Inhibition by colchicine of the mitogenic stimulation of lymphocytes prior to the S phase

Colchicine, vinblastine, and vincristine inhibit the mitogenic stimulation of lymphocytes by concanavalin A as measured by the incorporation of [3H]thymidine and the appearance of blast cells. The inhibitory effect of colchicine could not be accounted for by diminution in cell viability or by metaphase arrest of mitosis in the stimulated cells. Moreover, the inhibition of [3H]thymidine incorporation was not due to blockage of thymidine transport or inhibition of DNA synthesis inasmuch as addition of colchicine had no effect on cells in the S phase of the cell cycle. The time of inhibition was correlated with the kinetics of cellular commitment to lectin activation and the kinetic data indicated that colchicine blocks stimulation early in the sequence of events following addition of the mitogen. These findings support the hypothesis that cytoplasmic microtubular function plays a role in the commitment of resting cells to undergo mitotic division.     

The kinetics of cellular commitment during stimulation of lymphocytes by lectins

The kinetics of cellular commitment in the stimulation of lymphocytes by concanavalin A (Con A) has been analyzed by measurement of DNA synthesis, autoradiography, and histologic staining techniques. If the competitive inhibitor α-methyl-D-mannoside (αMM) is introduced into cultures of mouse spleen cells at various times after the addition of Con A, there is a gradual decrease in its capacity to inhibit the lectin-stimulated incorporation of [³H]thymidine. Addition of the saccharide 20 h after exposure of the cells to Con A had no effect on the level of the cellular response to the lectin. With increasing periods of contact with Con A, the percentage of blast cells and the percentage of [³H]thymidine-labeled blast cells increased in parallel with the total radioactive thymidine incorporated while the average number of autoradiographic grains per labeled blast cell remained relatively constant. These observations suggest that the rising level of [³H]thymidine incorporation results from an increase in the number of cells that respond to lectin stimulation and become refractory to inhibition with αMM. Once such cells become committed, they synthesize DNA at a rate independent of the length of exposure to the lectin. The combined results indicate that mouse splenic lymphocytes are heterogeneous in their capacities to respond to Con A and that different cells require different induction periods to be stimulated.     

Concanavalin A affects beta-tubulin mRNA expression during neuritic processes of mouse neuroblastoma N18TG2 cells in a different manner from colchicine

Addition of concanavalin A (Con A) to mouse neuroblastoma N18TG2 cells cultured with dibutyryl-cAMP which can stimulate neurite outgrowth, stopped the neuritic processes effectively. The extended neurites showed a gradual retraction for at least 8 hrs after addition of Con A, while addition of colchicine caused rapid retraction of the neurites. Immunocytochemistry showed that the addition of Con A did not disorganize the microtubules but the addition of colchicine did. The increase in beta-tubulin mRNA expression which was observed after cell culture and after stimulation by dB-cAMP was suppressed by the addition of Con A. Con A did not affect the beta-tubulin mRNA expression when the cells had already been cultured, while colchicine drastically decreased it. Thus, Con A appeared to affect the beta-tubulin mRNA expression in a different manner from colchicine, probably through inhibition of cell movement.     

Microtubules and lymphocyte responses: effect of colchicine and taxol on mitogen-induced human lymphocyte activation and proliferation

The role of microtubules in mitogen-induced human lymphocyte activation and proliferation was examined. The effect of colchicine, a microtubule-disrupting agent, was compared with taxol, a microtubule-stabilizing drug, and with isaxonine (N-isopropyl-amino-2-pyrimidine orthophosphate), a proposed microtubular-active drug. Lymphocyte proliferation, assessed by measuring the increase in the number of cells in mitogen-stimulated cultures, was completely suppressed by both colchicine and taxol (100 nM) whereas significant inhibition by isaxonine required much higher concentrations (5 mM). In order to characterize the inhibition, initial lymphocyte blast transformation and subsequent DNA synthesis were investigated. Neither colchicine nor taxol inhibited lymphocyte blast transformation assessed by quantitating the change in volume of the stimulated cells after a 24-hour incubation. In contrast, isaxonine (2-5 mM) suppressed blast transformation. Initial DNA synthesis, evaluated by measuring the cumulative incorporation of [3H]thymidine between 30 and 48 hours of culture, was inhibited in a concentration-dependent manner by both isaxonine and colchicine but not by taxol. Electron microscopic studies confirmed that both taxol and colchicine (10 nM) arrested the responding lymphocytes in mitosis, and that isaxonine inhibited initial activation. These results suggest that normal microtubule function is only necessary for cell division and that drug effects on blast transformation and initial DNA synthesis are unrelated to microtubules.     

Human recombinant interleukin-1 receptor antagonist inhibits lymphocyte blastogenesis induced by concanavalin A. Restorative effect of hrIL-1.

Interleukin-1 (IL-1), mainly produced by monocyte-macrophages, is a polypeptide cytokine with pleiotropic biological effects. IL-1 plays an important role in mediating immune response and inflammation. Recently a natural inhibitor to IL-1 has been discovered, interleukin-1 receptor antagonist (IL-1ra), produced by human monocytes cultured on adherent IgG which binds to the IL-1 receptors. In our study we found that the pretreatment of cells with serial dilutions of IL-1ra (250 ng/ml-2.5 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with Con A (10 micrograms/ml). IL-1ra did not have any effect on resting peripheral blood mononuclear cells (PBMC). Time course experiments show that IL-1ra at 250 ng/ml has its maximum inhibitory effect on lymphocyte blastogenesis when cells are pretreated 2 h before Con A. No effect was found when hrIL-1ra was added after Con A. Moreover, hrIL-1ra also inhibits the enhancing effects of exogenous hrIL-1 (400, 200, 100 and 50 ng/ml) on lymphocytes stimulated with Con A; while when hrIL-1ra was used on cells treated with only Con A, the inhibition was more pronounced. When PBMC were removed from monocytes, by adherence, the Con A-treated lymphocytes were not influenced by 2 h pretreatment of hrIL-1ra; while a strong inhibition was found when exogenous hrIL-1 was added at different concentrations. In addition, hrIL-1ra also inhibits the enhancing effect of hrIL-2 on lymphocyte DNA synthesis. In another set of experiments PBMC were pretreated with hrIL-1ra (250 ng/ml) for 2 h and then added LPs (10 ng/ml) and IL-1 alpha generation was determined using ELISA. In these experiments IL-1ra completely abolished the generation of IL-1 alpha. These data suggest that hrIL-1ra exhibits a dose-response inhibition of lymphocyte blastogenesis induced by Con A, probably through the down-regulation of IL-1 synthesis necessary as an early signal for T-cell activation and IL-2 production.     

Studies on the activation mechanism of human mononuclear leukocytes isolated by physical methods

Responder lymphocytes, accessory lymphocytes and monocytes were isolated using countercurrent centrifugal elutriation and free flow electrophoresis. The Concanavalin A (Con A) binding behaviour of the isolated cell populations was studied. Responder lymphocytes and accessory cells bound Con A by different mechanisms. Cells in all the isolated populations were able to interact directly with Con A. Con A binding to responder lymphocytes was inhibited by alpha-methylmannoside (alpha MM) and by a distinct plasma protein. Accessory lymphocytes and monocytes bound Con A even in the presence of 10% human plasma or 50 mM alpha MM. The plasma protein which inhibited interaction of responder lymphocytes with Con A also reduced lymphocyte proliferation, when resting monocytes were used as accessory cells. If, however, monocytes were used after activation by lipopolysaccharide no inhibition effect was observed. From the results we conclude that there is a distinct plasma protein which protects the organism against lymphocyte stimulation not controlled by accessory cells. This inhibitory plasma protein appears to be a euglobulin.     

The relationship between the disassembly of microtubules during G1 and the enhancement of DNA synthesis by colchicine in mouse fibroblasts stimulated with peptide growth hormones

By immunofluorescent staining to visualize the cytoplasmic microtubular cytoskeleton in mouse fibroblasts we have ascertained that after a relatively short exposure of cells to colchicine, microtubules remain disassembled for a prolonged period of time after cells are transferred to a colchicine-free medium. In contrast to the persisting effects of colchicine, a brief exposure of cells to nocodazole first induces the expected disruption of microtubules followed by regeneration of the cytoskeleton within a few hours after removal of extracellular drug. These results shed light on our previous finding that quiescent mouse fibroblasts first treated with colchicine and then transferred to colchicine-free medium exhibit an enhanced proliferative response to EGF and insulin, whereas cells treated in a similar manner with nocodazole show no enhancement of DNA synthesis stimulated by peptide growth hormones. We conclude that cytoplasmic microtubules must remain disaggregated during the prereplicative G1 period in order for cells to exhibit the enhancing effects of the microtubule-disrupting drugs on DNA synthesis.     

Time-dependent inhibition of tuberculin-induced lymphocyte DNA synthesis by a serine protease inhibitor

Diisopropylfluorophosphate (DFP), a group-specific irreversible inhibitor of serine proteases, has been shown to exert time-dependent inhibition of DNA synthesis of lymphocytes stimulated by three different B lymphocyte mitogens: purified protein derivative of tuberculin (PPD), endotoxin protein (EP), and lipopolysaccharide (LPS). The time-dependent inhibition profile found in B lymphocytes is absent in concanavalin A (Con A)-stimulated T lymphocytes. Structural analogs of DFP, which have lost the phosphorylating ability, are not inhibitory. Inhibition of DNA synthesis by DFP is reversible in the first 8 hr of mitogenic stimulation. Maximal and irreversible inhibition by DFP occurs around the 16th hour of stimulation. These data support the postulate that a mitogenesis-linked protease, or proteases, in B lymphocytes is absent in the resting cells but is made available several hours before the initiation of DNA synthesis in the late G1 phase of the cell cycle.     

Taxol protects the microtubules of concanavalin A-activated lymphocytes from disassembly by methylmercury, but DNA synthesis is still inhibited

We have shown previously that there is a good correlation between the degree of microtubule disassembly by methylmercury (MeHg) and the extent of inhibition of DNA replication in Concanavalin A (Con A)-stimulated mouse splenic lymphocytes. The purpose of this study was to determine if these two events are causally related and to examine the effects of MeHg-induced microtubule disassembly on earlier events of the stimulation process. We show that early steps constituting the activation pathway, such as the Con A-induced increase in Ca2+ influx and the expression of interleukin 2 receptor, are not inhibited by concentrations of MeHg that disassemble microtubules. RNA synthesis is not affected by short-term (3 h) treatment with MeHg, but longer treatment (24 h) inhibits RNA synthesis. In contrast, DNA synthesis is effectively inhibited by a 3-h treatment with MeHg. In lymphocytes treated with taxol, microtubules are not disassembled by MeHg; however, the inhibition of RNA and DNA synthesis persists. We conclude that the inhibition of nucleic acid synthesis by MeHg is not causally related to MeHg-induced microtubule disassembly.     

Antigenic stimulation of lymphocytes I. Calorimetric exploration of metabolic responses

The rate of evolution of beat, an index of overall metabolic activity, has been measured for lymphocytes undergoing simple culture as well as in the presence of the specific antigen, DNP-BSA (dinitrophenylated bovine serum albumin), of the mitogenic lectin Con A (concanavalin A), and of inhibitors of various aspects of cell culture. Unstimulated lymphocytes evolve heat initially at a decreasing rate which stabilizes after 3 days of culture. Cells stimulated with antigen increase thermogenesis after 2 days of exposure to a maximum at 4–5 days, on a schedule that parallels increases in DNA synthesis. Cells stimulated with Con A show early increases in heat output which precede the onset of increased DNA synthesis. Inhibition of DNA synthesis alone does not significantly inhibit heat output, whereas inhibition of protein and RNA synthesis has a profound effect, as does interference with the structure of microfilaments with cytochalasin B. The completely synthetic multivalent high molecular weight antigen, PEO-DNP₈₀ (dinitrophenylated polyethylene oxide) is capable of abolishing the response of the cells to DNP-BSA and to Con A as well as of directly inhibiting the output of heat by the cells.     

Loosening of cell cycle controls of human lymphocytes under the action of tumour promoter TPA

The effect of tumour promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on the cell cycle of human peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro was studied and it was found that TPA caused cells to accumulate in all the cell cycle phases. This accumulation took place preferentially at later culture passages, when lymphocytes stimulated by PHA alone stopped mainly in G0/G1 phases. Other effects of TPA were cell induction to enter higher DNA ploidy and to survive and even synthesize DNA under colchicine block of mitosis or under cytochalasin block of cytokinesis. In addition, in experiments in which a transitory block through the G1 phase of cell cycle was applied with use of aminopterin, we could show that a fraction of TPA-treated cells still entered the active phase of DNA synthesis. These findings suggest that TPA causes cell cycle controls to become loose, thereby enhancing adaptability of human lymphocytes to various hindrances in the course of cell cycle and eventually causing them to acquire characteristics known to be common for tumour cells.     

Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis

Microtubule disrupting drugs initiated DNA synthesis in serum-free cultures of nonproliferating fibroblast-like cells. The addition of colchicine to chick, mouse and human fibroblasts in serum-free medium stimulated thymidine incorporation at least twofold, with a half-maximal concentration of 1 X 10(-7) M. This stimulation represented up to 75% of the maximal stimulation by thrombin and was paralleled by an increase in the percentage of labeled nuclei. Other microtubule disrupting drugs showed similar stimulation, whereas lumicolchicine had no effect. Indirect immunofluorescent staining of tubulin showed a correlation between microtubule depolymerization and initiation of DNA synthesis by these drugs. A 2 hr treatment with 10(-6) M colchicine caused complete disruption of the microtubular network and stimulated thymidine incorporation (measured 28 hr later) to an even greater extent than continuous colchicine exposure. A similar 2 hr exposure to 10(-6) M colcemid also stimulated thymidine incorporation and led to a 50% increase in cell number. Taxol, a drug which stabilizes cytoplasmic microtubules, blocks initiation of DNA synthesis by colchicine, indicating that microtubule depolymerization is necessary for this initiation. To determine if microtubule depolymerization is involved in stimulation of DNA synthesis by other growth factors, highly purified human thrombin was added to cells with or without colchicine. In no case did colchicine plus thrombin increase DNA synthesis above that of the maximal stimulation by thrombin alone. Furthermore, pretreatment of cultures with taxol (5 micrograms/ml) inhibited approximately 30% of the stimulation of thymidine incorporation by thrombin. Together, these studies demonstrate that microtubule depolymerization is sufficient to initiate both DNA synthesis and events leading to cell division and suggest that microtubule depolymerization may be a required step in initiation of cell proliferation by growth factors such as highly purified human thrombin.     

Commitment and proliferation kinetics of human lymphocytes stimulated in vitro: Effects of colchicine on mitogen response

We have examined the effects of colchicine on concanavalin A (Con A)- and phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes and from the time course of proliferation have extracted the relative size of the responding cell population, the rate of entry of this population into S-phase, and the length of the lag period. Additions of colchicine at any time did not appear to influence the size of the responding population nor did it greatly affect the duration of the lag period. Only the rate at which the cell population enters initial S-phase is a function of the time of previous exposure to colchicine. Colchicine does not appear to inhibit the commitment of stimulated lymphocytes to enter the cell cycle. Rather, it merely serves to decrease the biochemical processes responsible for fixing a maximal rate of entry into S-phase.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.     

Effects of estramustine, a new anti-microtubule drug, on the induction of casein gene expression by prolactin

Estramustine, a new anti-microtubule drug, was added to the culture medium of rabbit mammary explants with lactogenic hormones. In the absence of the drug, prolactin with insulin and cortisol stimulated DNA synthesis and it induced beta-casein and beta-casein mRNA accumulation in the tissue. As opposed to other anti-microtubule agents such as colchicine, estramustine was unable to prevent prolactin actions. An examination of the mammary cells by immunofluorescence revealed that the microtubule network was significantly altered under the influence of estramustine. These data indicate that the integrity of microtubules is not required for prolactin to deliver its message to the mammary cell. These data also suggest that other anti-microtubule drugs such as colchicine which prevent prolactin action act through their binding to tubulin molecule unrelated to microtubule structures.     

Inhibition of lymphocyte activation by cyclosporin A: interference with the early activation of the membrane phospholipid metabolism in rabbit lymphocytes stimulated with concanavalin A, anti-rabbit immunoglobulin or the Ca2+ ionophore A 23187

Rabbit lymphocytes from the mesenteric lymph nodes were stimulated with concanavalin A, goat anti-rabbit immunoglobulin, or the Ca2+ ionophore A 23187. The stimulated incorporation of labeled uridine into RNA as well as of labeled thymidine into DNA was suppressed within a dose range of 40-1000 ng/ml cyclosporin A in both Con A-stimulated T lymphocytes and in anti-immunoglobulin-stimulated B lymphocytes, without affecting the resting cells. A 23187-stimulated rabbit lymphocytes proved to be more sensitive to cyclosporin A. At 40 ng/ml the immunosuppressive drug was effective in inhibiting elevated incorporation of labeled nucleosides into macromolecules in ionophore-stimulated cells. Cyclosporin A, at the same concentrations that were effective in inhibiting stimulated RNA and DNA synthesis, suppressed one of the earliest events occurring in stimulated lymphocytes, i.e., enhanced incorporation of unsaturated fatty acids into membrane phospholipids. Whereas cyclosporin A significantly inhibited the incorporation of arachidonic acid into phosphatidylcholine and phosphatidylethanolamine in concanavalin A-, anti-immunoglobulin-, and A 23187-stimulated cells, it proved to be ineffective in inhibiting the incorporation of arachidonate into phosphatidylinositol. The data indicate that cyclosporin A inhibits both T- and B-cell stimulation by interfering with a common target, e.g., the early activation of membrane phospholipid metabolism of rabbit lymphocytes.     

Advance toward S phase and retreat toward deeper "G0" states in resting 3Y1 cells with environmental changes

To elucidate conditions which affect the lag time for resting cells to enter S phase after serum stimulation, we used a wild-type 3Y1 rat fibroblast line and four temperature-sensitive mutants of 3Y1 (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203). Among these five lines, in only tsG125 cells was there an obviously prolonged lag time with increase in time in resting state at 33.8 degrees C. The resting wild-type 3Y1 cells, preexposed to 39.8 degrees C, also showed a prolongation of lag time. The prolongation in tsG125 had a certain limit. Preexposure to 39.8 degrees C before serum stimulation accelerated such prolongation in tsG125 to its limit, but did not change the limit, per se. Resting tsG125 cells stimulated by serum at 39.8 degrees C, did not enter S phase, yet they did advance toward S phase. When they were kept at 39.8 degrees C, they retreated toward a deeper resting state ("G0") with time. These retreats correlated with the decrease in stimulating activity in the culture media. About 20% of the resting tsG125 cells stimulated by serum at 39.8 degrees C were committed to enter S phase, when the extent of commitment was examined at 33.8 degrees C. Most of the tsG125 cells committed at 33.8 degrees C did not enter S phase, when the extent of commitment was examined at 39.8 degrees C. More cells were committed after stimulation at 33.8 degrees C than at 39.8 degrees C, when the test was done at 33.8 degrees C. We suggest that resting cells may be reversibly changed within range of resting states, in either direction, that is, advance toward S phase or retreat toward deeper "G0." These changes may be determined by alterations in the balance between synthesis and decay of the preparedness for the initiation of DNA synthesis caused by cellular response to environmental changes (e.g., medium activity, temperature, etc.). The ts defect in tsG125 may affect the cell cycle progression, both before and after commitment by serum.     

Inhibition of the mitogenic response to lipopolysaccharide (LPS) in mouse spleen cells by polymyxin B.

The addition of low doses of the cationic polypeptide antibiotic, polymyxin B (PB), to cultures of mouse spleen cells inhibits lipopolysaccharide-(LPS) induced DNA synthesis but not that stimulated by PPD, PHA, or Con A. Inhibition is stoichiometric; the mitogenic response is suppressed by 50% at a weight ratio of PB:LPS of 0.055 to 1. Furthermore, PB-LPS complexes have a much reduced mitogenic capacity. These complexes inhibit the mitogenic response of spleen cells to unmodified LPS but not to PPD, Con A, or PHA. The inhibitory activity of PB is less effective when added after LPS is mixed with responding cells, achieving 50% inhibition when addition is made at 4 to 6 hr. Time course experiments indicate that partial inhibition is a reflection of a lower rate of DNA synthesis. Thus, PB inhibition of LPS mitogenesis apparently occurs as a result of formation of PB-LPS complexes with reduced mitogenic capacity. Specific inhibition by the complexes of mitogenesis induced by native LPS suggests that the inactive complex may bind to B cells but is unable to trigger them.     

Changes in organization and microtubule assembly activity of the centrosome during lymphocyte stimulation

Changes in the organization of centrosomes in mouse splenic T lymphocytes stimulated by concanavalin A (con A) were examined by electron microscopy of serial sections. In both resting and stimulated lymphocytes the single centrosome consists of a pair of centrioles, satellite bodies, and pericentriolar material. In resting cell centrosomes the satellite bodies are preferentially associated with, and appear to be attached by short stalks to, one of the centrioles. The satellite bodies are the primary sites of microtubule termination in the resting cell centrosome. During stimulation by con A there is a several-fold increase in microtubule content. This is correlated with an overall increase in centrosome size, an apparent increase in the size and in the number of satellite bodies, and a redistribution of satellite bodies to occupy a position between the two centrioles. Increased numbers of microtubules are detected terminating on the satellite bodies and in the pericentriolar material of the stimulated cell centrosome. Microtubule assembly from centrosomes in vitro was assessed by electron microscopy using detergent-permeabilized lymphocytes that had been pretreated to remove endogenous microtubules and supplied with purified bovine brain tubulin. These studies indicate that satellite bodies are major sites of microtubule assembly in both resting and stimulated cell centrosomes and show that the centrosomes of stimulated cells assemble more microtubules in vitro than resting cell centrosomes. This parallels the increase in microtubule content in intact lymphocytes stimulated by con A and suggests that the changes in centrosome organization and microtubule assembly capacity that occur during stimulation are causally related.