Studies of the incorporation of [5-3H]uridine during activation and transformation of lymphocytes induced by phytohaemagglutinin. Dependence of the incorporation rate on uridine concentration at certain critical concentrations

1. Partially purified pig blood lymphocytes were stimulated to transform in culture with phytohaemagglutinin. Initial cell activation was assessed by measuring the increase of uridine incorporation into RNA induced by phytohaemagglutinin. The phytohaemagglutinin/serum ratio for this effect was similar to that required for transformation; however, no inhibition at high phytohaemagglutinin/serum ratios was found during cell activation. 2. Without replenishment of medium the pool of competitors with added uridine for incorporation fell to zero during 2 days of culture. At certain critical pool concentrations uridine itself could stimulate the rate of uridine incorporation. 3. Most of the tritium from [5-(3)H]uridine added at the initiation of culture had been incorporated into RNA by the end of the second day of culture; the subsequent loss of radioactivity preceded a fall in the total RNA content of cultures. 4. RNA was qualitatively examined on sucrose density gradients. In the first 30min. after the addition of phytohaemagglutinin, increased labelling occurred predominantly between the 28s and 4s regions of the gradients. On the second day of culture with phytohaemagglutinin mainly RNA sedimenting beyond the 28s region of gradients was labelled in 30min.     

Incorporation of [5-3H]uridine and attachment of cells to glass during activation of lymphocytes induced by phytohaemagglutinin

1. Shortly after the addition of phytohaemagglutinin to cultures, partially purified pig blood lymphocytes showed increased labelling of RNA by [5-(3)H]uridine and began to attach to the glass of the culture tubes. 2. In the presence of phytohaemagglutinin most cells became attached to glass during the first 5hr. of culture; those first attaching had a low mean RNA/DNA ratio. In the absence of phytohaemagglutinin cell attachment to glass was diminished. 3. During the first 5hr. of culture the rates of increase of radioactivity/unit of DNA of the attached and unattached populations in the presence of phytohaemagglutinin were of the same order and exceeded that of cultures without phytohaemagglutinin. 4. The increase of labelling in cultures to which phytohaemagglutinin was added after cells had sedimented to glass was greater than that occurring in cultures in which cells were resuspended before phytohaemagglutinin was added.     

Quantitative nucleic acid changes during phytohaemagglutinin-induced lymphocyte transformation in vitro. Dependence of the response on phytohaemagglutinin/serum ratio

1. Pig and human blood lymphocytes have been grown in culture without replenishment of medium, and stimulated to transform by phytohaemagglutinin. Quantitative nucleic acid changes during this process have been used as an index of transformation. 2. On the first day, cells attach to glass; then they detach and continue transforming. 3. The degree of transformation is dependent on the phytohaemagglutinin/serum ratio, and is independent of cell concentration within the range 0.5x10(6)-2.0x10(6) cells/ml. 4. At low phytohaemagglutinin/serum ratios there is no response; at high phytohaemagglutinin/serum ratios, inhibition appears after the cells have been cultured for a day.     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cells was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2-100 micrograms/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. These results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells which represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the 14C-labelled asialofetuin degradation. We can conclude that Ricinus lectin present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Myelin Basic Protein inhibits the calcium response to phytohaemagglutinin in human lymphocytes

Myelin Basic Protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation by phytohaemagglutinin.Pre-treatment of human lymphocytes with myelin basic protein results in a lower rising of cytosolic concentration of free calcium after stimulation with phytohaemagglutinin.This effect is dependent on myelin basic protein concentration and on the preincubation time of the protein with the cells. It is not due to a interaction between myelin basic protein and phytohaemagglutinin, but appears to be a consequence of the binding of the protein to the cell surface.The reduction of the rise of cytosolic calcium induced by phytohaemagglutinin is specific for the myelin basic protein because other proteins like albumin and protamine have no effect.     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cell was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2–100 μg/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. Theses results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells whihc represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the ¹⁴C-labelled asialofetuin degradation. We can conclude that Ricinus lectic present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Esterases and phosphatases in the leucocytes of rainbow trout, Salmo gairdneri Richardson

A preliminary report is made on the distribution of acid phosphatases and acid esterases in the cells of rainbow trout. A modified technique for the cytochemical demonstration of acid esterases is given, resulting in clearer visualization. Lymphocytes undergoing transformation in the presence of phytohaemagglutinin show a parallel decrease in enzyme localization for acid esterase and acid phosphatase as transformation in culture proceeds.     

Chromatography of human urinary erythropoietin and granulocyte colony-stimulating factor on insolubilized phytohaemagglutinin

Human urinary erythropoietin was adsorbed to phytohaemagglutinin coupled to agarose or porous glass and quantitatively eluted by a saturated solution of MgCl₂. This method provides a means of separating erythropoietin from several of its contaminants, presumably on the basis of its carbohydrate side chains. Erythropoietin which had been purified by chromatography on insolubilized phytohaemagglutinin was sufficiently free of toxicity to be assayable in tissue culture even when crude urine was used as a starting material.     

The role of zinc ions in the transformation of lymphocytes by phytohaemagglutinin

1. Incorporation of [(3)H]thymidine into DNA was inhibited by EDTA and diethylenetriamine-NNN'N'N'-penta-acetate but not by nitrilotriacetate even when Ca(2+) was present at more than twice the concentration of the chelators. 2. The inhibition caused by EDTA was most effectively reversed by Zn(2+) but also to a lesser extent by Cd(2+). Very little if any activation of the EDTA-inhibited system was obtained with Co(2+), Cu(2+), Fe(3+), Mn(2+) or Ni(2+) added alone. 3. Fe(3+) added to the Zn(2+)-activated lymphocytes in the presence of EDTA markedly increased thymidine incorporation. Addition of Cd(2+) prevented the inhibition of incorporation which occurred at high Zn(2+) concentrations. 4. If EDTA was added more than 15h after phytohaemagglutinin, the inhibition of incorporation was less than that obtained by its addition at zero time. If Zn(2+) was added later than 12h after EDTA and phytohaemagglutinin, the inhibition of incorporation by EDTA was not fully reversed. A study of the time-course of the effects of delayed additions of EDTA and Zn(2+) suggested that, on average, the cells required Zn(2+) between 20 and 30h after phytohaemagglutinin addition to allow the full rate of thymidine incorporation at 37h. 5. The increase in the rate of protein synthesis caused by phytohaemagglutinin was not inhibited by EDTA until about 8h. The further increase after this was totally inhibited by EDTA but this inhibition was fully reversible with Zn(2+). The rate of protein synthesis in EDTA-inhibited cultures at 40h was the same as that at 10h. 6. There was a close similarity between the effects of EDTA on lymphocyte stimulation and those reported by Kay et al. (1969) with low doses of actinomycin D.     

The effect of 12-O-tetradecanoyl-phorbol 13-acetate on the ribonuclease activity of circulating human lymphocytes.

12-O-Tetradecanoyl-phorbol 13-acetate is a very effective tumor promotor and inflammatory agent and can act as a mitogen for a subset of T lymphocytes. We report here that even short exposure of lymphocytes to 12-O-tetradecanoyl-phorbol 13-acetate changes the balance between the levels of neutral ribonuclease and ribonuclease inhibitor. The most dramatic change occurs in a B-lymphocyte-enriched population. We find that most, if not all, of the neutral ribonuclease activity in circulating lymphocytes is associated with this population and that this activity is lost with exposure to 12-O-tetradecanoyl-phorbol 13-acetate. Both 12-O-tetradecanoyl-phorbol 13-acetate and phytohaemagglutinin increase the level of ribonuclease inhibitor in T cells. However, phytohaemagglutinin has no effect on the ribonuclease or inhibitor level of the B-cell-enriched population.     

A comparison of the effects of phytohaemagglutinin and of calcium ionophore A23187 on the metabolism of glycerolipids in small lymphocytes.

1. The effects of phytohaemagglutinin and of a Ca2+ ionophore (A23187) on glycerolipid metabolism in lymphocytes from pig lymph nodes were compared (a) by studying the incorporation of [32P]Pi and [3H]glycerol, and (b) by following the redistribution of [3H]glycerol among the lipids caused by these agents in pulse-chase experiments. 2. Phytohaemagglutinin only stimulated 32P incorporation into phosphatidylinositol and, to a slight extent, phosphatidate. Removal of most of the extracellular Ca2+ somewhat decreased this response. 3. Ionophore A23187 stimulated the labelling of phosphatidate and phosphatidylinositol with 32P to a much greater extent than did phytohaemagglutinin: the increase in phosphatidate labelling, but not that of phosphatidylinositol, was almost abolished by the removal of extracellular Ca2+. 4. The combined effects of phytohaemagglutinin and ionophore appeared to be additive, rather than synergistic. 5. Treatment with ionophore A23187 somewhat decreased the total incorporation of [3H]glycerol into glycerolipids, possibly because it lowered cell ATP content. In these experiments di- and tri-acylglycerol behaved anomalously, triacylglycerol labelling being suppressed completely, whereas that of diacylglycerol was enhanced. The pulse-chase results revealed that triacylglycerol was converted into diacylglycerol in the ionophore-treated cells, and the availability of this diacylglycerol probably led to the enhanced labelling of phosphatidate and phosphatidylinositol in the these cells. 6. Thus an increase in intracellular Ca2+ concentration appeared to have three effects on glycerolipid metabolism: (a) slight inhibition of some metabolic step preceding phosphatidate synthesis, (b) inhibition of diacylglycerol acyltransferase and (c) activation of a triacylglycerol lipase. 7. In contrast, it seems likely that the only effect of phytohaemagglutinin is to stimulate phosphatidylinositol breakdown. 8. Pig polymorphonuclear leucocytes treated with ionophore A23187 showed metabolic changes that were similar to those demonstrated with lymphocytes. 9. A possible similarity is suggested between Ca2+-stimulated triacylglycerol lipase in lymphocytes and polymorphonuclear leucocytes and previous observations of enhanced triacylglycerol metabolism in stimulated cells whose metabolic functions involve membrane fusion.     

Chromatography of human urinary erythropoietin and granulocyte colony-stimulating factor on insolubilized phytohaemagglutinin.

Human urinary erythropoetin was absorbed to phytohaemagglutinin coupled to agarose or porous glass and quatitatively eluted by a saturated solution of MgCl2. This method provides a means of separating erythropoietin from several of its contaminants, presumably on the basis of its carbohydrate side chains. Erythropoietin which had been purified by chromatography on insolubilized phytohaemagglutinin was sufficiently free of toxicity to be assayable in tissue culture even when crude urine was used as a starting material.     

A method for obtaining mitotic figures from blood leucocyte cultures of rainbow trout, Salmo gairdneri

A method for obtaining dividing cells for chromosome analysis from blood cultures of the rainbow trout, Salmo gairdneri , is described. The effects of different phytohaemagglutinin and foetal calf serum concentrations, oxygenation of the cultures, and varying the initial volume of cell inoculum on the number of mitoses obtained are described and discussed.     

Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.     

Response of enzymes involved in the metabolism of polyamines to phytohaemagglutinin-induced activation of human lymphocytes.

The stimulation of lymphocyte ornithine decarboxylase and adenosylmethionine decarboxylase produced by phytohaemagglutinin was accompanied by an equally marked, but delayed, stimulation of spermidine synthase, which is not commonly considered as an inducible enzyme. In contrast with the marked stimulation of these biosynthetic enzymes, less marked changes were observed in the biodegradative enzymes of polyamines in response to phytohaemagglutinin. Diamine oxidase activity was undetectable during all stages of the transformation. The activity of polyamine oxidase remained either constant or was slightly decreased several days after addition of the mitogen. The activity of polyamine acetylase (employing all the natural polyamines as substrates) distinctly increased both in the cytosolic and crude nuclear preparations of the cells during later stages of mitogen activation. Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, although powerfully inhibiting ornithine decarboxylase, produced a gradual enhancement of adenosylmethionine decarboxylase activity during lymphocyte activation, without influencing the activities of the two propylamine transferases (spermidine synthase and spermine synthase).     

Micronucleus formation in different lymphocyte subpopulations in peplomycin-treated and control cultures

Spontaneous micronucleus formation and micronucleus induction by peplomycin in B and T lymphocytes was studied by a recently developed MAC (Morphology, Antibody, Chromosomes) method allowing the immunologic identification of different cell lineages. Blood samples from 3 healthy donors were cultured in the presence of phytohaemagglutinin and pokeweed mitogen. An increased frequency of micronuclei was observed in peplomycin cultures compared with controls. B cells were found to be more sensitive to peplomycin induction than were T lymphocytes. In control cultures, pokeweed mitogen yielded a higher frequency of micronuclei than did phytohaemagglutinin. In both pokeweed- and phytohaemagglutinin-stimulated cultures, B cells showed a higher frequency of micronuclei than did T cells. The relative proportion of mitotic B cells was equal in pokeweed and phytohaemagglutinin cultures. In peplomycin cultures, the proportion of B cells decreased as compared with control cultures.     

Enhanced transport of natural amino acids after activation of pig lymphocytes.

Na+-dependent uptake of the amino acids L-proline and L-methionine was greatly accelerated when pig lymphocytes were activated with phytohaemagglutinin or other mitogens. The increased influx was apparent after incubation with phytohaemagglutinin for 1 h, and reached a maximum after 24 h. The lymphocytes appear to possess at least three different transport systems for neutral amino acids with properties similar to, but not identical with, those described for other cells. The activity of a system resembling the A system of other cells was increased most dramatically after activation, its activity in unstimulated lymphocytes being extremely low or absent. A second Na+-dependent system, which has properties similar to those of the ASC system in other cells, but with a broader specificity for amino acids, was more active in unstimulated lymphocytes, and uptake by this system was also accelerated after incubation with phytohaemagglutinin. The activity of a third system, very similar to the L system in other cells, was increased to a much smaller extent after lymphocyte activation.     

Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes : II. Relative importance of glycolysis and oxidative phosphorylation on phytohaemagglutinin stimulation

The effect of inhibition of the oxidative phosphorylation of human blood lymphocytes in the presence and absence of phytohaemagglutinin has been investigated. It was found that the incorporation of inorganic phosphate into acid-soluble nucleotides is dependent on, though not a direct measure of oxidative phosphorylation. Optimal concentration for inhibition of oxidative phosphorylation with oligomycin and the uncoupler 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) were determined. Under conditions of maximally inhibited P_i incorporation into acid-soluble nucleotides (80%) and maximally increased oxygen consumption and lactate production (4–5 times), the stimulatory effect of phytohaemagglutinin on several glycolytic parameters could still be observed. Therefore, stimulation of cellular processes by PHA is still possible when energy is provided by glycolysis only.     

Mitogen treatment of permeabilized human T lymphocytes stimulates rapid tyrosine and serine phosphorylation of a 42 kDa protein

Three agents which are mitogenic for T lymphocytes (phytohaemagglutinin, monoclonal antibody UCHT 1 and 12-O-tetradecanoylphorbol-13-acetate) stimulated rapid phosphorylation of a 42 kDa protein in permeabilized T lymphocytes. Phosphorylation occurred on tyrosine and serine residues. A non-mitogenic monoclonal antibody (RFT11) did not stimulate phosphorylation of this protein. Furthermore, the dose response of 42 kDa protein phosphorylation and of mitogenesis to increasing amounts of phytohaemagglutinin were closely similar. We therefore propose that mitogen-stimulated phosphorylation of the 42 kDa protein is part of the mechanism for transduction of mitogenic signals in lymphocytes. To our knowledge, this is the first report of rapid, ligand-stimulated tyrosine protein phosphorylation in T lymphocytes.     

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.