Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.     

Arsenite induced sensitization and self-tolerance of Reuber H35 hepatoma cells

Our data show that a short incubation with arsenite (30–300 M) induces a biphasic change in ceSlular sensitivity towards a second exposure to arsenite. A transient sensitization was followed by the development of self-tolerance. Sensitization was measured using the step-down protocol; i.e., application of a high dose of arsenite pretreatment (100 or 300 M) followed immediately by incubation in a low dose of arsenite (1–30 M), with extensive rinsing in between. Whereas no effect of 1 and 3 M on cellular survival is observed without pretreatment, a large decrease in cell survival can be established when these low doses of arsenite are applied immediately after a 1 hr pretreatment with 100 or 300 arsenite.According to the step-down protocol, a high dose of toxic compounds is applied and is followed by prolonged incubation in a lower concentration of the initial toxic compound. This might be a more accurate model for studying the effects of toxic insults on cells and organisms in the manner in which they occur in their natural environment. The level of tolerance was determined by a 1 hr test treatment with 300 pM arsenite applied at different times after pretreatment. Using this fractionated treatment protocol, it was established that tolerance increases with the increasing time intervals between the sodium arsenite treatments, during the 6 hr studied.These observations suggest that sensitization gradually decreases, whereas tolerance develops. Furthermore, our data indicate that the condition of pretreatment determines the extent to which the early sensitivity increases, as well as the development of tolerance later on. A relatively high arsenite concentration leads to more sensitized cells, which are transformed into more tolerant cells in comparison with the effect of a lower arsenite concentration.     

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.     

Phosphorylation of histones is stimulated by phorbol esters in quiescent Reuber H35 hepatoma cells

Histones isolated from Reuber H35 rat hepatoma cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for possible alterations in phosphorylation. Incorporation of 32P orthophosphate into individual acid-extracted histones was monitored by autoradiography and scintillation counting of polyacrylamide gels or by reverse-phase high performance liquid chromatography. Treatment of quiescent H35 cells (arrested by serum starvation) with submicromolar doses of TPA resulted in a rapid and specific increase in phosphorylation of histones H2B and H1(0). Smaller increases in phosphorylation were observed for H4. No significant change in phosphorylation of the major H1 histones or H2A were observed after 1 h of treatment. The phosphorylation was TPA dose-dependent, with a maximum increase of approximately 14-fold for H2B, 11-fold for H1(0), and 2-fold for H4 achieved at 0.8 M TPA. The nonpromoting parent compound phorbol did not induce any of these changes. Furthermore, the mitogenic hormone insulin did not cause a similar pattern of histone phosphorylation, suggesting that the effect observed was not due to a general mitogenic response in the H35 hepatoma cells. Addition of 8-Br-cAMP also failed to reproduce the effect of TPA on histone phosphorylation, suggesting that cAMP-dependent protein kinases are not likely to be involved in mediating this response to TPA.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Characteristics of insulin binding to H35 hepatoma cells

Well differentiated hepatoma cells in culture exhibit insulin binding and insulin effects. We have studied insulin binding in control and in H35 hepatoma cells down-regulated with insulin. H35 cells were grown in monolayers in alpha MEM. Insulin binding was measured with A14 mono 125I labelled insulin 72 h after seeding. Binding was time, temperature and pH-dependent. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabelled insulin. Monolayers preincubated with 10 micrograms/ml unlabelled insulin for 24 h showed a decrease of 65% in the number of insulin binding sites. There was no change in affinity.     

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.     

Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt2cAMP. Treatment of H35 cells with cAMP or Bt2cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and a depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt2cAMP on radiation-induced cell death was studied during fractionated irradiation with 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt2cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that whereas cAMP had no effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2.     

Tumor-promoting phorbol esters stimulate the phosphorylation of ribosomal protein S6 in quiescent Reuber H35 hepatoma cells

The addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) to serum-starved quiescent Reuber H35 hepatoma cells results in a rapid 5- to 11-fold increase in the incorporation of 32Pi into a Mr = 32,000 ribosomal protein. The Mr = 32,000 protein was the major phosphorylated protein extracted from isolated 80 S ribosomes and was identified as the 40 S ribosomal protein S6 based upon its migration in two-dimensional gels. Insulin, which has been demonstrated to increase the phosphorylation of S6 in a number of cell lines, caused a 10- to 20-fold increase in the incorporation of 32Pi into this Mr = 32,000 ribosomal protein. S6 phosphorylation was dose- and time-dependent being detected as early as 5 min following the addition of 1.6 microM TPA. Maximal phosphorylation of ribosomal protein S6 was achieved by 60 min and remained elevated for at least 90 min in the presence of TPA. The 50% effective dose for TPA was estimated to be 0.14 microM. Based upon the altered migration of S6 in pH 8.5 urea-polyacrylamide gels, it was demonstrated that the increased 32Pi labeling of S6 by TPA was due to a net increase in the incorporation of phosphates into the S6 molecule. Non-tumor-promoting phorbol esters were ineffective in increasing the phosphorylation of S6. In whole cells, exogenously added 1 mM 8-bromoadenosine 3':5'-monophosphate failed to substantially increase phosphorylation of S6 suggesting that the TPA-induced phosphorylation of S6 occurs via a cyclic AMP-independent mechanism. The S6 amino acid residue phosphorylated in response to TPA was phosphoserine. A possible role for protein kinase C in the phosphorylation of ribosomal protein S6 is discussed.     

A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.     

Stimulation by glucagon and adenosine-3',5'-cyclic monophosphate of protein degradation in Reuber H35 hepatoma monolayers

Protein degradation in Reuber H35 hepatoma monolayers was measured as release of radioactive trichloroacetic acid-soluble material from intracellular protein labelled with [3H]leucine for 16 hr followed by 3-hr chase period. Proteolysis in this system was stimulated by physiological concentration of glucagon reaching a maximum at 10(-7) M with an increase of 30%. Dibutyryl cyclic AMP also had a stimulatory effect. When both glucagon and dibutyryl cyclic AMP were present at optimal concentrations, their effects were not additive suggesting that glucagon may act via the formation of cyclic AMP. In the presence of protein synthesis inhibitor, cycloheximide or puromycin, proteolysis remained responsive to glucagon. Glucagon counteracted the inhibitory effect of insulin on proteolysis.