Expression of differentiated functions in hepatoma cell hybrids. II. Aldolase

A study of aldolases in rat hepatoma clones and subclones has revealed that they synthesize all three forms of aldolase monomers: A (the ubiquitous glycolytic isozyme), B (the form characteristic of the liver) and C, and that in vitro–in vivo passage results in a reversible modulation in aldolase A activity. Three kinds of somatic hybrids, between rat hepatoma cells and either mouse fibroblasts or rat epithelial cells, have been studied. In each case, aldolase B, found only in the hepatoma parent, was absent in the hybrid cells. The absence of aldolase B in the somatic hybrids seems not to be due to trivial factors (species differences, inactivation of all hepatoma aldolase genes, increase in ploidy or loss of chromosomes); it is concluded that extinction of this differentiated function of the hepatoma parent reflects a genetic regulatory phenomenon.     

Establishment of a rat hepatoma cell line which has ornithine carbamoyltransferase activity and grows continuously in arginine-deprived medium

The epithelial cell line, H4-II-E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine-deprived medium. A multi-step selection procedure is described which selects from H4-II-E populations, cells with OCT activity which can grow in arginine-deficient, ornithine-supplemented media.     

Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells.

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinomas or hepatomas when injected in experimental animals.     

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.     

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.     

Glucocorticoid responsiveness of hepatoma cell hybrids

The dominance or recessiveness of glucocorticoid responsiveness and albumin production of different hepatoma cell lines were investigated using somatic cell hybrids. The majority of the intraspecific, intraorgan hybrids between glucocorticoid sensitive, tyrosine aminotransferase (TAT) non-inducible, albumin non-secreting parents and glucocorticoid resistant, TAT-inducible, albumin secreting parents were glucocorticoid sensitive, TAT non-inducible and albumin non-producing. The TAT inducibility was extinguished in interspecific, interorgan hybrids of TAT-inducible hepatoma and non-inducible Chinese hamster fibroblast parents. No TAT reexpression was observed among the many independent hybrid clones. The experiments provided evidence for the non-coordinate control of the expression of differentiated functions in hepatoma hybrid cells.     

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.     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures.

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal calf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous addition of 10(-2) M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and is noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal aclf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous additionof 10^?2 M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and its noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

Metabolism of N8-monoacetylspermidine in rat hepatoma cells. Investigation of its effect on the activity of L-ornithine decarboxylase

Recent evidence has indicated a role for the acetyl derivatives of polyamines, particularly N8-monoacetylspermidine, as activators of L-ornithine decarboxylase in rat hepatoma tissue culture (HTC) cells. This is in contrast with the well-described negative regulatory control of ornithine decarboxylase exerted by their non-acetylated counterparts. Because of the possibility of a rapid extracellular and intracellular catabolism of the acetyl derivatives of polyamines, the metabolism of N8-monoacetylspermidine and its effect on HTC cell ornithine decarboxylase have been investigated, under conditions which eliminate its extracellular catabolism. Differing from previous reports, we demonstrate that N8-monoacetylspermidine does not elevate ornithine decarboxylase activity when added at low concentrations to the culture medium of HTC cells. Higher concentrations decrease ornithine decarboxylase activity in a dose-dependent manner. This effect cannot be unambiguously attributed to the effect of the acetyl derivative itself, because of the presence in situ of a very active N8-monoacetylspermidine deacetylase, which generates spermidine intracellularly.     

Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells able to form functional bile canaliculi in vitro

A large number of hepatoma cell lines has been used to study expression and regulation of liver-specific function. However these cells, even the most differentiated, are morphologically far from hepatocytes. In no case is the typical hepatocyte cell polarity well maintained. Cell hybridization has been used as a potential means for turning on specific genes. From hybrids between well differentiated Fao rat hepatoma cells and WI 38 human fibroblasts, we have attempted to isolate segregated cells that are highly differentiated and polarized. Such cells, detected in aged cultures of only one hybrid (WIF12), were isolated by subcloning. One subclone, WIF12-1 was analyzed. Expression of liver-specific functions extinguished in the original hybrid is restored in all WIF12-1 cells at a very high level, similar to that of hepatocytes and 5-30 times higher that that of parental cells. Moreover human genes coding for liver-specific proteins (albumin, fibrinogen, and alcohol dehydrogenase) are actively expressed. WIF12-1 cells have acquired a polarized phenotype as attested by the presence of bile canaliculi between adjacent cells and by the asymmetrical localization of apical (Mg(2+)-ATPase, gamma-glutamyl transpeptidase) and basolateral membrane markers. The bile canaliculi formed are dynamic and functional structures, characterized by long periods of expansion followed by rapid contractions. The ability to polarize is a general and permanent property of WIF12-1 cells. These cells appear to constitute a valid model for the in vitro study of hepatocyte cell polarity, membrane domain formation and mechanisms of membrane protein sorting.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

Extinction of alpha-fetoprotein gene expression in somatic cell hybrids involves cis-acting DNA elements.

alpha-Fetoprotein (AFP), a liver-specific protein, is extinguished in somatic cell hybrids formed by the fusion of mouse hepatoma cells (BWTG3) with rat fibroblast cells (JF1). Our studies show that the extinction of mouse AFP expression in these somatic cell hybrids may involve at least two cis-acting regulatory domains, i.e., the enhancer elements and a tissue-specific promoter region, which are located in the 5'-flanking region of the AFP gene.     

Extinction, Retention and Induction of Serum Protein Secretion in Hepatoma-fibroblast Hybrids

The production of four serum proteins has been analysed in several hepatoma-fibroblast hybrids. Extinction of albumin and alpha-foetoprotein production occurs systematically in intra and interspecific (rat X mouse) hybrids derived from mouse hepatoma cells (BW). Similar hybrids derived from two related clones of rat hepatoma cells either do not produce albumin (Fa32-derived hybrids), as the BW-derived hybrids, or retain the capacity to produce it, but at a reduced rate (Fu5-derived hybrids); some differences in the control of albumin production thus seem to exist between clonal hepatoma cell lines. The mouse hepatoma cell hybrids retain the capacity to secrete transferrin at a reduced rate, and C3 (the third component of complement) at a high rate. Further analysis of C3 production in interspecific hybrids showed that both parental genomes actively contribute to C3 production: induction of C3 secretion is thus observed in these hybrids.     

Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.     

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.     

Gangliosides of hepatoma 27, normal and regenerating rat liver.

The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.     

Extinction of peroxisomal functions in hepatoma cell-fibroblast hybrids

Although peroxisomes are ubiquitous, differences in the number of organelles and in the expression of associated metabolic activities are observed, depending on the cell type. To investigate the control of peroxisomal activity in connection with cell differentiation, we constructed hybrids between two types of cells whose histogenetic origins dictate significant differences in peroxisomal activities: hepatoma cells and fibroblasts, with high and low expression, respectively, of peroxisomal functions. In these hybrids, extinction of the elevated activities that characterize liver cells is observed, in parallel with the well-documented extinction of differentiated functions. This suggests the existence in fibroblasts of a negative trans-acting regulation.     

Destabilization of the adenosine deaminase gene sequences in rat-rat somatic cell hybrids

Rat hepatoma cells amplified for adenosine deaminase (ADA) gene sequences show the amplified DNA on large, homogeneously staining regions (HSRs). The amplified cells are stable in the absence of selection for 12 mo without loss of ADA activity or gene sequences. However, in hybrids formed between an amplified cell line with a prominent HSR and a nonamplified cell line, rapid loss of ADA activity, as well as gene sequences, occurs. Karyotype analyses of the hybrids indicate that the HSR structures are no longer visible in a large percentage of the hybrid metaphase spreads and appear to have been replaced by DNA structures that resemble double minutes. Our data provide evidence that the extent of the breakdown of the HSR in the hybrids may be affected by the presence of an active adenosine kinase or the level of ATP in the cells and additional unidentified factors are present in the hybrids that affect the integrity of the HSR structure. There is no evidence for a specific trans-acting factor in nonamplified cells that regulates gene amplification.