liver Regeneration; Mice; Mice, Transgenic; Organ Size; Ploidies; Reference Values; Transforming Growth Factor alpha

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor-stimulated or transforming growth factor alpha-stimulated DNA synthesis by three- to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10(-10) M, and it was increased in a dose-dependent manner with maximal effects at 10(-8) M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor beta. Secondary mitogens (co-mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Culture Media; DNA; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Liver; Liver Neoplasms; Liver Regeneration; Male; Neuropeptides; Neurotensin; Rats; Rats, Inbred F344; RNA, Messenger; Tissue Distribution; Transforming Growth Factor alpha

Hepatocyte growth factor (HGF) is an important paracrine regulator for liver growth, whereas it inhibits growth of tumor cells including hepatocellular carcinoma. In contrast, transforming growth factor alpha (TGF alpha) is a factor which stimulates hepatocyte growth and causes hepatocellular carcinoma in an autocrine fashion. To determine whether TGF alpha is affected by HGF, we examined the regulation of TGF alpha expression in response to exogenous HGF in HepG2 cells. We first examined TGF alpha mRNA and protein in the presence of HGF and found nearly a 3.6-fold increase in mRNA and a 2.5-fold increase in TGF alpha protein. This induction was dose-dependent and followed delayed kinetics. Nuclear run-on experiments suggested that the mechanism for this induction was an increase in transcription of TGF alpha mRNA. These data suggest that HGF may modulate TGF alpha expression and the interaction of these cytokines may play an important role in liver diseases.

Topics: Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Kinetics; Liver; Liver Neoplasms; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor alpha (TGF-alpha) is a mitogenic polypeptide which acts on the epidermal growth factor receptor (EGFR). The aim of this study was to examine the expression of TGF-alpha in human hepatocellular carcinoma (HCC) and surrounding cirrhotic tissue, and to compare it with normal liver. Immunoreactive TGF-alpha was detected using two antibodies raised against its C terminus, a polyclonal antibody 26T and a monoclonal antibody Ab-2. In normal liver immunoreactive TGF-alpha was localised strongly to bile duct epithelium and weakly in occasional parenchymal cells but was notably absent from perisinusoidal and Kupffer cells. Eight out of twenty-eight (28%) cases of HCC expressed TGF-alpha as demonstrated by cytoplasmic staining with both antibodies and in four cases additional membrane immunoreactivity was demonstrated using 26T. However, where cirrhotic tissue surrounding TGF-alpha positive tumours was available for analysis immunoreactive TGF-alpha was detected in only 1/7 cases. TGF-alpha synthesis by malignant hepatocytes was supported by the detection of specific RNA by Northern blotting from two cases with TGF-alpha immunoreactivity. These results implicate bile duct epithelium as an important source of TGF-alpha in human liver. Furthermore, in HCC the expression of TGF-alpha in some cases, together with paucity of TGF-alpha immunoreactivity in surrounding cirrhotic tissue, suggests that TGF-alpha may play a role in continued cell proliferation in human hepatocarcinogenesis.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Liver; Liver Cirrhosis; Liver Neoplasms; Male; RNA, Messenger; Transforming Growth Factor alpha

Hepatitis B virus (HBV) infection is closely associated with the development of hepatocellular carcinoma (HCC) in many patients, but the mechanisms by which HBV contributes to HCC are not known. Transforming growth factor-alpha (TGF-alpha), a regulator of growth and regeneration in rat liver that can be found in high levels in some human cancers, theoretically could play such an intermediate role in the development of HCC.. The expression of TGF-alpha and its relation to the HBV antigens were evaluated in human HCC and adjacent nontumorous livers from 33 patients from the United States and China using immunoperoxidase staining of paraffin-embedded sections.. TGF-alpha was detected in HCC from 27 of 33 (82%) patients; the frequencies were similar in patients from the United States and China. TGF-alpha was detected in HCC more frequently in patients whose adjacent nontumorous livers had detectable hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) than in those whose adjacent livers lacked HBsAg and HBcAg. Detection of TGF-alpha was not affected by tumor size, histologic type, or grade. TGF-alpha was detected in adjacent nontumorous livers from 31 of 33 patients (94%). Coexpression at a high intensity of TGF-alpha and HBsAg in the same hepatocytes could be demonstrated by specific staining of consecutively cut sections for 17 of 33 patients (52%).. TGF-alpha is expressed at a high level in 82% of human HCC. Localization of HBsAg within the same hepatocytes as TGF-alpha suggests a possible interaction between HBV and TGF-alpha during hepatocarcinogenesis in humans. Stimulation of TGF-alpha expression could be part of a chain of events by which HBV contributes to the development of HCC in some patients.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Differentiation; China; Female; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity; Transforming Growth Factor alpha; United States

We studied the development of liver tumors in male transforming growth factor alpha (TGF-alpha) transgenic mice of the CD1 strain and examined the expression of the transgene by immunohistochemistry and in situ hybridization. Livers of 4-5-week-old transgenic mice contained areas of centribobular hypertrophy with low glucose-6-phosphatase activity. These areas progressively expanded, and hypertrophy and dysplasia became generalized in livers of mice at 10-12 months of age. The expression of the transgene, determined by either immunohistochemistry or in situ hybridization, was uneven in animals that were 10 weeks old or older. The positive hepatocytes formed patches with a predominant centrilobular distribution. We studied a total of 23 liver tumors (7 hepatocellular carcinomas and 16 adenomas) obtained from 11 mice at 13-15 months of age and from one 7-month-old animal which received zinc sulfate to induce the transgene. The carcinomas were well differentiated tumors, without glucose-6-phosphatase or gamma-glutamyltranspeptidase activity, that developed from the dysplastic parenchyma and occasionally within an adenoma. In all carcinomas and in 56% of the adenomas there was overexpression of the transgene in relationship to the surrounding tissue. The majority of the tumors that overexpressed TGF-alpha were alpha-fetoprotein positive, while alpha-fetoprotein staining was not detected in tumors (all adenomas) that did not show excessive transgene expression. We conclude that TGF-alpha functions as a promoter of liver carcinogenesis through its effect as an autocrine inducer of hepatocyte proliferation. Further, the data indicate that TGF-alpha overexpression may favor tumor progression.

Topics: Animals; Carcinoma, Hepatocellular; Gene Expression; Histocytochemistry; Hypertrophy; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Nucleic Acid Hybridization; Transforming Growth Factor alpha

The expression of transforming growth factor alpha (TGF-alpha) was examined in a human hepatoblastoma cell line, Hep G2, which does not contain hepatitis B virus (HBV) DNA, and in the cell line 2.2.15, which was formed by the transfection of Hep G2 cells with the complete HBV DNA, to study the possibility that HBV and TGF-alpha could function as co-factors in hepatocarcinogenesis. Northern blot hybridization of RNA extracted from these cell lines, with densitometric analysis, revealed expression of the TGF-alpha gene in the transfected cells at a level three times higher than in the nontransfected cells. Staining of the cells using a monoclonal antibody to TGF-alpha and the avidin-biotin-peroxidase immunohistochemical method revealed a much higher intensity of TGF-alpha staining in the transfected cell line. These findings show that the presence of HBV DNA appears to cause a significant up-regulation of the TGF-alpha gene. This effect on the TGF-alpha gene may be a mechanism by which HBV contributes to the etiology of hepatocellular carcinoma in some patients.

Topics: Carcinoma, Hepatocellular; Cocarcinogenesis; Hepatitis B virus; Humans; Liver Neoplasms; RNA, Neoplasm; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

We have previously demonstrated that BDS.1 cells are a minimal deviation rat hepatoma cell line that is hypersensitive to the anti-proliferative effects of glucocorticoids in vitro. When transplanted into athymic mice, exposure to dexamethasone reduced the initial growth rate and increased the latency time before detection of palpable BDS.1-derived tumors but did not affect the maximal growth rate, size and histology of the tumor. After collagenase dissociation, the in vitro growth of BDS.1-derived tumor cells was fully suppressed by dexamethasone. Exposure to insulin prevented the glucocorticoid inhibition of anchorage-independent growth of BDS.1 cell colonies in vitro and may therefore be one of the systemic factors that masks the long term in vivo growth response of glucocorticoids.

Topics: Animals; Body Weight; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Drug Antagonism; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Liver Neoplasms; Mammary Neoplasms, Animal; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Platelet-Derived Growth Factor; Rats; Transforming Growth Factor alpha; Tumor Cells, Cultured

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-beta (TGF-beta) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blood Platelets; Carcinoma, Hepatocellular; Epidermal Growth Factor; Humans; Liver Neoplasms; Plasminogen Inactivators; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.

Topics: Acute-Phase Proteins; alpha 1-Antichymotrypsin; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cytokines; Fibrinogen; Haptoglobins; Humans; In Vitro Techniques; Interleukin-1; Interleukin-6; Liver; Liver Neoplasms; Serum Albumin; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-alpha (TGF alpha), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGF alpha and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGF alpha, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are involved in the degradation of TGF alpha and EGF as well as insulin, and that the degradation of TGF alpha, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGF alpha may explain how the two factors exert differential effects in some systems, and degradation of TGF alpha by IDE would provide a possible mechanism for interaction between the insulin and TGF alpha/EGF signalling systems.

Topics: Bacitracin; Carcinoma, Hepatocellular; Endopeptidases; Epidermal Growth Factor; Humans; Insulin; Liver Neoplasms; Metalloendopeptidases; Protease Inhibitors; Transforming Growth Factor alpha; Tumor Cells, Cultured

A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia). The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed. TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors. Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha. In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.

Topics: ADP Ribose Transferases; Bacterial Toxins; Base Sequence; Binding, Competitive; Carcinoma, Hepatocellular; Cell Survival; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Escherichia coli; Exotoxins; Female; Gene Expression; Liver Neoplasms; Molecular Sequence Data; Ovarian Neoplasms; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Structure-Activity Relationship; Transforming Growth Factor alpha; Transforming Growth Factors; Tumor Cells, Cultured; Virulence Factors