Characteristics Of Human Hepatocellular Carcinoma Research Articles

DEN-Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma is the most frequent form of primary liver cancer, characterized by increasing incidence and high mortality. Animal models of hepatocellular carcinoma are widely used to study the biology of cancer and to test potential therapies. Herein, we describe how the rat model of DEN-induced hepatocellular carcinoma mimics the pathogenesis of hepatocellular carcinoma seen in humans, including liver damage, chronic inflammation, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and modulations of the liver’s immune microenvironment. Our results should help the hepatocellular carcinoma field to better tailor the use of the DEN-induced rat liver cancer model for testing specific experimental hypotheses or to perform preclinical testing. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease’s progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver’s inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.

Abstract 1636: Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B)

Abstract Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and predicted to become the leading cause of liver transplant in 2020 and of HCC in the next decades. There is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis due to this changing in HCC etiology. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape. Methods: We used in the whole study ASV-B mice: a transgenic mouse model (C57BL/6J) spontaneously developing a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20). Livers were characterized for angiogenesis and immune populations using immunostaining (IHC and IF), and qRT-PCR (LightCycler, Roche). To mimic NASH, ASV-B mice were exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. A second experiment was performed on ASV-B and C57BL/6J wild type mice, using control and Nash-inducing regimen, to confirm our first results. Results: ASV-B model showed an increase in liver volume and angiogenesis, HCC livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expressions, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weights. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and green regimens. Interestingly, by microscopic analysis, we observed liver steatosis and inflammation, in 100%, and 75% of the mice, respectively. These results indicate that the green regimen is the most suitable to induce NASH-underlyning disease in our transgenic HCC model. At the conference, we will show in the diet-variants, the immune landscape of the livers and the results of our ongoing second experiment. Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants mimicking NASH characteristics that could be used for drug testing. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Philippe Bonnin, Benoit Rousseau, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Armand de Gramont, Eric Raymond, Sandrine Faivre, Valérie Paradis, Clarisse Eveno. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1636.

C3HeB/FeJ Mice mimic many aspects of gene expression and pathobiological features of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Combined use of alcohol in conventional chemical-induced mouse liver cancer model improves the simulation of clinical characteristics of human hepatocellular carcinoma.

Liver cancer is the one of most common types of cancer and the 2nd cause of cancer-associated mortalities worldwide. Establishing appropriate animal models of liver cancer is essential for basic and translational studies. The present study evaluated the effects of the combined use of alcohol with a conventional chemical-induced mouse liver cancer model. The treatment of alcohol/diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) in the mice of experimental groups resulted in a series of pathological changes in the liver. Liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma were identified, and this method used less time (1–5 months) for inducement compared with the conventional chemical-induced method alone. In addition, murine α-fetoprotein (mAFP) was expressed throughout and ultrastructural features met the criteria for liver cancer. Fatty degeneration of pancreas, reduced blood glucose levels, and increased spleen weight were observed. These results indicated that an AFP-secreting hepatocellular carcinoma model of BALB/c mouse was successfully developed. The disease process and morphological changes met the criterion of the liver cancer process. Therefore the model developed in the present study may be an ideal animal model for studying the occurrence and development of liver cancer.

Expression of adiponectin and its receptors in human hepatocellular carcinoma tissues

Objective To investigate the relationship among adiponectin,its receptors (AdipoR1,AdipoR2 and T-cadherin) and the pathological characteristics of human hepatocellular carcinoma (HCC).Methods The expression of Adiponectin,AdipoRl,AdipoR2 and T-cadherin was detected by immunohistochemistry in 42 cases of HCC tissues (tumor hepatic tissues and pericarcinomatous tissues).Results The expression of Adiponectin (2.88 ± 1.06 vs.5.98 ± 0.95),AdipoR1 (2.36 ± 1.38 vs.5.43 ±1.04),AdipoR2 (2.29±1.55 vs.5.14±1.22) andT-cadherin (2.17±1.34 vs.5.21 ±1.18) was decreased in tumor hepatic tissues compared to pericarcinomatous tissues.The expression of Adiponectin (2.57±1.03 vs.3.19±1.03),AdipoR1 (1.81 ±1.44 vs.2.90±1.09),AdipoR2 (1.48±1.47 vs.3.10 ± 1.18) and T-cadherin (1.76 ± 1.34 vs.2.57 ± 1.25) in HCC tissues with tumor thrombi was lower than in cancerous tissues without tumor thrombi.Adiponectin (2.30 ± 1.16 vs.3.06 ±0.98) and AdipoR2 (1.20 ± 1.48 vs.2.63 ± 1.43) expression in HCC tissues with satellite lesions was lower than in cancerous tissues without satellite lesions.The expression of T-cadherin in large size (＞5 cm) tumor tissues (1.59 ± 1.33) was lower than in the small size (≤5 cm) tumor tissues (2.56 ± 1.23).Conclusion The decreased expression of Adiponectin and its receptors may play a role in the intrahepatic metastasis of HCC. Key words: Carcinoma, hepatocellular; Adiponectin

Generation of a mouse model for studying the role of upregulated RTEL1 activity in tumorigenesis

Regulator of telomere length 1 (RTEL1) is a DNA helicase protein that has been demonstrated to be required for the maintenance of telomere length and genomic stability. It has also been found to be essential for DNA homologous recombination during DNA repairing. Human RTEL1 genomic locus (20q13.3) is frequently amplified in multiple types of human cancers, including hepatocellular carcinoma and gastrointestinal tract tumors, indicating that upregulated RTEL1 activity could be important for tumorigenesis. In this study, we have developed a conditional transgenic mouse model that overexpress mouse Rtel1 in a Cre-excision manner. By crossing with a ubiquitous Cre mouse line, we further demonstrated that these established Rtel1 conditional transgenic mice allow to efficiently and highly express a functional Rtel1 that is able to rescue the embryonic defects of Rtel1 null mouse allele. Furthermore, we demonstrated that more than 70% transgenic mice that widely overexpress Rtel1 developed liver tumors that recapitulate many malignant features of human hepatocellular carcinoma (HCC). Our work not only generated a valuable mouse model for determining the role of RTEL1 in the development of cancers, but also provided the first genetic evidence to support that amplification of RTEL1, as observed in several types of human cancers, is tumorigenic.

Abstract 2415: TGF-ß signaling inactivation inhibits the formation of hepatocellular carcinomas induced by loss of p53

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most deadly forms of cancer and results from the accumulation of mutated or altered tumor suppressor genes and/or oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and downstream signaling components. Inactivation of the TGF-ß signaling pathway and mutation of TP53, the gene for p53, are common in HCCs suggesting they may cooperate in HCC formation. Thus, we assessed whether inactivation of TGF-ß signaling, by deletion of the TGF-ß receptor type II, TGFBR2, affects the formation of HCCs arising secondary to loss of p53. Aim: To determine if loss of TGFBR2 and p53 cooperate in vivo to affect HCC formation. Methods and Results: Albumin-cre (Alb-Cre) transgenic mice were crossed with mice homozygous for floxed Tgfbr2 (Tgfbr2flx/flx) and/or floxed Trp53 (Trp53flx/flx) to generate mice lacking TGFBR2 and/or p53 in the liver. Deletion of Tgfbr2 alone did not induce liver tumors, while inactivation of both Tgfbr2 and Trp53 resulted in a subset of mice (19%) that developed liver tumors by 65 weeks of age. Surprisingly, deletion of p53 alone, in the context of intact TGF-ß receptors, resulted in an increased number of mice developing tumors (38%), as well as increased morbidity (42 weeks of age). Interestingly, in both genotypes, Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx and Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt, the mice develop both hepatocellular carcinomas (HCC) and cholangiocarcinomas (CC), suggesting these tumors originate from a common liver stem cell population. Quantitative RT-PCR analysis of tumor and non-tumor tissue demonstrated a subset of Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt (p53 null) tumors express significantly increased levels of alpha-fetoprotein mRNA, a clinical marker for HCC. Additionally, tumors from the Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt displayed increased TGFβ-1 protein levels as compared to tumors from Trp53 and Tgfbr2 null mice. Furthermore, increased phosphorylated ERK1/2 expression was also present in the tumors from the p53 null mice and correlated with a slight up-regulation in beta-catenin expression, another common molecular event observed in human HCC. Conclusions: The Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx mouse model recapitulates many molecular features of human HCC and demonstrates that TGF-ß signaling is oncogenic in the setting of loss of p53 in the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2415. doi:10.1158/1538-7445.AM2011-2415

Genetic and epigenetic characteristics of human multiple hepatocellular carcinoma

BackgroundMultiple carcinogenesis is one of the major characteristics of human hepatocellular carcinoma (HCC). The history of multiple tumors, that is, whether they derive from a common precancerous or cancerous ancestor or individually from hepatocytes, is a major clinical issue. Multiple HCC is clinically classified as either intratumor metastasis (IM) or multicentric carcinogenesis (MC). Molecular markers that differentiate IM and MC are of interest to clinical practitioners because the clinical diagnoses of IM and MC often lead to different therapies.MethodsWe analyzed 30 multiple tumors from 15 patients for somatic mutations of cancer-related genes, chromosomal aberrations, and promoter methylation of tumor suppressor genes using techniques such as high-resolution melting, array-comparative genomic hybridization (CGH), and quantitative methylation-specific PCR.ResultsSomatic mutations were found in TP53 and CTNNB1 but not in CDKN2A or KRAS. Tumors from the same patient did not share the same mutations. Array-CGH analysis revealed variations in the number of chromosomal aberrations, and the detection of common aberrations in tumors from the same patient was found to depend on the total number of chromosomal aberrations. A promoter methylation analysis of genes revealed dense methylation in HCC but not in the adjacent non-tumor tissue. The correlation coefficients (r) of methylation patterns between tumors from the same patient were more similar than those between tumors from different patients. In total, 47% of tumor samples from the same patients had an r ≥ 0.8, whereas, in contrast, only 18% of tumor samples from different patients had an r ≥ 0.8 (p = 0.01). All IM cases were highly similar; that is, r ≥ 0.8 (p = 0.025).ConclusionsThe overall scarcity of common somatic mutations and chromosomal aberrations suggests that biological IM is likely to be rare. Tumors from the same patient had a methylation pattern that was more similar than those from different patients. As all clinical IM cases exhibited high similarity, the methylation pattern may be applicable to support the clinical diagnosis of IM and MC.

Expressional and Mutational Analysis of Pro-apoptotic Bcl-2 Member PUMA in Hepatocellular Carcinomas

Deregulation of apoptosis is involved in mechanisms of cancer development. PUMA is a pro-apoptotic member of the Bcl-2 family and mediates p53-dependent and -independent apoptosis. The aim of this study was to investigate whether alterations of PUMA protein expression and somatic mutations of PUMA gene are characteristics of human hepatocellular carcinoma (HCC). We analyzed expression of PUMA protein in 20 HCCs using immunohistochemistry. Also, we analyzed mutation of the Bcl-2 homology 3 (BH3) domain of PUMA gene, which is an important domain in apoptosis function of PUMA by single-strand conformation polymorphism (SSCP) in 69 HCCs. PUMA protein expression was detected in both HCC cells and non-tumor hepatocytes in all of the 20 HCCs. In 10 of these HCCs, cancer cells showed higher PUMA expression than non-tumor (cirrhotic) hepatocytes of the same patients; whereas in the remaining 10, cancer cells and non-tumor hepatocytes showed similar levels. Mutational analysis revealed no PUMA BH3 domain mutation in the 69 HCCs, suggesting that PUMA BH3 domain mutation is not a direct target of inactivation in hepatocellular cancer development. The increased expression of PUMA in malignant hepatocellular cells relative to that in non-tumor hepatocytes suggests that PUMA expression may play a role in HCC development.

Downregulating the expression of heparanase inhibits the invasion, angiogenesis and metastasis of human hepatocellular carcinoma

Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC.

Expressional analysis of anti-apoptotic phospho-BAD protein and mutational analysis of pro-apoptotic BAD gene in hepatocellular carcinomas

Background It has become clear that, together with proliferation, deregulation of apoptosis plays a pivotal role in tumourigenesis. BAD is a pro-apoptotic Bcl-2 family protein and regulates the intrinsic apoptosis pathway. Phosphorylation of BAD inhibits the apoptosis function of BAD. Aims To investigate whether alteration of the phospho-BAD protein and somatic mutation of BAD gene are characteristics of human hepatocellular carcinoma. Patients and methods We analysed the expression of phospho-BAD in 20 hepatocellular carcinomas by immunohistochemistry. Also, we analysed the BAD gene for the detection of somatic mutations by a single-strand conformation polymorphism assay in 69 hepatocellular carcinomas. Results Phospho-BAD expression in the non-tumour hepatocytes was seen in all of the hepatocellular carcinomas, while the expression in the cancer cells was observed in 15% (3 of the 20) of the hepatocellular carcinomas. There was no somatic mutation of BAD Bcl-2 homology 3 (BH3) domain in the 69 hepatocellular carcinomas. Conclusions The data showed that loss of phospho-BAD expression, but not BAD gene mutation, is a feature of hepatocellular carcinomas. The decreased expression of phospho-BAD in the hepatocellular carcinoma cells compared to the non-tumour hepatocytes suggests that loss of phospho-BAD expression may play a role in hepatocellular tumourigenesis.

Genome-wide semiquantitative microsatellite analysis of human hepatocellular carcinoma: discrete mapping of smallest region of overlap of recurrent chromosomal gains and losses

Recurrent chromosomal gains at 1q, 6p, 8q, and 17q, or losses at 1p, 4q, 6q, 8p, 9p, 13q, 16q, and 17p are common features of human hepatocellular carcinoma (HCC). For precise determination of the shortest region of overlap (SRO), 49 HCC obtained at the time of surgery or autopsy were subjected to comprehensive microsatellite analysis by using 400 markers distributed at almost equal distances throughout the 22 autosomes and X chromosomes. Each allele showing imbalance was subjected to comparative duplex polymerase chain reaction using a retained allele as an internal control to determine whether the imbalance was the result of chromosomal gain or loss. The following SRO of recurrent chromosomal gains and losses were determined: −1p36.22∼p36.33, D1S450-D1S2893, 5.0 mega–base pairs (Mbp); +1q23.3∼q25.3, D1S2878-D1S2619, 16.9 Mbp; −4q21.2∼q24, D4S2964-D4S1572, 23.0 Mbp; −6q23.3∼qter, D6S292-qter, 34.7 Mb; −8p22∼p23.1, D8S549-D8S550, 4.8 Mbp; +8q12.2∼q24.13, D8S260-D8S514, 61.8 Mbp; −13q13.3∼q22.1, D13S218-D13S156, 35.6 Mbp; −16q22.1∼qter, D16S503-qter, 26.7 Mbp; and −17p12∼pter, D17S921-pter, 14.2 Mbp. Contrary to our initial expectations, many HCC showed major deletions or additions of chromosome arms, so that a number of genes were included in the SRO. Although some putative oncogenes or tumor suppressor genes mapped in these SRO may be important, relative copy number changes of numerous other genes may affect pathogenesis of HCC.

Ubiquitous Activation of Ras and Jak/Stat Pathways in Human HCC

Although the natural history and pathologic characteristics of human hepatocellular carcinoma (HCC) are well documented, the molecular pathogenesis of HCC remains poorly understood. Here, we define the role for Ras and Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathways in human HCC. Promoter and genomic status of Ras and Jak/Stat inhibitors were assessed in 80 HCCs by methylation-specific polymerase chain reaction and microsatellite analysis. Activation of Ras and Jak/Stat signaling pathways was determined by DNA sequencing, Western blot, and immunoprecipitation analysis. Suppression of Ras and Jak/Stat pathways in HCC cell lines was evaluated by viability and apoptosis assays. Activation of Ras and Jak/Stat pathways was enhanced in all HCCs when compared with nonneoplastic surrounding and normal livers coincidently with the suppression of at least 1 Ras (RASSF1A and/or NORE1A) and 2 Jak/Stat inhibitors (cytokine-inducible SH2-protein [CIS]; suppressor of cytokine signaling [SOCS]1, 2, 3; and SH2-containing phosphatases [SHP1]). HCC associated with cirrhosis showed significantly higher frequency of RASSF1A, CIS, and SOCS1 promoter methylation than HCC without cirrhosis (P < .002, P < .02, and P < .02, respectively). Furthermore, aberrant methylation of NORE1A and SOCS3 promoters was observed only in a subclass of HCC with poor survival, suggesting that inactivation of these 2 genes might be involved in HCC progression. Combined treatment of HCC cell lines with Ras and Jak/Stat inhibitors as well as with the demethylating agent zebularine induced a strong apoptotic response. These data demonstrate the ubiquitous activation of Ras and Jak/Stat pathways in HCC and suggest the potential use of Ras and Jak/Stat inhibitors and demethylating agents as therapeutic modality for human liver cancer.

Discrete breakpoint mapping and shortest region of overlap of chromosome arm 1q gain and 1p loss in human hepatocellular carcinoma detected by semiquantitative microsatellite analysis

Recurrent chromosomal gain at 1q is one of the most common features of human hepatocellular carcinoma (HCC), but how the gain at 1q contributes to hepatocarcinogenesis is still unclear. To identify the target genes, precise determination of the shortest region of overlap (SRO) and of breakpoints is necessary. Similarly, the role of loss at 1p, which is also a major cytogenetic aberration in HCC, needs to be determined. Fifty HCCs were examined with the aid of 59 microsatellite markers distributed throughout both arms of chromosome 1. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Alleles showing imbalance were subjected to multiplex PCR, using a retained allele as an internal control, to determine whether the imbalance was the result of chromosomal gain or loss. The SRO of the gains was defined as D1S2878-D1S2619 (1q23.-q25.3, 16.9 Mb), which involved 36 cases (72%). Gains in the number of copies of certain oncogenes within this region seemed to be critical for the pathogenesis of HCC. In contrast, the centromeric breakpoints of these gains varied, but they tended to occur mainly in the pericentromeric region (26 of 50 cases, 52%). Rearrangement of specific genes associated with the gains is unlikely. On the other hand, the SRO of deletion was defined as D1S2893-D1S450 (1p36.32-p36.22, 5.1 Mb). Four known putative tumor-suppressor genes (TP73, RIZ1, NBL1/DAN, and CDKN2C) were outside the SRO, suggesting the presence of other candidate genes with critical roles in hepatocarcinogenesis.

The establishment and characterization of the first canine hepatocellular carcinoma cell line, which resembles human oncogenic expression patterns

BackgroundHepatocellular carcinoma (HCC) is one of the most worldwide frequent primary carcinomas resulting in the death of many cirrhotic patients. Unfortunately, the molecular mechanisms of this cancer are not well understood; therefore, we need a good model system to study HCC. The dog is recognized as a promising model for human medical research, namely compared with rodents. The objective of this study was to establish and characterize a spontaneous canine tumor cell line as a potential model for studies on HCC.ResultsHistomorphological, biochemical, molecular biological and quantitative assays were performed to characterize the canine HCC cell line that originated from a dog with a spontaneous liver tumor. Morphological investigations provided strong evidence for the hepatocytic and neoplastic nature of the cell line, while biochemical assays showed that they produced liver-specific enzymes. PCR analysis confirmed expression of ceruloplasmin, alpha-fetoprotein and serum albumin. Quantitative RT-PCR showed that the canine HCC cell line resembles human HCC based on the measurements of expression profiles of genes involved in cell proliferation and apoptosis.ConclusionsWe have developed a novel, spontaneous tumor liver cell line of canine origin that has many characteristics of human HCC. Therefore, the canine HCC cell line might be an excellent model for comparative studies on the molecular pathogenesis of HCC.

Biological function of the differentially expressed genes bax-inhibitor-1 and insulin-like growth factor I receptor in prostate carcinomas

Recurrent chromosomal gains at 1q, 6p, 8q, and 17q, or losses at 1p, 4q, 6q, 8p, 9p, 13q, 16q, and 17p are common features of human hepatocellular carcinoma (HCC). For precise determination of the shortest region of overlap (SRO), 49 HCC obtained at the time of surgery or autopsy were subjected to comprehensive microsatellite analysis by using 400 markers distributed at almost equal distances throughout the 22 autosomes and X chromosomes. Each allele showing imbalance was subjected to comparative duplex polymerase chain reaction using a retained allele as an internal control to determine whether the imbalance was the result of chromosomal gain or loss. The following SRO of recurrent chromosomal gains and losses were determined: −1p36.22∼p36.33, D1S450-D1S2893, 5.0 mega–base pairs (Mbp); +1q23.3∼q25.3, D1S2878-D1S2619, 16.9 Mbp; −4q21.2∼q24, D4S2964-D4S1572, 23.0 Mbp; −6q23.3∼qter, D6S292-qter, 34.7 Mb; −8p22∼p23.1, D8S549-D8S550, 4.8 Mbp; +8q12.2∼q24.13, D8S260-D8S514, 61.8 Mbp; −13q13.3∼q22.1, D13S218-D13S156, 35.6 Mbp; −16q22.1∼qter, D16S503-qter, 26.7 Mbp; and −17p12∼pter, D17S921-pter, 14.2 Mbp. Contrary to our initial expectations, many HCC showed major deletions or additions of chromosome arms, so that a number of genes were included in the SRO. Although some putative oncogenes or tumor suppressor genes mapped in these SRO may be important, relative copy number changes of numerous other genes may affect pathogenesis of HCC.

Impaired interaction of mutant thyroid hormone receptors associated with human hepatocellular carcinoma with transcriptional coregulators.

Thyroid hormone (T(3)) exerts its many biological activities through interaction with specific nuclear receptors (TRs) that function as ligand-dependent transcription factors at genes that contain a thyroid hormone response element (TRE). Mutant TRs have been detected in human hepatocellular carcinoma cell lines and tissue, but their contribution to carcinogenesis has remained unclear. The interaction of four such mutant TRs (J7-TRalpha1, J7-TRbeta1, H-TRalpha1, and L-TRalpha1) with transcriptional coregulators has now been investigated. With the exception of J7-TRalpha1, which in the absence of T(3) exhibited transcriptional silencing activity with a TRE-reporter gene construct in transfected cells, the mutant TRs had little effect (compared with that of wild-type receptors) on transcriptional activity of the reporter gene in the absence or presence of T(3), of the transcriptional corepressors SMRT, NCoR or of the transcriptional coactivator SRC. Electrophoretic mobility-shift assays revealed that, in the presence of T(3), the J7-TRss1 mutant did not interact with SRC, whereas J7-TRalpha1 and H-TRalpha1 exhibited reduced abilities to associate with this coactivator and L-TRalpha1 showed an ability to interact with SRC similar to that of wild-type TRalpha1. The dominant negative activity of the mutant TRs in transfected cells appeared inversely related to the ability of the receptors to interact with SRC. Whereas J7-TRss1, H-TRalpha1, and L-TRalpha1 did not interact with SMRT, and NCoR. J7-TRalpha1 bind to corepressors but failed to dissociate from them in the presence of T(3). These aberrant interactions between the mutant TRs and transcriptional coregulators may contribute to the highly variable clinical characteristics of human hepatocellular carcinoma.

Characteristic Histologic Features of Human Hepatocellular Carcinoma with Mutant p53 Protein

To characterize hepatocellular carcinoma (HCC) cells with mutant (m) p53 protein histologically, we examined 68 main nodules and 20 accessory lesions of 72 patients with HCC who underwent hepatic resection between October 1990 and September 1993. Some sections were fixed in periodate-lysine-paraformaldehyde, embedded in OCT compound, and stained with the mouse monoclonal antibody PAb1801 to m-p53 protein by the immunoperoxidase technique with avidin-biotin complexes. Other sections were fixed in 20% buffered formalin, embedded in paraffin, and stained with the mouse monoclonal antibody DO-1 to m-p53 protein in the same way. Lesions in which cells had nuclei stained for m-p53 protein were defined as being positive for the protein; 25 of the 68 main nodules and 14 of the 20 accessory lesions were positive. Large main nodules were more likely to be positive than small ones. Microscopic examination showed that a larger proportion of poorly differentiated main nodules than well differentiated nodules were positive. Larger proportions of main nodules with extracapsular invasion, septa, portal thrombi, or intrahepatic metastases were positive than main nodules without these features. Accessory lesions that seemed to be metastatic were almost all positive, but few accessory lesions that seemed to be of multicentric occurrence were positive. Our results suggest that lesions with m-p53 protein had a high grade of malignancy and metastasized readily.