Early events in thymocyte activation. II. Changes in nonhistone chromatin proteins induced by a thymus-dependent human serum factor.

Previously, it has been shown that a human thymus-dependent serum factor (SF), isolated from peripheral blood and acting on precursors of mature T lymphocytes, induces an increase in the synthesis of cyclic AMP and proteins in thymocytes. We have now investigated the action of SF on the incorporation of 3H-leucine and 32P-orthophosphate into nuclear proteins of thymocytes after 15 to 240 min of culture. SF induced a rapid increase in the synthesis and phosphorylation of nuclear proteins, especially in the phosphorylated nonhistone chromatin proteins (P-NHCP). Electrophoretic patterns in polyacrylamide gels of the P-NHCP fractions, extracted from the chromatin of the stimulated cells, showed that proteins with m.w. higher than 50 x 10(3) were synthesized to a larger extent as compared with unstimulated cells. These data suggest that SF acts specifically on the synthesis of P-NHCP and may in this way control DNA-template activity.     

Sera from both normal and thymectomized animals produce elevations of cyclic AMP in thymocytes.

Cyclic AMP content in mouse thymocytes has been measured after incubation either with sera or serum fractions from normal or thymectomized (Tx) mice and pigs or with a synthetic circulating pig thymic factor. Sera from both Tx and normal pigs and mice induced an increase in cyclic AMP in mouse thymocytes, whereas the synthetic pig thymic factor did not. It is concluded that the increase in cyclic AMP in mouse thymocytes should be used with caution for the evaluation of circulating thymic hormone levels.     

Thymosin-induced human serum factor increasing cyclic AMP.

Thymosin has virtually no in vitro effect on cyclic AMP levels in thymocytes and seems not to react with either the beta-adrenergic receptor or with the putative human serum thymic factor (SF) receptor. However, when thymosin is injected into patients lacking SF activity, it induces the appearance of a serum factor which increases cellular cyclic AMP levels in thymocytes in vitro. This factor appears to act on a membrane site distinct from the beta-adrenergic receptors.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.     

Gangliosides do not raise cyclic AMP levels during inhibition of lymphocyte mitogenesis

The effect of bovine brain gangliosides on the intrathymocyte levels of cyclic AMP as a potential mediator of ganglioside action has been studied. Commercial tri-, and disialogangliosides, at 2.5 to 5 μM were found to produce a rapid and profound increase (eg., 10 fold within 2 min by trisialoganglioside). When the preparations were purified on Florisil, this effect on cyclic AMP content was lost, but not the immunoinhibitory potency of the ganglioside (as tested on Concanavalin A induced DNA synthesis). The water soluble “ganglioside associated protein” fractions separated from commercial di- and trisialo gangliosides by Florisil chromatography did not alter the cyclic AMP levels of thymocytes. Previous reports of an effect of commercial gnagliosides on the enzymes of cyclic AMP metabolism in nervous tissue should be re-evaluated.     

Serum modification of cyclic nucleotide phosphodiesterase forms independent of protein synthesis.

Addition of 10% fetal calf serum to BHK cells made quiescent by maintenance for 48 hours in sub-optimal serum (0.5%) caused rapid changes in cyclic AMP phosphodiesterase activity (increased maximum velocity and affinity) even in the presence of inhibitors of protein synthesis. Activity changes were associated with an alteration in the number of forms of cyclic AMP phosphodiesterase identified by Agarose gel filtration. Three forms of cyclic nucleotide phosphodiesterase were apparent after serum addition whereas only two forms were resolved in quiescent BHK cells. The initial rapid increase in cyclic AMP phosphodiesterase activity seen when serum was added to quiescent cells was followed temporally by a much slower increase in cyclic AMP phosphodiesterase activity that could be prevented by cycloheximide or actinomycin D.     

Action of phenylephrine on protein synthesis in liver cells.

The alpha-adrenergic agonist phenylephrine was found to inhibit protein labelling from [3H]valine in isolated liver cells. This effect is only observable under conditions of partial Ca2+ depletion and in cells displaying maximal rates of protein labelling, i.e. cells isolated from fed animals or from starved animals when incubated in the presence of alanine. The ability of phenylephrine to inhibit protein labelling at near-saturating concentrations of the amino acid precursor indicates that this alpha-agonist actually decreases the rate of protein synthesis. The possibility that phenylephrine acts by making cellular Ca2+ availability further limiting can be ruled out, since alanine stimulates protein labelling under conditions of severe Ca2+ depletion obtained by pretreatment of the cells with EGTA. The following observations indicate that the phenylephrine action may be mediated by an increase in cellular cyclic AMP content: (1) a close relationship was found between the abilities of phenylephrine to inhibit protein labelling and to increase cyclic AMP content; (2) cyclic AMP mimics the phenylephrine action only in cells partially depleted of Ca2+; (3) the alpha 1-antagonist prazosin, which inhibited the phenylephrine-mediated increase in cyclic AMP, also abolished the effect on protein synthesis.     

Adenylic acid catabolism in thymocytes of the regenerating thymus in mice

The T-cell non-peptide mitogenic factor isolated from the thymus stimulates thymus regeneration in mice previously treated with hydrocortisone. [8-14C]AMP catabolism in cortisone resistant thymocytes of mice has been investigated. Incorporation of radioactivity into hypoxanthine in cortisone resistant thymocytes is found to increase as compared with the total thymocyte population. Accumulation of labeled AMP catabolites in the form of hypoxanthine grows considerably after in vitro incubation of cortisone-resistant thymocytes with the non-peptide T-cell mitogenic factor. A large proportion of [8-14C]AMP catabolite radioactivity incorporated into cortisone-resistant thymocytes is excreted into the medium as hypoxanthine. It is supposed that hypoxanthine accumulation abrogates limitation of thymocyte DNA synthesis inhibited by relative excess of dGTP.     

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.     

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.     

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.     

Effects of adenosine 3'':5''-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts.

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.     

Differential effects of intracellular calcium elevating agents on adrenergic-stimulated cyclic nucleotide and melatonin synthesis in rat pinealocytes.

In this study, the role of elevation of intracellular Ca2+ and activation of protein kinase C on adrenergic-stimulated cyclic nucleotide accumulation and melatonin synthesis in rat pinealocytes was investigated. It was found that whereas KCl, ionomycin, and ouabain, three Ca(2+)-elevating agents, had a potentiating effect on adrenergic-stimulated cyclic AMP response, their effects on melatonin synthesis were inhibitory. Similar inhibition was also observed when dibutyryl cyclic AMP was used to stimulate melatonin synthesis. By determining intracellular Ca2+ directly, it was found that the enhancing effects of these agents on the cyclic AMP response but not their inhibitory effects on melatonin synthesis paralleled their abilities to elevate intracellular Ca2+. In comparison, activation of protein kinase C significantly enhanced the adrenergic-stimulated cyclic AMP response and, to a lesser degree, the adrenergic-stimulated N-acetyltransferase and melatonin levels. These results indicate that (i) Ca(2+)-elevating agents have opposite effects on adrenergic-stimulated cyclic AMP and melatonin production; (ii) a post cyclic AMP event of importance to melatonin synthesis is inhibited by these agents; and (iii) the mechanism of inhibition may not be directly related to their effect on intracellular Ca2+.     

Growth hormone, cyclic nucleotides and the rapid control of translation in heart muscle.

Perfused rat heart incorporated L-[14C]tyrosine into protein at a constant rate for up to 75 min. A purified bovine growth-hormone preparation (1 mug/ml) stimulated the incorporation to a new constant rate that was more than three times the control rate by 10 min after hormone addition to perfusate. The hormone, however, did not alter the intracellular tracer amino acid pool, and the relationship of this to the aminoacyl-tRNA precursor pool is discussed. It is concluded that the increased incorporation largely reflected a rapid increase in protein synthesis at the ribosomes. Measurements of cyclic nucleotide contents during the perfusion showed that these appeared to vary in a systematic way during the perfusion. This strands in contrast with the constant values given by several other parameters measured in this preparation. Futher, the cyclic nucleotide variation seems to be independent of external effectors. The steady-state performance of the heart correlates more closely the [cyclic AMP]/[cyclic GMP] ratio than with the content of the individual cyclic nucleotides. At 10 min after the addition of growth hormone a slight decrese in cyclic AMP content and a large decrease in cyclic GMP were found, suggesting that the hormone's effect in stimulating protein synthesis may be mediated by a decrease in cyclic nucleotide concentrations or an increase in the [cyclic AMP]/[cyclic |p] ratio. The findings are also consistent with an intracellularly directed role for these nucleotides, and the possibility that the cyclic nucleotide changes are an indirect result of growth-hormone action is discussed.     

Effect of thyroliberin on the concentration of adenosine 3'':5''-phosphate and on the activity of adenosine 3'':5''-phosphate-dependent protein kinase in prolactin-producing cells in culture.

1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.     

The roles of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocyte proliferation

Both the parathyroid hormone (PTH) and the calcium ion increase the cellular content of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), promote the initiation of deoxyribonucleic acid synthesis and stimulate the proliferation of rat thymocytes maintained in vitro. The ability of cyclic AMP to serve as the mediator of the mitogenic actions of both PTH and calcium is established by the fact that cyclic AMP itself stimulates cell proliferation in the absence of PTH and extracellular calcium. Neither PTH nor calcium appear to raise the cellular cyclic AMP level by increasing the nucleotide's synthesis by adenylate cyclase (formerly adenyl cyclase); PTH concentrations as high as 50 μg per ml of medium do not increase the enzyme's activity (in the presence or absence of calcium) and mitogenic calcium concentrations inhibit it. PTH also does not directly affect isolated thymocyte phosphodiesterase, but mitogenic calcium levels inhibit the enzyme's activity. Additional experiments show that it is calcium which raises the cyclic AMP level in cells treated with PTH, and some possible calcium-mediated mechanisms by which the hormone could elevate the cellular cyclic AMP levels are discussed. Thus, the mitogenic action of PTH is primarily mediated by calcium while cyclic AMP is the ultimate implementor of the hormonal action. However, calcium has a dual role and evidence is presented which indicates that besides raising the cellular cyclic AMP level, it also controls the operation of cyclic AMP's mitogenic end-reaction.     

Adenosine receptor-mediated accumulation of cyclic AMP-induced T-lymphocyte death through internucleosomal DNA cleavage.

Incubation of mouse thymocytes with adenosine and its receptor site agonist, 2-chloroadenosine, induced a pronounced increase in the intracellular cAMP level and resulted in internucleosomal DNA fragmentation followed by cell lysis. Similar DNA fragmentation was induced in peripheral T-lymphocytes prepared from spleen cells but to a lesser extent than in the thymocytes. The DNA fragmentation in both thymocytes and splenic T-lymphocytes was prevented by the addition of actinomycin D and cycloheximide, indicating that this process required mRNA and protein synthesis. The inhibition was accompanied by a reduction in cell lysis as judged by the release of lactate dehydrogenase into the medium. Involvement of cAMP accumulation in inducing DNA fragmentation was supported by the results of experiments with cAMP analogs such as dibutyryl cAMP and 8-bromo-cAMP, and cAMP level-raising drugs including forskolin, cholera toxin, and isobutylmethyxanthine. The latter agents induced pronounced or sustained elevation of cellular cAMP followed by internucleosomal DNA cleavage in T-lymphocytes. These results suggest that adenosine receptor-mediated accumulation of cyclic AMP regulates T-lymphocyte death through inducement of internucleosomal DNA cleavage.