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.     

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.     

A selective system for hepatoma cells producing gluconeogenic enzymes.

Gluconeogenesis is a liver-specific pathway which permits the synthesis of phosphorylated sugars from oxaloacetate, pyruvate, amino acids, or trioses. The absolute requirement for glucose or an alternative hexose which characterizes most mammalian cells probably reflects an inablility to perform gluconeogenesis rather than to generate sufficient energy by respiration alone. Cells of diverse histogenetic origins have been tested in glucose-free medium, supplemented with oxaloacetate or with dihydroxyacetone. The only cells able to grow are well-differentiated hepatoma cells which produce the relevant gluconeogenic enzymes: phosphoenolpyruvate carboxykinase, fructose diphosphatase, and triokinase. Reconstruction experiments demonstrate that glucose-free media permit the selective growth of cells producing gluconeogenic enzymes. These media should be useful for analysis of reexpression of differentiated functions in somatic cell hybrids and for the isolation of mutants.     

Hormone-sensitive carbohydrate metabolism in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of hepatocytes from adult rats and Fao hepatoma cells in the presence of polyethylene glycol. These hybrids were called hepatocytoma cells. The preservation of liver-specific enzyme activities and metabolic functions was studied in the hybrid clone 1E3. 1) The proliferating hepatocytoma cells formed monolayers presenting morphological similarity to primary cultures of hepatocytes. 2) In contrast to Fao hepatoma cells, activities of all gluconeogenic key enzymes were preserved at normal or reduced levels. 3) Lactate-dependent glucose formation was maintained at a state reduced to 36% of the gluconeogenesis in hepatocytes; no glucose formation was detected in Fao hepatoma cells. 4) The activity of the liver-specific glucokinase was reduced in hepatocytoma cells, but it was still present in contrast to Fao cells. The liver-specific isoenzyme pyruvate kinase type L was replaced by the isoenzyme type M2. 5) Gluconeogenic and glycolytic enzyme activities were regulated in hepatocytoma cells by glucagon (0.1 microM) and by insulin (0.1 microM). 6) The genome of hepatocytoma cells and its expression were stable for at least one year, when spontaneously dedifferentiating cells were removed by recloning in hypoxanthine-aminopterine-thymidine (HAT) medium.     

Differentiation is not restored in hybrids between independent variants of a rat hepatoma

Crosses have been undertaken between cells of three independent clones of dedifferentiated rat hepatoma variants to investigate whether "complementation" leading to restoration of the original differentiation would occur. Hybrids were examined between ten days and two months after fusion for the presence of intracellular albumin and for their ability to proliferate in glucose-free medium where survival requires activity of the liver-specific gluconeogenic enzymes. In none of the three possible crosses involving the three variants was evidence of reexpression of hepatic functions obtained.     

Effect of glucocorticoids on fetoprotein production by an established cell line from Morris hepatoma 8994

Glucocorticoids at concentrations equal to or higher than 10^?7M lead to an increase of alpha-fetoprotein production by an established cell line from Morris hepatoma 8994. These cells also secreted alpha_M-fetoprotein into the culture medium but only after addition of at least 4×10^?7M hydrocortisone or 5×10^?8M dexamethasone. The effects on both fetoproteins were observed in spite of a decrease of cell multiplication and an increase of cell detachment.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Rat liver nuclear N-acetyltransferases: Separation of two enzymes with both histone and spermidine acetyltransferase activity

Two similar histone acetyltransferases have been separated from rat liver nuclei and purified 500-fold. Both enzymes also acetylate spermidine and spermine but diamines are not acetylated. Both enzymes preferentially acetylate histone 3; among the remaining histones H2A and H2B are good substrates, whereas H1 and histone 4 are poor substrates. Apparent Michaelis constants for spermidine were about 2 × 10^?4m; apparent Michaelis constants for acetyl coenzyme A were 1.5 × 10^?5 and 10^?5m for enzymes A and B, respectively. At low concentrations DNA inhibits histone acetylation by enzyme A (50% inhibition at 25 μg/ml DNA). Enzyme B is relatively insensitive to DNA. This suggests the possibility of separate intranuclear localization of the two enzymes.     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

The antagonistic effect of dexamethasone and insulin on alpha-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma.

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.     

The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique

Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities.From 4 × 10⁸ to 5 × 10⁸ cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

Re-expression of hepatic functions in mouse hepatoma X rat hepatoma hybrids

Abstract. Neonatal hepatic functions are selectively extinguished in hybrids between mouse hepatoma cells, that express only fetal hepatic functions, and rat hepatoma cells expressing neonatal as well as fetal functions. A search for hybrid cells reexpressing these neonatal functions was undertaken to determine; (1) whether the selective extinction of neonatal functions is reversible and at what frequency, and (2) whether the reexpression of neonatal functions would be accompanied by modifications in the expression of fetal functions. The criterion used to obtain hybrids showing reexpression was glucose-free medium (G-) where growth requires the presence of the extinguished gluconeogenic enzymes. Even though the parental cells are of the same histotype it proved difficult to obtain re-expression. Survivors in G- were obtained only from hybrids containing a greater than Is complement of rat chromosomes; they reexpress not only gluconeogenic enzymes but also basal tyrosine aminotransferase activity, and the fetal hepatic function a-fetoprotein continues to be expressed in most of the clones. All survivors in G- display a significant loss of chromosomes and this loss concerns essentially mouse chromosomes.     

l-Glutamic acid,a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [³H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10^?5 M) modified the spontaneous release of exogenous [³H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [³H]dopamine newly synthesized from [³H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10^?6-1 × 10^?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [³H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca²⁺ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca²⁺-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.     

Re-expression of hepatic functions in mouse hepatoma X rat hepatoma hybrids

Neonatal hepatic functions are selectively extinguished in hybrids between mouse hepatoma cells, that express only fetal hepatic functions, and rat hepatoma cells expressing neonatal as well as fetal functions. A search for hybrid cells reexpressing these neonatal functions was undertaken to determine; (1) whether the selective extinction of neonatal functions is reversible and at what frequency, and (2) whether the re-expression of neonatal functions would be accompanied by modifications in the expression of fetal functions. The criterion used to obtain hybrids showing re-expression was glucose-free medium (G) where growth requires the presence of the extinguished gluconeogenic enzymes. Even though the parental cells are of the same histotype it proved difficult to obtain re-expression. Survivors in G- were obtained only from hybrids containing a greater than 1s complement of rat chromosomes; they reexpress not only gluconeogenic enzymes but also basal tyrosine aminotransferase activity, and the fetal hepatic function alpha-fetoprotein continues to be expressed in most of the clones. All survivors in G- display a significant loss of chromosomes and this loss concerns essentially mouse chromosomes.     

Expression of differentiated functions in hepatoma cell hybrids: selection in glucose-free media of segregated hybrid cells which reexpress gluconeogenic enzymes.

Selective glucose-free media have been used to study the reexpression of liver-specific gluconeogenic enzymes in rat hepatoma X mouse lymphoblastoma somatic hybrids. The utilization for gluconeogenesis of dihydroxyacetone or oxaloacetate requires two enzymes: fructose diphosphatase as well as either triokinase for the former or phosphoenolpyruvate carboxykinase for the latter. By sequential selection with these substrates, the reexpression of the three gluconeogenic enzymes has been dissociated. The reexpression of these enzymes is correlated with the loss of mouse chromosomes. In addition, the characterization of the parental forms of aldolase B, another liver-specific enzyme, shows that reexpression corresponds to the simultaneous production of the rat and mouse enzymes. These results demonstrate the chromosomal origin of extinction and suggest that activation of mouse silent genes which accompanies reexpression can occur without loss of the parental determinations. The hypothesis that determination involves regulatory rather than structural genes is discussed.     

Epidermal growth factor: Characteristics of specific binding in membranes from liver,placenta, and other target tissues

Epidermal growth factor, a 6,400-dalton polypeptide from the mouse submaxillary gland, binds specifically to cells and membranes derived from a variety of human, rat, mouse, and bovine tissues. Liver, placenta, skin, cornea, and cultured chondrocytes, Hela cells, and Chang liver cells bind large amounts of epidermal growth factor, whereas fat cells, resting and lectin-stimulated human peripheral lymphocytes, mouse thymocytes, cultured rat hepatoma cells, and mammary cells from virgin and pregnant mice bind little or no epidermal growth factor. The binding site for epidermal growth factor is distinct from receptors for other anabolic peptides such as insulin, nerve growth factor, and growth hormone. The binding of epidermal growth factor is rapid and reversible. The rate constant of association is approximately 10⁶ mole^?1 sec^?1, the rate constant of dissociation is about 6 × 10^?4 sec^?1, and the apparent equilibrium dissociation constant is about 10^?9m. Trypsin at low concentrations (50–200 μg/ml) destroys the receptor site for epidermal growth factor. The binding of epidermal growth factor by membranes is not accompanied by appreciable degradation of the peptide present in the medium or of that bound to the membranes. Use can be made of the high affinity and specificity of membranes for epidermal growth factor to measure by a competitive binding assay as little as 200 pg of EGF per ml (3 × 10^?11m).     

Effect of ortho-dihydroxyphenols on growth and protein pattern of callus cultures from Prunus avium

Excised shoot discs from Prunus avium L. were cultivated on a modified Murashige and Skoog medium for 4 weeks under long day conditions. The basal medium was supplemented with IAA and benzylaminopurine. Further addition of 5.7 × 10^?5 resp, 11.4 × 10^?5M catechin and 4.25 × 10^?5 resp. 8.5 × 10^?5M chlorogenic acid resulted in a highly significant promotion of callus proliferation. Proteins and enzymes were separated by isoelectric focusing. The supply of both polyphenols resulted in a higher number and intensity of protein bands including esterases as compared to controls.