S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA.     

Recycling of 5''-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.     

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.     

State of hepatitis B viral DNA in a human hepatoma cell line.

PLC/PRF/5, a tissue culture cell line isolated from a human hepatocellular carcinoma and producing hepatitis B surface antigen, was studied for the presence of hepatitis B virus (HBV)-specific DNA and RNA. PLC/PRF/5 cell DNA accelerated the rate of reassociation of HBV [32P]DNA, and quantitative experiments indicated that the cells contained approximately four copies of viral DNA per haploid, mammalian cell DNA equivalent. PLC/PRF/5 DNA accelerated the rate of reassociation of all individual restriction endonucleases HincII and HaeIII fragments of HBV [32P]DNA, indicating that DNA from all regions of the viral genome is present in the cells. This suggests that these cells contain at least most, and possibly all, of the viral genome. Digestion of PLC/PRF/5 cell DNA with restriction endonuclease HindIII (an enzyme found not to cleave the DNA of any HBV isolate so far examined) yielded only three fragments, all larger than virion DNA, which contained HBV DNA base sequences, suggesting that HBV DNA is integrated in high-molecular-weight DNA at three different sites in these cells and that there is no viral DNA in an episomal form. PLC/PRF/5 cell [32P]RNA was found to hybridize with all restriction fragments of HBV DNA adequately tested, indicating that at least most, and possibly all, of the viral DNA in these cells is transcribed.     

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dexamethasone on kininogen production by a rat hepatoma cell line

T Kininogen and High Molecular Weight Kininogen were characterized in the cell culture medium of Fao cells, a highly differentiated cell line derived from the Reuber H35 rat hepatoma. Immunoreactive T Kininogen and High Molecular Weight Kininogen identified by direct and specific RIAs were indistinguishable from standard kininogens. Immunoreactive T Kininogen was further identified by HPLC analysis of T kinin released after trypsin hydrolysis of the cell culture medium. The basal release rate of T kininogen was ten-fold higher than that of High Molecular Weight Kininogen. T Kininogen was not stored within the cells contrary to High Molecular Weight Kininogen. The production of the two kininogens in the cell medium was stimulated by dexamethasone up to five times in a dose-dependent manner. The specific antiglucocorticoid compound RU 38486 did not alter the basal rate of kininogen release by Fao cells, but abolished the stimulation by dexamethasone, indicating that dexamethasone exerts a true glucocorticoid type effect.     

Spontaneous apoptosis in a rat hepatoma cell line and its inhibition by dexamethasone

Cultures of dedifferentiated rat hepatoma Rab1-5-1 cells exhibit spontaneously a high level of mortality during the exponential growth phase. We demonstrate that these cells die by apoptosis, showing chromatin condensation and internucleosomal DNA fragmentation. Cells of the original H4II cell line and of its differentiated and dedifferentiated derivatives also die by apoptosis, but only in heavily confluent cultures. We evaluated mortality with time in Rab1-5-1 cultures by establishing growth curves, including quantification of floating cells, and conclude that up to half of the cells in a culture are lost to apoptosis. The production of apoptotic cells is abolished by the presence of 10(-6) M dexamethasone and this inhibition is reversible in 48 hours. Rab1-5-1 cells that spontaneously die by apoptosis with high frequency represent a novel model to investigate factors that regulate the spontaneous frequency of death, and to study the nature and the kinetics of commitment to the apoptotic pathway.     

Cytophotometric analysis of glycogen,protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G₂/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N⁶,O²-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G₀/G₁ and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Synthesis and secretion of albumin by a synchronized rat hepatoma cell line.

The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.     

Stable ornithine decarboxylase in a rat hepatoma cell line selected for resistance to alpha-difluoromethylornithine

Ornithine decarboxylase (ODC) is extremely unstable in mammalian cells. This unusual characteristic facilitates rapid fluctuations in the activity of this enzyme in response to variations in its biosynthesis. Unfortunately, very little is known about the mechanism or regulation of this ODC-specific proteolytic pathway. This study describes the production and characterization of a variant of the rat hepatoma HTC cell line that is strikingly deficient in this pathway. This cell variant was induced by selection for growth in stepwise increasing concentrations (up to 10 mM) of the irreversible ODC inhibitor, alpha-difluoromethylornithine (DFMO). Resistance to this inhibitor appears to result from a combination of elevated (10X) ODC biosynthesis and inhibited degradation, producing greater than a 2000-fold increase in the level of ODC protein. In these variant cells (DH23b) inhibition of protein synthesis by cycloheximide did not result in rapid loss of enzyme activity or ODC protein determined by radioimmunoassay. Pulse-chase studies with [35S]methionine confirmed that this enzyme was not preferentially degraded, even when spermidine was added to the media. ODC purified from the variant cells was found to be identical to the control cell enzyme in size, isoelectric point, substrate binding kinetics, and sensitivity to the inhibitor DFMO. Also, as in the control cells, a major fraction of the ODC molecules extracted from DH23b cells was shown to be phosphorylated on a serine residue. The inability to detect physical or kinetic differences between the parent and the variant cell ODC suggests that the unusual stability of ODC in this cell is associated with a defect in a cellular mechanism for ODC-specific degradation.     

The hormonal induction of gamma glutamyltransferase in rat liver and in a hepatoma cell line

Molecular and Cellular Biochemistry - Gamma glutamyltransferase (GGT) is a membrane-bound enzyme that is involved in glutathione metabolism and aminoacids uptake. GGT activity is stimulated by a...     

An expression system for human apolipoprotein B100 in a rat hepatoma cell line

We have designed an in vitro expression system for human apolipoprotein (apo) B. A full-length human apoB minigene was constructed from cDNA and genomic apoB clones and inserted into a vector where its expression was directed by the cytomegalovirus promoter. The apoB minigene was expressed in a rat hepatoma cell line, McA-RH7777. Human apoB100, which is the ligand for the low density lipoprotein receptor, was secreted in low density lipoprotein or very low density lipoprotein particles, depending on the composition of the medium. A protein with the mobility of apoB48, a structurally related protein involved in cholesterol metabolism, was also produced from the human apoB minigene. This in vitro expression system for human apoB will enable investigators to identify which domains of this protein are involved in processes such as lipoprotein assembly and secretion. This system should also allow investigators to identify definitively the domain in apoB that enables the protein to bind to the low density lipoprotein receptor.     

Cytophotometric analysis of glycogen, protein and DNA of a glycogen-storing rat hepatoma (N13) cell line.

This study examines the behavior of glycogen-storing rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2'-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Utilization of an established rat hepatoma cell line for mutation studies

A selected strain of rat hepatoma cells was evaluated for its utility in the assay of mutation. We demonstrate that these cells have practical and theoretical advantages over most other mammalian cells utilized for the mutation assay at the HGPRT locus. Characteristics of the H4IIE cell line which enhance the ability of the HGPRT assay to detect mutation include: intrinsic metabolic capabilities; small cell size, and lack of mobility; colony-forming efficiency of 85–95%; a background mutation level between 0.9 and 3 mutants per 10⁵ viable cells; and the ability to proliferate in medium containing 5% fetal bovine serum and 5% horse serum. The optimal expression period for the maximum frequency of mutants was investigated using UVC as the mutagenic agent, and found to plateau after 8–10 days. The metabolic capacity of this cell line was demonstrated using 2-aminoanthracene and cyclophosphamide, two types of mutagens requiring biotransformation for activity. We conclude that the use of the H4/HGPRT system could prove valuable in the mutagenic screening of suspect environmental agents.     

Non-enzymatic DNA methylation by S-adenosylmethionine results in the formation of minor thymine residues and 5-methylcytosine from cytosine

It was found that nonenzymatic DNA methylation proceeds in water solution in the presence of S-adenosylmethionine (AdoMet). The main reaction products are thymine and 5-methylcytosine residues. It was shown that labelled thymine residues are formed also upon DNA incubation in the presence of [methyl-14C]methionine as well as [methyl-14C]cobalamine. Only cytosine reacts with AdoMet resulting in thymine production. AdoMet may be a potential mutagen that induces GC----AT transitions during DNA replication in the cell.     

Immunohistochemical and biochemical studies on DNA ligase from a rat liver parenchymal cell line

We have recently obtained a monospecific antibody against calf thymus DNA ligase composed of a single Mr = 130,000 polypeptide. Immunohistochemical studies using the antibody and immunoperoxidase detection methods indicated that DNA ligase in a rat liver parenchymal cell line (BB) is localized essentially in nucleus. The specific activity of DNA ligase from growing BB cells was more than 10-fold higher than that from rat hepatocytes. The molecular forms of DNA ligase in these cell-free extracts were also analyzed.     

Partial characterization of a glucocorticoid suppressible mitogenic activity secreted from a rat hepatoma cell line hypersensitive to the antiproliferative effects of glucocorticoids

We have previously shown that glucocorticoids suppress the proliferation of Fu5 hepatoma cells and have selected subclones which are either hypersensitive (BDS1) or resistant (EDR3) to the antiproliferative effects of dexamethasone, a synthetic glucocorticoid. BDS1 cells externalize a glucocorticoid suppressible mitogenic activity (denoted GSM) which stimulated [3H]thymidine incorporation in quiescent, serum-starved Balb/c 3T3 cells. Glucocorticoid treatment of BDS1 cells reduced the secreted levels of GSM activity by approximately 20-fold in comparison to untreated cells. The GSM activity was constitutively secreted from a glucocorticoid receptor minus variant (EDR3) demonstrating that the suppression of this mitogenic activity is a new glucocorticoid hormone response which required a functional receptor. GSM activity was sensitive to sulfhydryl reducing agents or trypsin, stable to heat and acid treatments and fractionated in gel filtration columns with a native molecular weight of approximately Mr 30,000. The persistence of this size for mitogenic activity after electrophoretic fractionation in nonreducing sodium dodecyl sulfate-poly-acrylamide gels suggested that the GSM activity is comprised of a single protein. Total secreted protein isolated from untreated BDS1, but not dexamethasone-treated BDS1, stimulated 3T3 cells to grow in transformed-appearing large colonies in soft agar and to display multiple layering and elongated spindle-like morphology on solid substratum. The addition of both insulin and EGF to conditioned medium protein isolated from glucocorticoid-treated BDS1 cells restored full induction of 3T3 cell anchorage-independent growth while insulin restored full and EGF partial mitogenic stimulation of these fibroblasts. These results suggest that the GSM activity acts in a pathway common to that of insulin or EGF in fibroblasts.