Expression of differentiated functions in dexamethasone-resistant hepatoma cells

Abstract. The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.     

Expression of differentiated functions in dexamethasone-resistant hepatoma cells

The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.     

THE FUNCTION OF HEAT-SHOCK PROTEINS IN STRESS TOLERANCE

We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid-resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance. Our experiments show that clone 2 cells were not protected from the killing effect of heat by pretreatment with sodium arsenite, whereas Faza 967 cells were. These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells. Stable heat-resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat-inducibility of hsp68. These results suggest that several hsps are needed to protect the critical biological processes at high temperature. The heat-resistant hepatoma cells also became resistant to several anticancer drugs. The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P-glycoprotein. Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P-gp may participate in protection against environmental stress such as heat.     

Enhancement of aflatoxin B1 cytotoxicity in differentiated rat hepatoma cultures by a prior glucocorticoid treatment of the monolayer.

The cytotoxic effect of aflatoxin B₁ on cultures of a differentiated rat hepatoma cell line, Faza 967, has been evaluated by scoring the surviving colonies two weeks after briefly exposing the freshly plated cells to the mycotoxin. At the lowest concentration, aflatoxin B₁ exhibits no toxicity, unless the cultures have been pretreated with dexamethasone. HF-1, an hepatoma hybrid cell line exhibiting extinction of the hepatic functions and HF1-4, its subclone, that reexpresses all of these functions, have been compared. A 6hrs exposure to 60ng/ml aflatoxin B₁ is not toxic for HF₁ even after an hormonal treatment, while dexamethasone enhances the effect on HF₁-4. Glucocorticoïds have been shown previously to induce, in the differentiated clones, the hydroxylation of bile acid - a cytochrome P-450-mediated reaction ; in contrast, 3-methylcholanthrene, an inducer of benzopyrene hydroxylase in hepatoma cultures, is without effect on bile acid metabolism and on aflatoxin B₁ cytotoxicity. These results suggest that in the differentiated hepatoma cells, aflatoxin B₁ is converted into a cytotoxic metabolite by a glucocorticoïd-induced monooxygenase belonging to the cytochrome P-450-related group.     

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.     

Angiotensinogen production by rat hepatoma cells in culture and analysis of its regulation by techniques of somatic cell genetics

Angiotensinogen was synthesized by cells derived from the Reuber H35 rat hepatoma. Independent clones produced similar amounts of angiotensinogen, which corresponded to about four times more than expected for normal hepatocytes. The protein was secreted rapidly but could be visualized within cells using immunofluorescence. For one clone, it is shown that maximal angiotensinogen synthesis occurred during mid-exponential growth. Somatic cell genetics techniques have been used to investigate the regulation of angiotensinogen expression. Eleven clones of dedifferentiated variant hepatoma cells that failed to produce most or all of the liver specific proteins analyzed including albumin fell into two groups: Seven clones produced only 1-3% as much angiotensinogen as the differentiated clones, and four showed a reduction to 10-30%. Clones of the latter class were the only ones among the eleven analyzed that retained the potential to give rise to revertants, showing restoration of the differentiated state. All revertants fully restored angiotensinogen production, but only some of them re-expressed albumin. Somatic hybrids between differentiated hepatoma cells and one of the variants showed a substantial reduction in angiotensinogen production, whereas for some clones, albumin synthesis was fully maintained. These results show that regulation of the expression of angiotensinogen and of a second serum protein, albumin, was independent and that angiotensinogen synthesis was a faithful indicator of the general differentiation profile of all classes of clones.     

Heat-shock response of rat hepatoma variant cells

The effect of mild heat shock on protein synthesis was examined in differentiated and dedifferentiated, glucocorticoid-sensitive and resistant clones of H4IIEC3 rat hepatoma cells by one- and two-dimensional gel electrophoresis of [35S]methionine-labeled proteins. Among the major heat-shock proteins, five were induced in all hepatoma clones. Certain members of the HSP70 family and the corresponding mRNAs were only slightly inducible in the glucocorticoid-resistant variants, but were strongly inducible in the sensitive ones. Three other proteins lacked heat inducibility in the dedifferentiated clones. The constitutive level of one major heat-shock protein was elevated in all dedifferentiated variants. These results show that the stage of differentiation influences the expression of heat-shock genes of hepatoma cells. We found no correlation between the elevated constitutive or induced level of heat-shock proteins and heat resistance.     

Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells.

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin . The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.     

Efficient one-step selection of hepatoma cell variants of a variety of phenotypes by use of aflatoxin B1

We have developed a method to select for rat hepatoma cells that fail to express hepatocyte-specific functions. Well-differentiated cells descended from the H4IIEC3 hepatoma line express aldrin epoxidase (AE) activity, an indicator of the liver-specific forms of cytochromes P450 and, concurrently, are able to activate the procarcinogen aflatoxin B1 (AFB1) into highly toxic metabolites. Thus, differentiated hepatoma cells are highly sensitive to AFB1, while dedifferentiated derivatives, which fail to express AE activity, are resistant. Exposure of differentiated Fao cells to 10 microM AFB1 for 24 h permits the isolation, at a frequency of 5 x 10(-5), of resistant colonies that exhibit strongly reduced AE activity. Strikingly, various morphological types can be observed. In more than 90% of the colonies, cells are morphologically similar to the original differentiated cells and accumulate all liver-specific mRNAs examined in amounts comparable to Fao cells. Moreover, they are able to carry out gluconeogenesis, as judged by their capacity to grow in glucose-free medium. For a minor fraction of colonies, the cells exhibit nonhepatic morphology. These cells fail to express three or more of the liver functions and are not able to proliferate in glucose-free medium. Our results demonstrate that the use of AFB1 constitutes a simple and efficient single-step selective method for obtaining variant hepatoma cells of a wide variety of phenotypes.     

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.     

Dedifferentiated variants of a rat hepatoma: analysis by cell hybridization

Two independent dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma, produce trans-acting diffusible substances(s) that extinguish the expression of liver-specific proteins when hybridized with a well-differentiated cell line of the same origin (Fao and Fu5-5, respectively). H5 x Fao hybrids show total and stable extinction of four liver functions and clonal variability in the expression of three others. FaoflC2 x Fu5-5 hybrids are initially flat (like FaoflC2 cells), and die in glucose-free medium where survival requires expression of hepatic gluconeogenic enzymes, but then evolve to hepatoma-like and finally round morphology; these latter cells express all liver functions analyzed including the gluconeogenic enzymes. Two exceptional clones that remained flat long enough for complete analysis showed extinction of all hepatic functions not expressed by FaoflC2 cells. We conclude that this transitory extinction reflects the action and then loss of extinguishing factor(s) contributed by FaoflC2. When crossed with BW1-J mouse hepatoma cells. FaoflC2 causes stable extinction of mouse aldolase B. We propose that production of extinguishing factor(s) is the rule for dedifferentiated variants.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.