Effect of colchicine, vinblastine and cytochalasin B on human lymphocyte transformation by phytohemagglutinin

The effect of colchicine, vinblastine, and cytochalasin B has been investigated on the phytohemagglutinin (PHA) induced transformation and DNA synthesis of human lymphocytes. The three drugs produced, at an appropriate concentration, inhibition of DNA synthesis and lymphocyte transformation, if added prior to PHA. Inhibitory concentrations of cytochalasin B were no longer effective in preventing DNA synthesis if added 2 h after exposure to PHA; on the other hand, colchicine and vinblastine were effective even if they were added 16 h after PHA. Studies of lymphocyte aggregation to beads of Sepharose with chemically bound PHA suggest that the effects of these drugs do not seem to lie primarily on blocking PHA binding to the lymphocyte membrane, but rather on a subsequent step(s).     

鲻鱼的染色体组型研究

本文采用体内注射ＰＨＡ制备肾细胞染色体标本的方法，对鲻鱼的染色体组型进行了研究，其结果２ｎ＝４８，ＮＦ＝４８，型公式为４８ｔ。     

Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

IL 2 alone is mitogenic only for Tac-positive lymphocytes in human peripheral blood

To investigate the ability of interleukin 2 (IL 2) alone to induce proliferation of resting human lymphocytes, we stimulated human peripheral blood mononuclear cells (PBMC) with an immunopurified preparation of IL 2 or with phytohemagglutinin (PHA). Proliferation and the percent of cells expressing IL 2 receptors were assessed over 6 days of culture. Regardless of the stimulus, the percent of cells bearing an IL 2 receptor paralleled the amount of proliferation, and proliferation was inhibited by an anti-IL 2 receptor monoclonal antibody (anti-Tac). When stimulated by IL 2 alone, less than 8% of PBMC expressed an IL 2 receptor after 24 hr of culture. Stimulation by IL 2 caused both proliferation and IL 2 receptor expression to increase over the entire culture period (routinely to 75,000 cpm and 50% respectively). When colchicine was added (to inhibit cell division), the percent of cells bearing an IL 2 receptor did not increase. IL 2 alone also induced proliferation of PBMC depleted of accessory cells, with the same kinetics but reduced peak response. Both accessory cells and supernatants that showed IL 1 but not IL 2 activity augmented this proliferation 50 to 100%. In contrast to the effect of IL 2, 25 to 50% of PBMC stimulated by PHA expressed an IL 2 receptor after 24 hr of culture. PHA-induced proliferation and IL 2 receptor expression peaked early in the culture period (routinely to 100,000 cpm and 50% respectively within 3 days), and colchicine did not inhibit the early induction of IL 2 receptors on PBMC. Our findings indicate that unlike PHA, IL 2 induces proliferation of PBMC (or PBMC depleted of accessory cells) by expanding the small percentage of cells in a resting population that already express IL 2 receptors. IL 2 does not appear to induce IL 2 receptors on cells previously lacking this molecule. We also find that IL 1 can enhance the response to IL 2 alone.     

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.     

两性型天然雌核发育彭泽鲫染色体组型的研究

彭泽鲫是一种两性型天然雌核发育群体。染色体组型分析结果表明：雌、雄鱼的染色体数目均为１６６。它们的核型组成是：３２条中部着丝点染色体、４０条亚中部着丝点染色体、１８条亚端部着丝点染色体和７６条端部着丝点染色体。臂数（ＮＦ）为２３８。本研究结果与其它雌核发育类型的鲫鱼有明显的差别。     

Tubulin Polymerization Modulates Interleukin-2 Receptor Signal Transduction in Human T Cells

Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R β -chain and enhanced the association of the IL-2R γ -chain with cytoskeletal tubulin. The IL-2R β -chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level.     

Tubulin polymerization modulates interleukin-2 receptor signal transduction in human T cells

Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R ss -chain and enhanced the association of the IL-2R beta -chain with cytoskeletal tubulin. The IL-2R beta-chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO 11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level.     

Increased spindle resistance to antimicrotubule agents in women of high risk for meiotic nondisjunction

Summary Spindle sensitivity of phytohemagglutinin (PHA)-stimulated lymphocytes to three antimicrotubule drugs was compared in two groups of women who differ in their predisposition to meiotic aneuploidy: young women of low-risk age (ranging from 22 to 34 years) and middle-aged women of high-risk age (ranging from 40 to 52 years). Numerical sensitivity values for the antimicrotubule drugs, colchicine, podophyllotoxin, and vinblastine were obtained for each woman by recording the percentage of fully arrested metaphases out of the total metaphase cell population, i.e., cells exhibiting short, thick, and condensed chromosomes with sister chromatids clearly separated at their distal parts. Sensitivity increased linearly with increasing drug concentrations and was highly correlated with youth: its rate was significantly higher for women of the low-risk group. In addition, dividing lymphocytes of young mothers (26–33 years old) of Down syndrome children revealed significantly lower sensitivity to colchicine and podophyllotoxin than those of all young women of the low-risk group and similar sensitivity to that of the middle-aged women, i.e., the high-risk age group. The data are consistent with the theory that factors involved in meiotic nondisjunction may be concurrently operating in somatic cells. These factors presumably shift the equilibrium between tubulin and microtubules towards microtubules stabilization and thereby affect some of their functions.     

Cytokinetic basis for the impaired activation of lymphocytes from patients with primary intracranial tumors

Patients with malignant brain tumors have a variety of immunologic abnormalities, including the impaired responsiveness of peripheral blood lymphocytes (PBL) to mitogens and alloantigens. We further investigated this impairment of lymphocyte reactivity by employing the techniques of limiting dilution analysis and cytokinetic analysis. PBL preparations from patients have approximately six times fewer phytohemagglutinin (PHA)-responsive cells than PBL from normal subjects. Similar results were obtained with purified T cell preparations. Cytokinetic analysis of PHA-induced [3H]thymidine incorporation employing colchicine blocking of mitosis demonstrated that the number of first generation cells entering the S-phase of mitosis for each 24-hr period was less for PBL from patients than for PBL from normal individuals. First generation responding cells from patients and normal subjects entered DNA synthesis at the same time (48 to 72 hr). Cytokinetic analysis over a period of 168 hr demonstrated that whereas PBL from normal individuals demonstrated second generation responding cells, PBL from the majority of patients did not, thus indicating a defect in their ability to undergo clonal expansion. Measurement of interleukin 2 (IL 2) activity in culture fluids from PHA-activated PBL from normal subjects and patients revealed significantly lower IL 2 levels in culture fluids from PBL from patients. The addition of various concentrations of lectin-free IL 2 to PBL from patients stimulated with PHA did not restore responsiveness to normal values. There was no difference between the levels of interleukin 1 (IL 1) produced by lipopolysaccharide-activated monocytes from normal subjects and patients. Overall, these results suggest that an intrinsic defect exists in T cells obtained from brain tumor patients that renders them unable to enter into normal mitogen-induced blastogenesis.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

美丽硬仆骨舌鱼核型分析

美丽硬仆骨舌鱼(Scleropages formosus)是一种古老的具有很高经济价值的观赏鱼类。为了解该鱼的细胞遗传背景,采用胸腔注射植物血球凝集素(PHA)和秋水仙素的方法,以头肾为材料,空气干燥法制片,对美丽硬仆骨舌鱼的染色体进行了分析研究。结果表明,美丽硬仆骨舌鱼的二倍体染色体数目为50,核型公式为2n=2m+8sm+8st+32t,臂数(NF)为60。这一核型符合低等鱼类的基本特征,研究结果可为美丽硬仆骨舌鱼的种质标准和系统演化等提供基础数据。     

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.     

Changes in the organization and biosynthesis of cell surface acidic sugars during the phytohemagglutinin-induced blast formation of human T-lymphocytes

Use of cell electrophoresis combined with specific enzymes and varying ionic strength revealed a topological change of acidic sugars in lymphocyte membrane treated with a T-cell mitogen, phytohemagglutinin (PHA). The suggested alterations were an early translocation of hyaluronic acid to the cell periphery within 15 min of PHA addition and, 4 h later, the appearance of chondroitin sulphate in T-lymphocytes, but not in B-lymphocytes. As the contribution of chondroitin sulfate to the electrophoretic mobility increased with time up to 24 h, that of sialic acid decreased conversely. Several agents which block blast formation (2 mM ethylene glycol bis-β-aminoethylethyl-N,N,N′,N′-tetraacetic acid, 2 × 10⁻⁷ M ouabain, 0.1 μg/ml colchicine and 1 μg/ml cytochalasin B) also blocked the translocation of hyaluronic acid at the same concentrations. Chemical analysis of [¹⁴C]glycosaminoglycans by means of gel filtration followed by paper chromatography revealed a four-fold enhancement of the biosynthesis of chondroitin sulfate C after PHA stimulation. The presence of chondroitin sulfate in the cell periphery was also detected electrophoretically in T-cell type leukemia cells (MOLT-4B). These results suggest that the reorganization of glycosaminoglycans may be one of the membrane changes associated with blast formation of lymphocytes.     

Serum and lymphocyte activation by phytohaemagglutinin (PHA)

Activation of rat lymph-node cells in homologous serum was assessed by measuring the enhanced labelling of cells with ³H-uridine produced by PHA during a 4–6 h culture period. The degree of activation was proportional to the ratio of PHA to serum macromolecules (non-dialysable molecules) over a wide range of macromolecule concentrations. The possibility that PHA activates cells indirectly by reacting with a serum macromolecule which normally inhibits activation, was examined. No activation was produced by incubating cells in macromolecule-depleted media in the absence of PHA. Some activation was produced in macromolecule-depleted media at low PHA concentrations, but the extent of activation was only 39% of that produced in the presence of macromolecules. The results indicate that serum macromolecules both buffer cells against reaction with PHA and facilitate either the reaction of cells with PHA or the immediate cellular response to that reaction.     

鲀形目3种鱼的染色体组型分析

采用胸腔注射植物血球凝集素(phytohemagglutinin,PHA)及秋水仙素溶液,取活体头肾细胞经低渗、固定、空气干燥法,分析比较了中华单角鲀(Monacanthus chinensis)、黄鳍东方鲀(Takifuguxanthopterus)、红鳍东方鲀(T.rubripes)的核型。结果表明,3种海水鱼中期染色体均为二倍体,未发现异型性染色体、随体和次缢痕。其核型如下:中华单角鲀的核型为2n=34(34t),臂数:NF=34;黄鳍东方鲀的核型为2n=44(12m+8sm+24t),臂数:NF=64;红鳍东方鲀的核型为2n=44(14m+6sm+24t),臂数:NF=64。中华单角鲀的核型与后两者存在较大差异。同时,将此3种鱼的核型与前人报道的其他鲀形目鱼类核型作了比较。     

Genetic study of the resistance of mouse somatic cells to colchicine (high resistance level)

The studies of the high level of colchicine resistance of mouse L cells have shown that two mutagens (EMS and NMM) do not induce cell variants resistant to 8 microgram/ml of colchicine in the population of mouse heteroploid L-53 cells (subline of L cells, the level of colchicine resistance 140) and that colchicine resistance of L-53 cells gradually diminishes when cells are propagated in non-selective conditions: after 1 month it diminishes 2-fold, after 3 month--9-fold. The extent of the decrease of the drug resistance was the same in 6 independent cultures obtained from the inoculum of 200 cells and in control cultures propagated by large quantities of cells. These data coincide with the results of the previous studies of lower level of colchicine resistance. In both studies the frequency of the occurrence of colchicine resistant variants in selective medium was about 2.10(-4). These data are consistent with the hypothesis that colchicine resistance of mouse L cells is not due to a gene mutation.     

Differences between cystic fibrosis and normal cells in the degree of satellite association

Summary The degree of satellite association was found to be significantly higher in phytohemagglutinin (PHA)-stimulated lymphocytes from cystic fibrosis (CF) patients than from those of control individuals. PHA-stimulated lymphocytes from obligatory heterozygotes for the CF mutant allele showed an intermediate degree of satellite association. The degree of satellite association was estimated by the frequency of cells exhibiting associations, by the number of associations per cell, and by the number of chromosomes in an association. The differences in the degree of satellite association were dependent on the concentration of colchicine used for cell arrest. These findings may assist in developing a diagnostic method for the early identification of heterozygotes for the CF allele and for prenatal detection of CF homozygous fetuses.This paper is based on a portion of a dissertation to be submitted by Y. Ravia in partial fulfilment of the Ph. D. requirements in the Graduate School of Tel Aviv University     

Modulation of membrane drug permeability in Chinese hamster ovary cells

The kinetics of colchicine uptake into Chinese hamster ovary cells have been investigated and found to be consistent with an unmediated diffusion mode. A variety of compounds such as local anesthetics and non-ionic detergents as well as drugs such as vinblastine, vincristine, daunomycin and actinomycin D potentiate the rate of colchicine uptake into these cells and into colchicine resistant mutants. In all cases, higher concentrations of these compounds were required to stimulate colchicine uptake in the colchicine resistant mutants than in the cells of the parental line. This stimulation was observed also in the uptake of puromycin, a structurally and functionally different drug. These stimulatory agents did not, however, cause the cells to become nonspecifically leaky since the uptake of 2-deoxy-d-glucose was unaffected. In addition, the activation energy of colchicine uptake was unaltered in the presence of stimulating agents, implying that they were not causing colchicine to enter the cells via a different mechanism. The results are compatible with the view that these compounds are membrane-active, and are able to stimulate an increased rate of unmediated diffusion of colchicine into the cells. It appears that a mechanism for the regulation of passive permeability is modified in the resistant mutants.     

Effect of some drugs on colchicine uptake by colchicine-sensitive and resistant cells

The effect of several agents on 3H-colchicine, uptake by L cells and resistant to colcemide and colchicine L-53 cells was studied. Vinblastin to which L-53 cells are cross-resistant increases labeled colchicine uptake by L and L-53 cells 3- and 8-fold, respectively. The substances which decrease ATP level in the cells (olygomycin, etc.) enhance colchicine uptake by L and L-53 cells 2--4-fold. In the presence of these substances colchicine uptake by resistant cells is more intensive than by sensitive L cells. The structural analogue of colchicine, lumicolchicine, inactive in binding the microtubular protein tubulin enhances colchicine uptake by L and L-53 cells to about equal degree.