Neoplastic Liver Cells Research Articles

DNA methylation mediated downregulation of histone H3 variant H3.3 affects cell proliferation contributing to the development of HCC

Chromatin alterations brought by histone variants and modifications potentially regulate gene transcription from tumor initiation to progression. Histone H3.3 variant is one such epigenetic player important for disease progression and development. Though many studies have implicated H3.3 role in cancer progression and metastasis, its regulation, importance of specific modifications and chaperones have been not understood yet. We report DNA methylation mediated downregulation of histone H3 variant H3.3 in HCC and a concomitant increase in the level of the H3.2 variant. The loss of H3.3 in cancer tissues correlates with a decrease in the histone modifications associated with active transcription like H3K9/K14/K27Ac and H3K4Me3. The ectopic overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) as observed in HCC tissues. Notably, knockdown of P150, a subunit of CAF-1 leads to a cell cycle arrest in S-phase in a neoplastic rat liver cell line, possibly due to the decrease in the histone levels necessary for DNA packaging. Remarkably, modulation of H3.3 in pre-neoplastic rat liver cells lead to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Our data suggests, inhibition of DNA methylation and histone deacetylation leads to the restoration of histone H3 variant expression in tumor cells.

Immunohistochemical study of hepatocyte paraffin 1 antibody, monoclonal antibody 31, and cytokeratin 7 in primary and secondary liver carcinomas

Background Primary hepatic malignant epithelial neoplasia includes several different types of tumors, among which includes cholangiocarcinoma (CC), the second most frequent cancer of the liver after hepatocellular carcinoma (HCC). However, the most common hepatic epithelial malignant neoplasms are metastatic adenocarcinomas (AC). Hepatocyte paraffin 1 antibody (Hep Par 1) is a monoclonal antibody that reacts with normal and neoplastic liver cell. Anti-epithelial cell adhesion molecule monoclonal antibody 31 (MOC-31) is an antibody directed against a cell surface glycoprotein. Cytokeratin 7 (CK7) is expressed in normal and neoplastic epithelia. Aim To study the usefulness of the immunohistochemistry of Hep Par 1, MOC-31, and CK7 in differentiating primary and secondary carcinomas in the liver. Materials and methods The present study included 45 specimens from cases of primary and metastatic liver tumors, including 15 primary HCCs in the liver, four intrahepatic CCs, and 26 metastatic ACs in the liver. They were studied to evaluate Hep Par 1, MOC-31, and CK7 expression. Results The sensitivity of Hep Par 1 for HCC in the study group was 73.3%, whereas its specificity was 100%. The sensitivity of MOC-31 for AC in the study group was 96.4%, whereas its specificity was 91%. The sensitivity of CK7 for CC in the study group was 100%, whereas its specificity was 54%. Conclusion Hep Par 1 has very sensitive and specific immunostaining for HCC, MOC-31 is a useful marker for AC with high sensitivity and specificity, and CK7 is highly sensitive for CC but its specificity is low. A combination of Hep Par 1 and MOC-31 is the most useful combination of two antibodies for the distinction between HCC and AC.

Induction of Transcriptional Gene Silencing by Expression of shRNA Directed to c-Myc P2 Promoter in Hepatocellular Carcinoma by Tissue-Specific Virosomal Delivery.

Double-stranded RNA-mediated transcriptional gene silencing (TGS) has shown promising results over posttranscriptional gene silencing (PTGS) due to its long term and heritable nature. Various research groups have shed light on different mechanisms by which TGS operate. Some of these include histone modification, DNA methylation, or restriction of RNA polymerase binding onto the target gene's promoter. This serves as an added advantage since permanent c-Myc inactivation is critical for suppressing hepatocellular carcinoma (HCC). Inability to target cancer cells specifically, without affecting the normal cells, has been one of the biggest drawbacks of an effective cancer therapy. Therefore, we aimed to overcome this barrier by first generating tumor-specific transcriptional units expressing TGS inducing shRNAs against c-Myc's P2 promoter only in neoplastic liver cells. Secondly, we coupled this TGS inducing system with Sendai fusion virosomes for liver-specific delivery to minimize nonspecific side effects in vitro.

NF-kB-regulated exosomal miR-155 promotes the inflammation associated with arsenite carcinogenesis

In the cancer microenvironment, extracellular communication allows various types of cells to coordinate and execute biological functions. Emerging evidence indicates that exosomes, as mediators of cell communication, are involved in tumor progression. Little is known, however, about the mechanism by which exosomal miRNAs regulate inflammatory infiltration in arsenite-induced liver cancer. The present research aimed to determine if miRNAs secreted from arsenite-transformed human hepatic epithelial (L-02) cells are transferred into normal L-02 and THLE-3 cells, which are functionally active in the recipient cells. The results show that the exosomes from arsenite-transformed L-02 cells enhance miR-155 expression and the pro-inflammatory properties of normal L-02 and THLE-3 cells. Transformed cells transfer miR-155 into normal L-02 cells via exosomes. The inhibition of NF-κB by siRNA and inhibitor, which reduces miR-155 levels in exosomes derived from transformed L-02 cells, blocks inflammation. Arsenite-transformed cells secrete exosomes to enhance inflammation, but the inhibition of the synthesis of exosomes fails to stimulate inflammation. miR-155 is involved in exosome-mediated intercellular communication between neoplastic and normal liver cells. In addition, miR-155, IL-6, and IL-8 were over-expressed in the serum of arsenite exposure group. And there was a positive correlation between miR-155 and IL-6 or IL-8 levels. Further, exosomal miR-155 was up-regulated in the serum of arsenite exposure group. Thus, these results show that exosomes derived from transformed L-02 cells transfer miR-155 to surrounding cells, which induces pro-inflammatory activity of normal liver cells. The findings support the concept that exosomal miRNAs are involved in cell–cell communication during carcinogenesis induced by environmental chemicals.

Determination and Characterization of Tetraspanin-Associated Phosphoinositide-4 Kinases in Primary and Neoplastic Liver Cells.

Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells.

A study of structural differences between liver cancer cells and normal liver cells using FTIR spectroscopy

Since liver cancer seriously threatens human health, it is very urgent to explore an effective method for diagnosing liver cancer early. In this study, we investigated the structure differences of IR spectra between neoplastic liver cells and normal liver cells. The major differences of absorption bands were observed between liver cancer cells and normal liver cells, the values of A2955/A2921, A1744/A1082, A1640/A1535, H1121/H1020 might be potentially useful factors for distinguishing liver cancer cells from normal liver cells. Curve fitting also provided some important information on structural differences between malignant and normal liver cancer cells. Furthermore, IR spectra combined with hierarchical cluster analysis could make a distinction between liver cancer cells and normal liver cells. The present results provided enough cell basis for diagnosis of liver cancer by FTIR spectroscopy, suggesting FTIR spectroscopy may be a potentially useful tool for liver cancer diagnosis.

The effect of sodium selenite on liver growth and thioredoxin reductase expression in regenerative and neoplastic liver cell proliferation

Selenium in supra-nutritional doses is tumour-preventative in animal models and in humans. In this work, we have compared the effect of sodium selenite on tumour growth in a rat hepatocarcinogenesis model with the effect of sodium selenite on the regeneration of liver mass after partial hepatectomy. In the tumour model, 5 μg/mL sodium selenite in the drinking water reduced the rate of tumour growth for up to 12 months after initiation; the volume fraction of liver cancers was 14 ± 4% with a mean bromodeoxyuridine-index of 19 ± 11% in the treated rats compared to a volume fraction of 26 ± 7% with a mean bromodeoxyuridine-index of 42 ± 27% in the control rats. Despite its efficacy in reducing tumour growth, 5 μg/mL sodium selenite treatment did not affect the gain of liver mass or the rate of cell proliferation after partial hepatectomy. In the regenerating livers, the activity of the cytosolic selenoenzyme thioredoxin reductase (TrxR1) was briefly and transiently increased, an increase further potentiated by sodium selenite. TrxR1 was selectively over expressed in proliferating liver tumours in relation to the surrounding liver tissue in the tumour model, as shown using immunohistochemistry analyses. We suggest that sodium selenite is a suitable candidate for liver cancer prevention in patients with chronic liver diseases that are dependent on sustained liver regeneration due to its differential effects on neoplastic and regenerative cell proliferation. Furthermore, the over expression of TrxR1 in liver neoplasia can only partly be explained by increased growth.

Étiologies et évolution des hépatites granulomateuses : étude rétrospective de 21 cas consécutifs

Purpose To assess the etiologies and outcome of liver granulomatosis. Methods We analyzed all consecutive liver granulomatosis diagnosed in our internal medicine department from 2000 to 2008. Results Among 471 liver biopsies, 21 disclosed evidence of liver granulomatosis (4.5%), in sixteen women (76%) and five men, with a median age of 41 years. Thirteen were caucasians (62%). At the time of diagnosis, six (28.5%) had isolated abnormal liver function tests, and fifteen (71.4%) presented with clinical manifestations. The underlying cause was identified in 18 cases (85.7%). Eleven (52.3%) were systemic diseases: five (23.8%) primary biliary cirrhosis, two (9.5%) primary sclerosing cholangitis, two (9.5%) common variable immunodeficiency, one (4.7%) Sjögren's syndrome, and one (4.7%) Behçet's disease. Two (9.5%) patients had sarcoidosis. Three (14.3%) liver granulomatosis were of infectious origin (tuberculosis, schistosomiasis, and hepatitis C virus), two (9.5%) were neoplastic (Hodgkin's lymphoma and liver cell adenoma), and three (14.3%) were idiopathic. With a median of 38 months of follow-up, four patients (19%, two common variable immunodeficiency and two sarcoidosis) developed portal hypertension, independently of cirrhosis. One patient died of cryptococcosis. Conclusion In accordance with other European studies, systemic diseases are the main causes of hepatic granulomas. Liver granulomatosis related to common variable immunodeficiency and sarcoidosis are at risk of portal hypertension.

Re-expression of tumorigenicity after attenuation of human synaptotagmin 13 in a suppressed microcell hybrid cell line

Human chromosome 11p11.2 contains a putative liver tumor suppressor locus that was identified using a microcell hybrid cell line-based model of tumor suppression. Transcription mapping of suppressed microcell hybrid cell lines suggests that human SYT13 represents a strong candidate for the 11p11.2 tumor suppressor gene. Other evidence suggests that the putative 11p11.2 tumor suppressor gene (SYT13 or some other) may modulate the tumorigenic potential of neoplastic liver cell lines through direct or indirect regulation of the rat Wt1 tumor suppressor gene. To characterize a functional role for SYT13 in liver tumor suppression, we employed RNAi to attenuate SYT13 expression in a suppressed microcell hybrid cell line (GN6TF-11neoCX4). SYT13-attenuated cells display aggressive phenotypic properties that are similar to or indistinguishable from the parental tumor cells (GN6TF), including altered cellular morphologies, disrupted contact inhibition, elevated saturation densities, restoration of anchorage-independent growth and increased tumorigenicity in vivo. Moreover, SYT13 attenuation and re-expression of tumorigenicity in GN6TF-11neoCX4-derived cell lines was accompanied by a significant decrease of Wt1 expression. In contrast, the phenotypic properties of scrambled-control cells were similar to the suppressed microcell hybrid cells and Wt1 expression was unaffected. These observations combine to establish that: i) human SYT13 functions as a liver tumor suppressor gene that complements a molecular defect in GN6TF rat liver tumor cells resulting in a normalized cellular phenotype in vitro and suppression of tumorigenicity in vivo; ii) RNAi-mediated attenuation of SYT13 expression restores the neoplastic phenotype of GN6TF-11neoCX4 microcell hybrid cells, consistent with the function of a liver tumor suppressor gene; and iii) loss of Wt1 expression is important for the re-establishment of tumorigenic potential by GN6TF-11neoCX4 microcell hybrid cells after attenuation of SYT13.

Novel endothelial cell markers in hepatocellular carcinoma

Hepatocellular carcinoma is characterized by hypervascularity and a propensity for vascular invasion. Detailed analysis of complementary DNA (cDNA) microarray global gene expression data and further validation on a smaller independent sample set by reverse transcription-polymerase chain reaction established the presence of two endothelial gene clusters in hepatocellular carcinoma. Cluster I, consists of 20 cDNA clones, representing 15 unique genes. Cluster II consists of nine unique genes. The expression of the cluster I genes appeared to be significantly upregulated in hepatocellular carcinoma compared with normal liver, cirrhotic liver, or nontumor liver tissues adjacent to the hepatocellular carcinoma. The pattern of gene expression of cluster I genes correlated positively with the ‘proliferation gene cluster’ and ‘stromal cells cluster 2‘. Expression of cluster II genes, in contrast, was not significantly different between hepatocellular carcinoma and non-neoplastic liver tissues. Studies conducted to localize the protein products of these genes by immunohistochemical staining of tissue arrays with up to 350 cores of tissues, and by in situ hybridization led to the discovery of novel sinusoidal endothelial cell markers in hepatocellular carcinoma: podocalyxin-like and regulator of G protein signaling-5. Our results underscore fundamental differences not only between neoplastic vs non-neoplastic liver cells but also between the hepatic sinusoidal endothelium of hepatocellular carcinoma and normal liver.

Cellular retinol-binding protein-1 in hepatocellular carcinoma correlates with β-catenin, Ki-67 index, and patient survival

The cellular retinol-binding protein-1 (CRBP-1) plays a key role in the esterification and intercellular transfer of retinol. By in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy (CLSM), we show that, in normal liver, CRBP-1 is strongly expressed in the cytoplasm of hepatic stellate cells (HSCs) and myofibroblasts (MFs) with only low CRBP-1 levels in hepatocytes. By contrast, in 196 hepatocellular carcinoma (HCC) specimens CRBP-1 expression in MFs was down-regulated in 83%. Patients with high CRBP-1 expression in MFs had a significantly higher 2-year survival as compared with patients with low CRBP-1 expression (52% vs. 29%, respectively; P = .034). An aberrant nuclear CRBP-1 accumulation resulting from cytoplasmic invagination was found in 29% of HCCs. Nuclear CRBP-1 staining correlated positively with a favorable tumor stage (Okuda stage I; P = .01) and negatively with the Ki-67 + proliferation fraction (PF). A Ki-67 + PF of ≥10% was associated with a lower 2-year survival probability as compared with patients with a Ki-67 + PF of <10% (12% vs. 40%, respectively; P = .015). Prognosis did not correlate with the nuclear β-catenin expression. There was, however, a close correlation between nuclear CRBP-1 inclusions and nuclear β-catenin staining in HCCs ( P = .008), suggesting a cross talk between CRBP-1 and the Wnt/wingless signal transduction pathway. In conclusion, our findings demonstrate that CRBP-1 detection may be useful for the discrimination between nonneoplastic and neoplastic liver cells and suggest that modulation of CRBP-1 expression in HCCs contributes to tumor growth and progression via retinoid-mediated signaling and disruption of cellular vitamin A homeostasis. (H epatology 2003;38:470-480.)

Function of a genetically modified human liver cell line that stores, processes and secretes insulin.

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic beta cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for beta cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic beta cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic beta cells and become a therapy for the treatment of type I diabetes.

New Synthetic Derivatives from Peroxisomicine A1 and their Biological Activity

Peroxisomicine A1 (PA1) is a dimeric anthracenone obtained from plants of the genus Karwinskia. Toxicological studies have demonstrated a selective toxicity of peroxisomicine A1 on various human tumor cell lines in vitro. For this reason it has been considered as a possible antineoplastic agent. This article describes the chemical manipulation of this compound with the purpose of finding a possible relationship between the structure and biological activity. Derivatization procedures on the phenolic OH groups, carbonyl group and aromatic ring are presented. Six new compounds were obtained and purified by several chromatographic methods and the pure products were identified and characterized by UV, IR, MS, and 1 H and 13 C NMR. To evaluate the biological activity of the derivatives, the selective cytoxicity of the compounds on normal and neoplastic human liver cells lines and their effect on catalase activity were analyzed. Cytotoxicity of compounds 2 - 5 suggests that phenolic groups are necessary for the biological action reported for PA1. On the other hand, compound 6, substituted on the aromatic ring, did not show a significant difference in the selective cytotoxicity assay when compared to peroxisomicine A1.

Increased levels of cytosolic thioredoxin reductase activity and mRNA in rat liver nodules

Background/Aims : Thioredoxin reductase, a redox active enzyme, is induced in several tumors. This study focuses on the presence of and subcellular localisation of thioredoxin reductase in a tumor model where neoplastic lesions are selected by their resistance to the toxic effects of the promotor. Methods : Liver nodules produced by intermittent feeding of 2-acetylaminofluorene to male Wistar rats were analyzed for thioredoxin reductase (TrxR) activity and mRNA. Results : This activity was increased 3.5-fold in the cytosol but decreased 60% in the mitochondrial fraction compared to the liver of age-matched untreated animals. Only traces of activity were observed in the microsomal, plasma membrane and nuclear fractions from normal liver or nodules. The level of TrxR mRNA was 3-fold higher in nodules than in normal rat liver. Furthermore, the total level of SH groups in homogenates was 2-fold higher in the case of the nodules. Conclusions : These findings indicate that the thioredoxin system makes an important contribution to the resistant phenotype of the neoplastic liver cell, which conveys a growth advantage of significance for tumor progression.

Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholine and dehydroepiandrosterone.

Preneoplastic liver foci and neoplasms of different morphological phenotypes were induced in rats with N-nitrosomorpholine (NNM; 120 mg/l in drinking water for 7 weeks) and the peroxisome proliferator dehydroepiandrosterone (DHEA; 0.6% in the diet for up to 84 weeks). Preneoplastic glycogen storage foci (GSF) occurred mainly upon treatment with NNM, and amphophilic cell foci (APF) were mainly observed in rats treated with DHEA alone or in combination with NNM. The 2 types of lesions belong to 2 different cellular lineages, the glycogenotic/basophilic lineage and the amphophilic lineage, which are characterized by distinct patterns of alterations in key enzymes of energy metabolism. Whereas in GSF enzymes of glucose metabolizing pathways were modified (increase in glucose-6-phosphate dehydrogenase and pyruvate kinase, decrease in glucose-6-phosphatase), APF mainly demonstrated alterations in mitochondrial enzymes (increase in cytochrome c oxidase, succinate dehydrogenase and glycerol-3-phosphate dehydrogenase) and, to a lower extent, in peroxisomal enzymes (increase in peroxisomal hydratase and acyl-CoA oxidase). The alterations in enzyme expression reflect an insulinomimetic effect in GSF and a thyromimetic effect in APF. Neoplasms resulting from APF show a more differentiated phenotype than those arising from GSF. We suggest that the different and in many aspects opposite effects of the 2 carcinogens on key enzymes of distinct pathways of energy metabolism modulate the process of neoplastic liver cell transformation and result in phenotypically different preneoplasias and neoplasias reflecting different cellular lineages.

Apoptosis in the liver and its role in hepatocarcinogenesis.

Apoptosis seems to be the predominant type of active cell death in the liver (type I), while in other tissues cells may die via biochemically and morphologically different pathways (type II, type III). Active cell death is under the control of growth factors and death signals. In the liver, endogenous factors, such as transforming growth factor beta 1 (TGF-beta 1), activin A, CD95 ligand, and tumor necrosis factor (TNF) may be involved in induction of apoptosis. Release and action of these death factors seems to be triggered by exogenous signals such as withdrawal of hepato-mitogens, food restriction, etc. During stages of hepatocarcinogenesis, not only DNA synthesis but also apoptosis gradually increase from normal to preneoplastic to adenoma and carcinoma tissue. Also, in human carcinomas, birth and death rates of cells are several times higher than in surrounding liver. (Pre)neoplastic liver cells are more susceptible than normal hepatocytes to stimulation of cell replication and of cell death. Consequently, tumor promoters may act as survival factors, i.e., inhibit apoptosis preferentially in preneoplastic and even in malignant liver cells, thereby stimulating selective growth of (pre)neoplastic lesions. On the other hand, regimens favoring apoptosis and lowering cell replication may result in selective elimination of (pre)neoplastic cell clones from the liver. Finally, we have studied the first stage of carcinogenesis, namely the appearance of putatively initiated cells after a single dose of the genotoxic carcinogen N-nitrosomorpholine (NNM). Most of these cells were found to be eliminated by apoptosis, suggesting that initiation, at the organ level, can be reversed at least partially by preferential elimination of initiated cells. These events may be regulated by autocrine or paracrine actions of survival factors.

Karyotype evolution of the clonal rat liver cell line CL 52 during progression in vitro and in vivo

Liver cell line CL 52, derived from a diethylnitrosamine-treated rat at the stage of preneoplasia/early neoplasia, had an inconspicuous 2n karyotype when analyzed 6 months after in vitro propagation. Malignant progression was accompanied by cytogenetic alterations of chromosomes 1, 3, and 11. In addition, trisomy of chromosomes 4, 6, and 7 led to a significant reduction of the tumor latency period after retransplantation. During 4 years of cytogenetic observation, the once clonal 2n population showed a characteristic karyotype evolution: loss of diploidy, occurrence of polyploid sidelines, deletions followed by unbalanced rearrangements, clonal diversification, and selection of the in vitro most rapidly growing or in vivo most malignant cell type. The karyotype alterations in the four sublines of CL 52 are discussed with special reference to oncogenesis-related genes assigned to the involved rat chromosomes 1, 3, 11, 12, 4, 6, 7, 10. The observed karyotype evolution of this cell line exemplifies genetic/chromosome instability of carcinogen-induced preneoplastic/early neoplastic liver cells and provides a tool for analyzing, under controlled conditions, stage-dependent sequences of molecular genetic alterations in liver carcinogenesis.

Cytochemical detection of a class 3 aldehyde dehydrogenase in human hepatocellular carcinoma.

The development of hepatocellular carcinoma in rodents treated with different chemical compounds is associated with the appearance in the cytosol of neoplastic liver cells of an unusual aldehyde dehydrogenase isozyme of class 3 (ALDH-3) which is very active with aromatic aldehydes. This tumor-associated isozyme is readily detected by enzyme cytochemistry using the substrate benzaldehyde with NADP as coenzyme. To determine whether human hepatocellular carcinomas express ALDH-3, the activity of this isozyme was examined in frozen sections from 68 echo-guided human liver biopsies. In 54 cases the guided biopsy was performed on one or more nodules suggestive for hepatocellular carcinoma found at ultrasonography within the liver parenchyma. The remaining 14 patients were affected by chronic active hepatitis or cirrhosis. An intense enzymatic activity was ascertained in 5 out of 36 hepatocellular carcinomas. In non-neoplastic liver, in macroregenerative nodules and in metastatic adenocarcinomas enzymatic activity was not detectable. ALDH-3-positive tumors were typical hepatocellular carcinomas (histological grade II and III). These results suggest that ALDH-3 is a phenotype associated with malignancy in human liver tumors.

Elimination of preneoplastic rat liver cells via induction of apoptosis by transforming growth factor β1

Transforming growth factor-B1 (TGFBI) is a potent growth inhibitor and inducer of apoptotic cell death in hepatocyte cultures and in regressing rat liver. Therefore, TGFBI might provide a powerful new agent for therapy of liver cancer. First we determined endogeneous hepatic TGFBI levels through acid ethanol extraction and quantification in the CC164-bioassay. The concentration of the TGFBI polypeptide in female Wistar rats appeared constant during cyproterone acetate-induced hyperplastic growth of the liver and during the subsequent regression of liver hyperplasia by apoptosis. In hepatocellular carcinoma (obtained after a 17 months treatment of male Wistar rats with the peroxisome proliferator nafenopin, 100mg/kg b.w./day) TGFBI levels were not different from that in the surrounding tissue. This suggests that (pre)neoplastic liver cells still might respond to large doses of exogeneously supplied TGFBI. Then we tested the possible therapeutic benefit of TGFBI. Hepatocarcinogenesis was initiated by a single dose of 7,12-dimethylbenz(a)anthracene (130mg/kg b.w.) in female Wistar rats. After recovery for few weeks rats received eight i.v. applications of TGFB1 (40~g /kg b.w./day) which induced apoptosis more in preneoplastic than in unaltered cells. AS a consequence, number and size of preneoplastic liver foci decreased considerably. Coadministration of the anti-estrogen tamoxifen (8mg/kg b.w./day) exerted an overadditive effect on the growth inhibiting and apoptosis stimulating activities of TGFBI in liver preneoplasia. In conclusion, TGFBI in combination with tamoxifen eliminates preneoplastic hepatocytes through apoptosis and may provide great benefit for cancer therapy.

Number of simultaneously expressed enzyme alterations correlates with progression of N-ethyl-N-hydroxyethylnitrosamine-induced hepatocarcinogenesis in rats.

Preneoplastic and neoplastic liver cell lesions, induced by EHEN (N‐ethyl‐N‐hydroxyethylnitrosamine) in rats, were investigated to establish the numbers of simultaneously expressed altered enzyme phenotypes within the lesion cells. The lesions were divided into 5 classes on the basis of altered expression in one or more of the following 5 enzymes: glutathione S‐transferase placental form, glucose‐6‐phosphate dehydrogenase, glucose‐6‐phosphatase, adenosine triphosphatase, and γ‐glutamyl transpeptidase. Class 1 lesions contained cells expressing one altered enzyme. Similarly, class 2, 3, 4 and 5 lesions had cells simultaneously expressing 2, 3, 4, and 5 enzyme alterations, respectively. Four histopathological categories of lesions, ACF (altered cell foci) (274 lesions), HN (hyperplastic nodules) (47 lesions), HCC (hepatocellular carcinomas) (99 lesions) and THC (transplanted hepatocellular carcinomas) (5 lesions) were studied. Proliferation potential was assessed in terms of 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation. The distribution profiles of classes 1 to 5 showed a clear reciprocal change from low class (1 to 2 enzymes) predominance in ACF to high class (4 to 5 enzymes) predominance in HN. Increase of BrdU labeling indices was clearly correlated with progression from HN to HCC. Only a small population of class 5 ACF showed a high BrdU labeling index, indicating particular potential for further development. Thus, the stages of EHEN‐induced neoplasia were found to be characterized by gradual increase in the number of altered enzyme phenotypes, with acquisition of proliferative potential being associated with further progression towards malignant conversion.