Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3′ region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine.

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Biochemical microanalysis of alpha-glucosidase activity in preneoplastic and neoplastic hepatic lesions induced in rats by N-nitrosomorpholine

Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawley rats by oral administration of N-nitrosomorpholine (NNM) for 7 weeks at a concentration of 200 mg/l of drinking-water (stop model). Using a laser dissection technique and biochemical microanalysis, the activity of the lysosomal enzyme alpha-glucosidase was measured in glycogen storage foci emerging early, and in mixed or basophilic cell populations (foci and carcinomas) appearing later during hepatocarcinogenesis. In the liver tissue of normal appearance in both untreated controls and NNM-treated animals a slight gradient of alpha-glucosidase activity was observed leading from relatively high activities in zone 1 to lower activities in zone 3 of the liver lobule. In preneoplastic glycogen storage foci a considerable relative reduction in alpha-glucosidase activity was detected, suggesting that a decrease in the hydrolytic glycogen degradation contributes to the disturbance in phosphorylytic glycogen breakdown observed earlier in the majority of the glycogenotic foci. In contrast with glycogen storage foci, mixed and basophilic cell foci and particularly hepatocellular carcinomas showed a marked increase in alpha-glucosidase activity compared with that of normal liver tissue. The gradual enhancement in enzyme activity appeared to be closely related to the reduction in glycogen initially stored in excess during the later stages of hepatocarcinogenesis. The results support the concept that a fundamental shift in carbohydrate metabolism is characteristic of neoplastic transformation of hepatocytes.     

Decreased density of beta1-adrenergic receptors in preneoplastic and neoplastic liver lesions of F344 rats

There is some evidence that rodent hepatocarcinogenesis is accompanied by changes in the adrenergic responsiveness of liver cells to catecholamines. In this study, immunohistochemical expression of beta1-adrenergic receptors (beta1-ARs) has been examined in spontaneous and chemically induced preneoplastic and neoplastic liver lesions of female and male Fischer 344 rats. An antibody specific for beta1-AR subtype was used. The study was carried out on archival formalin-fixed and paraffin-embedded livers from rats used in a previous study of hepatocarcinogenesis. One control group given distilled water by gavage, and two experimental groups, one initiated with a single dose of diethylnitrosamine (DEN) and one initiated with DEN and continuously treated with phenobarbital (PB) were examined. Rats were sacrificed after 2, 4, 8 and 21 months of experimentation. All types of liver putative preneoplastic lesions examined (basophilic, glycogen-retaining, or mixed cell foci) show a lower density of beta1-ARs than the surrounding normal liver parenchyma, either in control and in DEN-treated or DEN+PB-treated rats. No immunostaining is detectable in several altered cell foci. Hepatocellular adenomas and hepatocellular carcinomas also show a very low density of beta1-ARs, extensive areas completely devoid of beta1-ARs being mingled with areas showing a weak immunostaining.     

Hepatocarcinogenesis in human liver

Due to the development of the imaging techniques and liver surgery, pathologists are encountered more frequently with preneoplastic liver lesions. Well-defined stages of human hepatocarcinogenesis have been distinguished recently. Dysplastic foci represent the earliest stage of this process. Small-cell dysplastic foci are tumor precursors, but the large-cell form of this lesion does not progress further. The next stage is the dysplastic nodule, this larger lesion can be recognized by imaging techniques and gross examination of the specimen. Low- and high-risk forms are distinguished based on the level of cytological and structural atypia. The small hepatocellular carcinomas have a diameter of less than 2 cm by definition. The small HCC of indistinctly nodular type is equivalent of in situ carcinomas in other organs and designated sometimes as early HCC. The small HCC of the distinctly nodular type can be interpreted as advanced cancer despite its small size. The distinction between these lesions can be facilitated by ancillary techniques. The so-called capillarization of the liver sinusoids during the progression is characterized by the increased expression of endothelial markers as CD31 and CD34. Immunostaining for CD44, beta-catenin and p53 has prognostic value. Molecular biological techniques reveal gradual epigenetic and DNA changes during the process of hepatocarcinogenesis. Global gene expression profiling of hepatocellular carcinomas may result in a new classification of this tumor and can reveal new potential therapeutic targets.     

Histochemical and stereological analysis of putative preneoplastic hepatic lesions

Histochemistry is a valuable tool in the analysis of altered hepatic foci. These lesions contain alterations in the activities of certain enzymes, including gamma-glutamyl transpeptidase (GGT), placental glutathione-S-transferase (PGST), glucose-6-phosphatase (G6Pase), and ATPase, or in certain cellular functions, such as the ability to store iron. The appearance of altered hepatic foci has been found to correlate with the later appearance of hepatocellular carcinomas in rodents. The markers PGST and GGT are the most sensitive at detecting altered hepatic foci induced by most chemicals, but are unable to detect altered hepatic foci induced by some agents, such as peroxisome proliferators. Other markers, such as ATPase or G6Pase, should therefore be used in combination with PGST or GGT in identifying altered hepatic foci. The strain of rat used and the type of diet fed also influence the number of altered hepatic foci induced and the enzyme markers seen. The number of foci per cm2 and the diameters of altered hepatic foci in histochemically-stained tissue sections can easily be quantified. The number of foci per cm2, however, does not give a reliable estimate of the number of altered hepatic foci induced because larger altered hepatic foci are more likely to be transected. The equations of quantitative stereology therefore should be used to transform the data to obtain the number of foci induced per cm3 or per liver, the average volume of individual foci, and the percent of the liver volume occupied by altered hepatic foci. In conclusion, the use of histochemistry to identify preneoplastic lesions and the use of quantitative stereology to estimate their number and volume allow accurate and sensitive quantitation of altered hepatic foci.     

Incidence and growth of methylcholanthrene-induced tumors in mice with altered immunological status

Summary BALB/c mice were treated s.c. with 3-methyl-cholanthrene (MCA), and tumor incidence and growth were followed for 9 months. Immunological status of mice was altered by various treatments. Thymectomized, lethally irradiated, bone marrow reconstituted mice served as T-cell deficient recipients. In order to suppres natural killer (NK)-cell/macrophage functions some mice were injected with silica particles; to enhance these functions some mice were given Corynebacterium parvum (CP). Silica and CP were given simultaneously with MCA to test their influence on the presumed function of surveillance of tumor incidence, and also 2 months after MCA to test their influence on the growth of greater numbers of transformed host cells. Almost all mice developed tumors at the inoculation site and at the end of the observation period there was no difference in tumor incidence among 9 experimental groups. However, in T-cell deficient mice we observed shorter tumor duration and earlier death than in normal mice. Silica particles appeared to enhance tumor growth but the differences compared to normal controls were not significant. A single injection of CP simultaneously with MCA caused earlier tumor appearance but also slowed its growth. In contrast, CP given 2 months after MCA significantly delayed the appearance of the tumors. In regard to the tumor growth immunosuppression had stronger effects in males than in females; the opposite was true for immunostimulation treatments. We concluded that immunological status does not influence long-term tumor incidence, but that both T-cell and NK-cell/macrophage compartments strongly influence the parameters of growth of chemically induced tumors, i.e., the immune and natural resistance mechanisms do not influence the frequency of de novo arising tumors but both can slow down tumor growth.Postgraduate and pregraduate students, University of Zagreb, Faculty of Medicine, Zagreb, Yugoslavia     

Genetic resistance to chemical hepatocarcinogenesis in the DRH rat strain

The carcinogen-resistant inbred rat strain DRH established from closed-colony Donryu rats by use of selective brother-sister mating over 20 generations under continuous feeding of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) maintains a highly resistant phenotype without carcinogen exposure for many years. We reported that the clonal expansion of preneoplastic glutathione S-transferase-P(GST-P)-positive foci induced by 3'-Me-DAB was less extensive in the liver of DRH rats than in the liver of susceptible strains, such as Donryu and F344, although levels of DNA adducts were comparable among these rats. Comparative studies of the events after initiation indicate that DRH rats are constitutionally less prone to cellular damage caused by continuous administration of 3'-Me-DAB than are parental Donryu rats. Consequently, the reduced growth response of the liver during the promotion stage may contribute to the low susceptibility to development of liver tumors. Genetic analysis of (F344 x DRH)F2 rats identified two quantitative trait loci, Drh1 on chromosome 1 and Drh2 on chromosome 4, which provide resistance to the development of GST-P-positive preneoplastic foci induced by 3'-Me-DAB during the early stage of its administration. The resistance to progression to hepatocellular carcinoma is affected solely by Drh2. These observations indicate that at least two genetic loci are critically involved in the steps leading to chemical hepatocarcinogenesis. The DRH rat is a useful experimental model with which to study genetic susceptibility and resistance to chemically induced liver cancers.     

Time-Dependent Effect of Hypoxia on Tumor Progression and Liver Progenitor Cell Markers in Primary Liver Tumors

Background & Aims

Expression of liver progenitor cell (LPC) characteristics has been proposed as a negative prognostic marker in primary liver tumors. Hypoxia has been linked to activation of the Notch pathway which is responsible for activation and proliferation of LPCs and hypoxia-induced LPC activation has been shown in hepatocellular carcinoma. Our aim was to elucidate the time-dependent effects of hypoxia on the LPC niche in hepatocellular carcinoma which could aid in determining a safe time frame for use of hypoxia inducing therapies.

Methods

We used dimethyloxaloylglycine to mimic a hypoxic reaction in mice by stabilizing hypoxia-inducible factor 1 alpha at three distinct time points in diethylnitrosamine induced hepatocarcinogenesis. LPC, metastasis and Notch pathway markers were determined by quantitative PCR and (immune)histochemistry (heamatoxillin-eosin, reticulin, Sirius red and cytokeratin 19 staining).

Results

Activating the hypoxia inducible pathway early in hepatocarcinogenesis resulted in an increased incidence of both cholangioma and hepatocellular lesions, associated with high expression of LPC, metastatic and Notch pathway markers. Adversely, activating the hypoxic response during tumor development resulted in decreased incidence of hepatocellular lesions and increased cholangioma incidence, with an unaltered gene expression profile of LPC-, Notch pathway- and metastatic markers. A hypoxic insult at advanced stages of hepatocarcinogenesis severely increased the expression of LPC characteristics, however without increased expression of actors of the Notch pathway and metastatic markers and minor changes in incidence of hepatocellular and cholangioma lesions.

Conclusion

Our results indicate that increased hypoxia at the onset of tumor development has detrimental effects on tumor progression; patients with HCC developed in a background of fibrosis/cirrhosis might therefore represent a more difficult treatment group. In contrast, hypoxia during tumor development appears to favor tumor outcome, highlighting the importance of early detection. Finally, hypoxia in advanced stages resulted in increased expression of LPC characteristics indicating poor outcome.     

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.     

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Five simian virus 40 (SV40)-hepatocyte cell lines were examined for tumorigenicity and the effect of in vitro passage on the expression of four liver-specific genes (albumin, transferrin, alpha 1-antitrypsin, and phosphoenolpyruvate carboxykinase), two oncogenes (c-Ha-ras and c-raf), and two genes associated with hepatocarcinogenesis (alpha-fetoprotein and placental-type glutathione-S-transferase). At low passage (12 to 22), all five cell lines expressed the four liver-specific genes at levels similar to those in the liver and were not tumorigenic or were weakly tumorigenic. At high passage (33 to 61), the cell lines formed carcinomas, and four out of five cell lines produced primary tumors that metastasized. At least two cell lines produced well-differentiated hepatocellular carcinomas that expressed liver-specific RNAs. Levels of expression of liver-specific genes changed with time in culture. Some of the changes in liver-specific gene expression in the tumor tissue (such as for the phosphoenolpyruvate carboxykinase gene) paralleled those that occurred with in vitro passage, while other changes (such as for the albumin gene) did not parallel those that occurred with in vitro passage. Correlations between enhanced expression of c-Ha-ras and tumorigenic potential and between the process of SV40 immortalization and induced expression of c-raf and glutathione-S-transferase-P were observed. Induction of alpha-fetoprotein was detected with in vitro and in vivo passage only in the CWSV14 cell line and was paralleled by diminished albumin expression. In conclusion, we developed a model system with five SV40-hepatocyte cell lines, tumors induced by them, and tumor cell lines to examine changes in gene expression that accompany the progression from a normal cell to a hepatocellular carcinoma. Because the SV40-hepatocyte cell lines and tumor cell lines remain highly differentiated and vary in the magnitude of expression of specific genes, they can be used to study the molecular mechanisms regulating gene expression, in particular those regulating specific genes associated with differentiation.     

Tumor cells do not arise frequently

Summary A hypothesis greatly influencing thinking and experimental work in tumor immunology is that many tumor cells arise daily in an organism. However, relatively low numbers of tumours become clinically manifest. This discrepancy has intially led to the hypothesis that the great majority of these potential tumors is killed by immune surveillance mechanisms. After some time, however, serious objections were raised against this function of the immune system. When the interest in immune surveillance faded away, investigators started to study natural resistance against the de novo arising tumor cells. In this paper the data on natural resistance (and immune surveillance) are reviewed. These data lead to the conclusion than an efficient natural resistance that could kill many de novo arising tumor cells is lacking. Taken together the data suggest that no tumors arise when immune surveillance and/or natural resistance are absent. This implies that tumor cells probably do not arise frequently. Acceptance of this conclusion leads to a reappraisal of the role of immune surveillance and natural resistance against tumor cells. This reappraisal will mean a shift from (a) the hypothesized failsafe mechanisms to (b) mechanisms that may or may not kill rarely arising tumor cells, depending for instance on the antigenicity of the tumor cells and their sensitivity to tumoricidal mechanisms.     

5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result.

The 5-methylcytosine content of nuclear DNA from nuclear hepatocellular tissues was determined during various phases of hepatic regeneration and carcinogenesis. DNA from premalignant nodules and primary hepatocellular carcinomas induced by exposure to acetylaminofluorene, as well as PHC induced by diethylnitrosamine was undermethylated by 20%, 45%, and 32.5% respectively. Since a 12.5% hypomethylation occurred during the DNA synthetic phase of hepatic regeneration, the effect of cell proliferation on DNA-methylation in malignancies was examined in transplantable hepatocellular carcinomas. The DNA from two transplantable hepatocellular carcinoma lines was less methylated than predicted rates of cell division in these tumors. This finding suggested that an aberration in endogenous DNA methylation may occur during neoplastic transformation.     

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring during hepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 100 kDa dimer composed of 54-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusion, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.     

Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells.

N-myc2 and insulin-like growth factor II (IGF-II) are coordinately overexpressed in the great majority of altered hepatic foci, which are the earliest precancerous lesions observed in the liver of woodchuck hepatitis virus carrier woodchucks, and these genes continue to be overexpressed in hepatocellular carcinomas (HCCs). We have investigated the function of these genes in woodchuck hepatocarcinogenesis by using a woodchuck liver epithelial cell line (WC-3). WC-3 cells react positively with a monoclonal antibody (12.8.5) against woodchuck oval cells, suggesting a lineage relationship with oval cells. Overexpression of N-myc2 in three WC-3 cell lines caused their morphological transformation and increased their growth rate and saturation density in medium containing 10% serum. Removal of serum from the medium increased cell death of the N-myc2-expressing lines, whereas cell death in control lines was minimal. The death of N-myc2-expressing WC-3 cells was accompanied by nucleosomal fragmentation of cellular DNA, and DAPI (4',6-diamidino-2-phenylindole) staining revealed condensation and fragmentation of the nuclei, suggesting that N-myc2-expressing WC-3 cells undergo apoptosis in the absence of serum. In colony regression assays, conducted in the absence of serum, control colonies were stable, while N-myc2-expressing colonies regressed to various degrees. Addition of recombinant human IGF-II to the serum-free medium blocked both cell death and colony regression in all the N-myc2-expressing lines. Therefore, coordinate overexpression of N-myc2 and IGF-II in woodchuck altered hepatic foci may allow cells which otherwise might die to survive and progress to hepatocellular carcinoma.     

Early Bioenergetic Changes in Hepatocarcinogenesis: Preneoplastic Phenotypes Mimic Responses to Insulin And Thyroid Hormone

Biochemical and molecular biological approaches in situ have provided compelling evidence for early bioenergetic changes in hepatocarcinogenesis. Hepatocellular neoplasms regularly develop from preneoplastic foci of altered hepatocytes, irrespective of whether they are caused by chemicals, radiation, viruses, or transgenic oncogenes. Two striking early metabolic aberrations were discovered: (1) a focal excessive storage of glycogen (glycogenosis) leading via various intermediate stages to neoplasms, the malignant phenotype of which is poor in glycogen but rich in ribosomes (basophilic), and (2) an accumulation of mitochondria in so-called oncocytes and amphophilic cells, giving rise to well-differentiated neoplasms. The metabolic pattern of human and experimentally induced focal hepatic glycogenosis mimics the phenotype of hepatocytes exposed to insulin. The conversion of the highly differentiated glycogenotic hepatocytes to the poorly differentiated cancer cells is usually associated with a reduction in gluconeogenesis, an activation of the pentose phosphate pathway and glycolysis, and an ever increasing cell proliferation. The metabolic pattern of preneoplastic amphophilic cell populations has only been studied to a limited extent. The few available data suggest that thyromimetic effects of peroxisomal proliferators and hepadnaviral infection may be responsible for the emergence of the amphophilic cell lineage of hepatocarcinogenesis. The actions of both insulin and thyroid hormone are mediated by intracellular signal transduction. It is, thus, conceivable that the early changes in energy metabolism during hepatocarcinogenesis are the consequence of alterations in the complex network of signal transduction pathways, which may be caused by genetic as well as epigenetic primary lesions, and elicit adaptive metabolic changes eventually resulting in the malignant neoplastic phenotype.     

Proteomic analysis of liver tissue from HBx‐transgenic mice at early stages of hepatocarcinogenesis

The hepatitis B virus X‐protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx‐transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2‐DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC‐MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3‐ketoacyl‐CoA thiolase, intestinal fatty acid‐binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx‐mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.