Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice.

Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.

DNA polymerase activity in muscle cultures.

Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion. Tne experiments described demonstrate that myogenic cells prevented from fusing have low levels of DNA polymerase activity. This is consistent with the notion that, in myogenic cultures, there is a population of mononucleated cells, the myoblasts, which have withdrawn from the mitotic cycle before fusion.

Hepatocyte differentiation in vitro: initiation of tyrosine aminotransferase expression in cultured fetal rat hepatocytes.

A fetal rat hepatocyte culture system has been used to study the molecular mechanisms of tyrosine aminotransferase (TAT) gene expression during development. It has previously been shown that TAT activity can be detected in 19-d, but not 15-d, gestation hepatocytes on the first day of culture (Yeoh, G. C. T., F. A. Bennett, and I. T. Oliver. 1979. Biochem. J. 180:153-160). In this study enzyme activity, synthesis, and mRNA levels were determined in hepatocytes isolated from 13-, 15-, and 19-d gestation rats maintained in culture for 1, 2, or 3 d and exposed to dexamethasone. TAT expression is barely detectable in 13-d gestation hepatocytes even after 3 d in culture. Hepatocytes isolated from 15-d gestation fetuses have undetectable levels of enzyme activity and synthesis on the first day of culture; both can be assayed by days 2 and 3. TAT mRNA levels in these hepatocytes, measured by hybridization with a specific cDNA, increase substantially during culture. TAT activity, synthesis, and mRNA are evident on the first and subsequent days of culture in 19-d gestation hepatocytes. Transcription measurements in isolated nuclei indicate that the increase in TAT mRNA in 15- and 19-d gestation hepatocytes is associated with an increase in transcription of the gene. Immunocytochemical studies demonstrated that the increase in TAT expression correlated with an increase in the proportion of hepatocytes expressing the enzyme, rather than a simultaneous increase in all hepatocytes. These results support the proposal that a subpopulation of 15-d fetal hepatocytes undergo differentiation in culture with respect to TAT.

Tyrosine aminotransferase gene expression in a temperature-sensitive adult rat liver cell line.

Regulation of tyrosine aminotransferase gene expression was studied in an adult rat hepatocyte line (RALA255-10G) which is temperature sensitive for the maintenance of the differentiated liver phenotype. Glucocorticoid hormones such as cortisol were necessary for expression of the aminotransferase gene. In the absence of these steroids, enzyme synthesis, activity, and mRNA accumulation were virtually abolished. In the presence of cortisol at 33 degrees C, RALA255-10G cells showed characteristics of malignant transformation and contained little tyrosine aminotransferase activity, synthesized low levels of this enzyme, and produced low levels of enzyme mRNA. At 40 degrees C, cells maintained in the presence of cortisol regained the normal, differentiated phenotype, and tyrosine aminotransferase synthesis and mRNA accumulation were greatly increased. This increase in aminotransferase synthesis paralleled the increase in the enzyme mRNA. However, after a temperature shift-up, tyrosine aminotransferase activity was increased only for the first 2 days, probably due to thermal inactivation of this enzyme at 40 degrees C. Dibutyryl cAMP alone was not sufficient to induce expression of the tyrosine aminotransferase gene, but it enhanced the induction caused by cortisol. Immunocytochemical studies revealed that the enhanced expression of the tyrosine aminotransferase gene at 40 degrees C and in the presence of cortisol or cortisol plus dibutyryl cAMP resulted from an increase in both the number of cells producing this enzyme and the quantity of tyrosine aminotransferase synthesized per cell.

Transformation-induced alterations in the regulation of tyrosine aminotransferase expression in fetal rat hepatocytes: changes in hormone inducibility and the DNase-hypersensitive site.

Regulation of expression of tyrosine aminotransferase (TAT) is examined in two cell lines (FRL) obtained by chemical transformation of cultured fetal hepatocytes derived from 19-day rat fetuses (FL19). Steroid induction of TAT is unaffected by transformation, while the response to cyclic AMP is attenuated. Consequently a synergistic response elicited by the simultaneous exposure of normal fetal hepatocytes to the inducers is almost abolished in FRL cells. FL19 and FRL are similar with respect to the negative effect of insulin on steroid induction, which is a response restricted to prenatal liver. Detailed examination of chromatin reveals that the attenuated effect of cyclic AMP is consistent with the lack of the DNase I-hypersensitive site located at about the cyclic AMP response element of the TAT promoter. From the studies, we conclude that transformation results in the modification of some aspects of TAT regulation, while others have been retained, and reflects the fetal pattern which is observed in normal embryonic hepatocytes.

Gene expression in clonally derived cell lines produced by in vitro transformation of rat fetal hepatocytes: isolation of cell lines which retain liver-specific markers.

The pattern of gene expression in fetal hepatocytes transformed in culture with a hepatocarcinogen (FRL cells) is studied with respect to a range of markers which are either developmentally regulated and/or shown to be expressed at high levels in hepatoma cells. The relative abundance of the respective mRNAs is determined and immunocytochemistry is used to detect the respective proteins in cultured cells. When compared with its normal counterpart, FRL cells retain the expression of transferrin, alpha 1-acid glycoprotein, gamma-glutamyltranspeptidase, and tyrosine aminotransferase at near normal levels, while expression of the liver-specific isoenzymes of pyruvate kinase (L form) and aldolase (B form) is reduced. The cell lines are different in that they fail to express albumin, alpha-fetoprotein, thiostatin and alpha 2-macroglobulin, and they express high levels of M2-pyruvate kinase and aldolase A, markers often found in abundance in hepatoma cells. Therefore transformation has resulted in different effects on different genes. Furthermore, it is of interest to find that the cells coexpress both forms of the pyruvate kinase isoenzymes which does not occur in the normal developing hepatocyte. These results indicate that it is possible to use this model to study changes which accompany transformation of fetal rat hepatocytes. The resulting cell lines have a stable phenotype and retain the changes which result from transformation even after extended passaging. This facilitates comparisons between the precursor cell and the tumor cell, both of which can be maintained under controlled conditions which exist in culture.

Transformation-induced changes in transferrin and iron metabolism in myogenic cells.

The uptake of transferrin and iron by cultured myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus (tsLA24) was compared with that of normal developing myogenic cells which were proliferating at the same rate as the transformed cells. The mechanism of transferrin and iron uptake was the same in the transformed cells as in normal myogenic cells and involved receptor-mediated endocytosis of transferrin. However, there were differences in transferrin receptor numbers and receptor function. The number of receptors in transformed cells was more than twice as great as in the normal cells largely due to increased surface receptor numbers. Despite this, the rate of iron uptake increased by only 20% in the transformed cells due to less efficient cycling of the transferrin receptors and less efficient release of iron from transferrin to intracellular sites. Some internalized iron was released from the transformed cells still bound to transferrin. A fast and a slow rate of transferrin exocytosis were identified in transformed cells, as in normal cells, indicating that there were at least two intracellular pathways for transferrin. The fast pathway predominated in the transformed cells, compared with an equal importance of the two pathways in the normal cells.

Factors affecting incorporation of (14C) leucine into albumin and transferrin by the liver in the postnatal rat.

The mechanisms responsible for the increase in incorporation of radioactive amino acid into albumin, transferrin and total soluble liver protein which occurs in the immediate postnatal period in the rat was investigated in rats delivered surgically in the last 2 days of gestation. The in vivo incorporation of [14C]leucine into the proteins in the liver was low at birth, but increased rapidly during the first half hour after delivery and then more gradually during the subsequent 4.5 h. Neonatal adrenalectomy had no effect on this pattern of results. Intraperitoneal administration of an amino acid supplement had little effect on [14C]leucine incorporation immediately after birth but increased incorporation at 0.5 h and eliminated the second phase of rising incorporation values between 0.5 and 5 h. The in vitro incorporation of 14C into albumin, transferrin and total protein by slices of the liver from animals immediately after delivery was as great as with slices from animals 5 h after delivery. It is concluded that the initial increase in synthesis of proteins in the liver in the first 0.5 h after delivery is probably due to an increase in the supply of metabolic energy due to improved oxygenation of the rats and that the slower increase in protein synthesis between 0.5-5.0 h results from an improved supply of amino acids to liver cells. It is unlikely that changes in the secretion of adrenal hormones in involved.

Transferrin receptors and iron uptake during erythroid cell development.

Experiments were performed to determine the level of transferrin receptors and rate of transferrin-bound iron uptake by various immature erythroid cell populations. Developing erythroid cells from the rat and mouse foetal liver at various stages of gestation were studied. In addition Friend leukaemic cells grown in culture were examined. The transferrin receptor level of Friend cells was similar to that of erythroid cells from the mouse foetal liver. During erythroid cell development the transferrin receptor level increased from about 300,000 per cell at the early normoblast stage to reach a maximum of about 8000,000 per cell on intermediate normoblasts. Further maturation of intermediate normoblasts was accompanied by a decline in the number of transferrin receptors, reaching a level of 105,000 in the circulating reticulocyte. The rate of iron uptake from transferrin during erythroid cell development was found to correlate closely with the number of transferrin receptors. In each of the immature erythroid cell populations studied the rate of iron uptake was about 36 iron atoms per receptor per hour. These results indicate that the level of transferrin receptors may be the major factor which determines the rate of iron uptake during erythroid cell development.

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures.

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.

Expression of alpha-fetoprotein by differentiating fetal rat hepatocytes in vitro.

Previous studies have established that, under appropriate conditions, fetal rat hepatocytes will differentiate in culture. This is characterized by the acquisition and loss of enzyme markers which are observed during liver development in vivo. The expression of alpha-fetoprotein (AFP), which declines during normal development, is examined in cultured hepatocytes derived from 15- and 19-day gestation rats. Secretion of AFP and relative levels of AFP mRNA and gene transcription were measured. Initially, AFP expression was greater in 15-day gestation hepatocytes, and in both instances AFP secretion and AFP mRNA decreased during culture. The decline in AFP expression by 15-day gestation fetal hepatocytes in vitro was not significantly altered by various manipulations of the culture conditions. It is proposed that cultured fetal hepatocytes continue to differentiate in vitro by repressing AFP expression while the expression of other liver-specific genes is being initiated. The fetal hepatocyte culture model therefore adequately reflects in vivo changes in developmentally regulated liver-specific genes.

The effect of dexamethasone on albumin production by fetal rat hepatocytes in culture.

Hepatocytes derived from 15 and 19-day gestation rats synthesize and secrete albumin during culture. Albumin secretion is maintained when the culture medium is supplemented with dexamethasone but declines in its absence. The fall in secretion rate correlates with the level of albumin messenger RNA in the respective cultures. Even when dexamethasone is present, the level of albumin production in 19-day gestation hepatocytes is 6 to 7 times greater than that observed in hepatocytes derived from 15-day gestation rats. Immunocytochemical studies were undertaken to establish whether the difference in secretion rate was due to a difference in the amount of albumin produced by all the hepatocytes of the respective cultures or whether there were fewer hepatocytes which were capable of synthesizing albumin in the less mature liver. The results indicate that albumin production is reduced in all hepatocytes when cultured in the absence of dexamethasone.

Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes.

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.

The effect of iron status on glyceraldehyde 3-phosphate dehydrogenase expression in rat liver.

The influence of iron status on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene transcription, mRNA levels and distribution was determined in whole liver samples from adult Wistar rats. While iron loading did not alter GAPDH expression, iron deficiency evoked a 2.3-fold increase in the steady-state level of liver GADPH mRNA, but did not affect gene transcription or intracellular localisation of the message. Therefore, the over-expression of GAPDH mRNA in iron deficiency is probably due to increased message stability.

Levels of 2,3-diphosphoglycerate in Friend leukaemic cells.

Most cells are thought to contain trace amounts of 2,3-diphosphoglycerate (DPG), as it acts as a cofactor in the interconversion of 2-phosphoglycerate and 3-phosphoglycerate by the glycolytic enzyme phosphoglyceromutase. DPG is synthesized from 1,3-diphosphoglycerate by the action of diphosphoglycerate mutase. Lowry et al. reported levels of 29 mumol DPG per kg wet weight brain tissue which is approximately 3 pmol per 10(8) cells, assuming that 1 g of brain tissue contains 10(9) cells. In contrast, erythroid cells contain 50-100 nmol DPG per 10(8) cells, depending on the species and the stage of development. This is of the order of a 1,000-fold more DPG compared with non-erythroid cells. In red cells DPG concentration modulates the binding of oxygen to haemoglobin. I show here that erythroid precurser cells also contain markedly raised levels of DPG.

Effect of 5-bromodeoxyuridine on the appearance of the liver isoenzyme of pyruvate kinase.

Hepatocyte cultures derived from 15-day foetal rats produce the liver form of pyruvate kinase (EC 2.7.1.40) only after 3 days of culture. The appearance of the liver form of the enzyme can be blocked by the addition of 5-bromodeoxyuridine on day 2 of culture, but not by addition on day 3 of culture. The reversibility of the action of 5-bromodeoxyuridine was shown when the inhibitor was added on day 2 and removed on day 4. By day 6 of culture the liver form of pyruvate kinase was detectable. The specificity of the action of 5-bromodeoxyuridine was monitored by following changes in the closely related embryonic form of the enzyme as a control. This was unaltered by the inhibitor.

A requirement for DNA synthesis in foetal hepatocyte differentiation. Effect of cytosine arabinoside on the appearance of the liver isoenzyme of pyruvate kinase.

In hepatocyte cultures derived from 15-day-old foetal rats, the appearance of the liver (L) form of pyruvate kinase is blocked when cytosine arabinoside is added on the 2nd day of culture. When added on the 3rd day of culture, the inhibitor of DNA synthesis does not prevent the appearance of the enzyme. If cytosine arabinoside is added on the 2nd day of culture and removed on the 4th day, the enzyme is detected by the 6th day of culture. The specificity of the action of cytosine arabinoside for the L form of pyruvate kinase is in contrast with the lack of effect observed on total protein synthesis and the activity of the embryonic (M2) form of the enzyme.

Phorbol myristate acetate (PMA) fails to prevent the appearance of markers associated with muscle and liver differentiation in culture.

The tumour promoter PMA has been shown to both prevent and induce differentiation of a variety of cell types in culture. The reason for its paradoxical effects is not known. However, it is clear that PMA alters the cell membrane and therefore it is possible that PMA may only be effective in instances where differentiation is accompanied by changes to the cell membrane e.g. myoblast fusion during myogenesis. In this study, its effects on myoblast fusion as well as the appearance of the muscle specific isoenzyme of creatine phosphokinase (M-CPK) which is not fusion dependent is examined. It is shown that M-CPK accumulates in myogenic cultures exposed to PMA although fusion is prevented. PMA is also tested in foetal rat hepatocytes which differentiate and acquire the enzyme tyrosine aminotransferase during culture. There is no evidence which suggests that this change is membrane dependent. The tumour promoter does not prevent the accumulation of tyrosine aminotransferase in cultured foetal rat hepatocytes.