Hyperthermia induces apoptosis in thymocytes.

Mild hyperthermia (43 degrees C for 1 h) induces extensive double-stranded DNA fragmentation and, at a later time, cell death in murine thymocytes. The cleavage of DNA into oligonucleosome-sized fragments resembles that observed in examples of apoptosis including radiation-induced death of thymocytes. Following hyperthermia, incubation at 37 degrees C is necessary to detect DNA fragmentation, although protein and RNA synthesis do not seem to be required. Two protein synthesis inhibitors, cycloheximide and emetine, and two RNA synthesis inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, do not inhibit DNA fragmentation or cell death in heated thymocytes at concentrations which significantly block these effects in irradiated thymocytes. We have used this difference in sensitivity to show that the DNA fragmentation induced in thymocytes which are irradiated and then heated seems to be caused only by the heating and not by the irradiation.     

Activation of mouse lymphocytes inhibits induction of rapid cell death by x-irradiation

A distinctive property of the resting lymphocyte is its ability to die rapidly in interphase after x-irradiation. Suspensions of thymocytes and peripheral lymphocytes from BALB/c mice were irradiated with doses ranging from 10 to 10,000 rad (0.1 to 100 Grays), and their viability was measured by eosin dye exclusion at intervals through 3 days of culture. After an initial latent period of a few hours, viability declined exponentially in a dose-dependent fashion. Doses as low as 20 rad caused some lymphocytes to die rapidly. After 1000 rad, 90% of the cells became nonviable in 15 to 20 hr and 99% in 25 to 35 hr. Peripheral lymphocytes showed a somewhat earlier loss of viability than did thymocytes, and were killed especially rapidly by 10,000 rad. Enriched T cells and B cells were killed by irradiation at equal rates, and medullary thymocytes were killed at the same rate as the whole thymocyte population. In contrast with resting cells, T and B lymphocytes activated by mitogens were not subject to such rapid induction of cell death. Irradiation with 1000 rad reduced the viability of activated cells by only 50% at a time when less than 1% of nonstimulated lymphocytes remained alive. Similarly, cloned lines of antigen-specific helper and cytotoxic T cells showed only a delayed and slow loss of viability after receiving 1000 rad. The state of activation can therefore be a significant determinant of the immunologic consequences of irradiation.     

Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death

Dexamethasone and corticosterone kill mouse thymocytes, as measured by eosin uptake, after several hours of in vitro incubation. This killing requires RNA and protein synthesis, because it is inhibited by cycloheximide, emetine, or actinomycin D. An earlier event than cell death is the extensive fragmentation of nuclear DNA into oligonucleosomal subunits; this fragmentation also requires RNA and protein synthesis. The DNA cleavage results from the action of an endonuclease that preferentially cleaves DNA in the linker regions between nucleosomes. This endonuclease is found constitutively in the nuclei of thymocytes and some other cells, and requires calcium and magnesium ions for its activation; if isolated fresh thymocyte nuclei are incubated with these ions, as much as 77% of their DNA is cleaved within 90 min. Thus, the protein for which synthesis is necessary for glucocorticoid-induced thymocyte death is not the endonuclease itself, but is in some way involved in its activation; we suggest that it may be part of a system for transporting calcium into the nucleus. The endonuclease is inhibited by zinc, which also prevents thymocyte death. It appears that glucocorticoids cause thymocyte death by activating an enzyme that rapidly and extensively degrades DNA. This "death from within" is biochemically and morphologically different from toxic or accidental cell death, such as that induced by azide, heat, or antibody and complement treatment. Although mature T cells also contain the endogenous endonuclease, they lack the glucocorticoid-inducible mechanism for activating it, and are thus glucocorticoid-resistant.     

Rapid turnover of endogenous endonuclease activity in thymocytes: effects of inhibitors of macromolecular synthesis

Previous work has shown that inhibitors of protein or mRNA synthesis block endonuclease activation in thymocytes undergoing programmed cell death. In the present study we used isolated nuclei to investigate the effects of cycloheximide and actinomycin D, inhibitors of protein and mRNA synthesis, respectively, on endogenous endonuclease activity in thymocytes. We observed a rapid loss of Ca2(+)-dependent endonuclease activity in nuclei isolated from thymocytes treated with these inhibitors. In contrast, pretreatment of cells with antipain and leupeptin, inhibitors of proteases, prevented the depletion of endonuclease activity in the nuclei, suggesting that proteolysis was involved. The effects of cycloheximide and actinomycin D were mimicked by incubating thymocytes with treatments known to exert their effects via activation of protein kinase C. Our results suggest that endonuclease activity in thymocyte nuclei undergoes rapid, spontaneous turnover. Agents interfering with macromolecular synthesis may therefore block DNA fragmentation in thymocytes by depleting nuclei of endogenous endonuclease activity.     

Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.     

12-O-tetradecanoylphorbol 13-acetate induces DNA cleavage at linker regions in mouse thymocytes

A phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), induced the cleavage of nuclear DNA at linker regions in cultured mouse thymocytes. Similar DNA fragmentation was induced by 1-oleoyl-2-acetyl-glycerol, a synthetic diacylglycerol, but not by 4 alpha-phorbol-12,13 didecanoate. The DNA fragmentation was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine dihydrochloride, an inhibitor of protein kinase C, as well as actinomycin D and cycloheximide. It appears that TPA induces DNA cleavage through activation of protein kinase C and synthesis of yet unidentified protein(s). That the inhibition of DNA fragmentation was accompanied by a reduction in cell lysis suggests a causal relationship between DNA fragmentation and cell death.     

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.     

Radiation-induced formation of apoptotic bodies in rat thymus

The process of interphase death of thymocytes in whole-body X-irradiated rats were studied. Cell size distribution analysis indicates that cell fragments (= apoptotic bodies) appeared in the thymus and increased in number depending on dose (200-1000 R) and time (2-6 hr) after irradiation with corresponding decrease in normal-size thymocytes. Occurrence of nuclear fragmentation in association with the cellular fragmentation was proved with cytofluorometric determination of DNA content in individual cells. Scanning electron microscopic observations also revealed extensive fragmentation of cells in the irradiated rat thymus. The results show clearly that cells as well as nuclei fragment rapidly into smaller pieces of various sizes in the irradiated rat thymus as commonly observed with apoptosis.     

DNA fragmentation induced in macrophages by gliotoxin does not require protein synthesis and is preceded by raised inositol triphosphate levels.

We have shown that the immunomodulating agent gliotoxin induces DNA fragmentation in macrophages characteristic of programmed cell death or apoptosis (Waring, P., Eichner, R. D., Mullbacher, A., and Sjaarda, A. (1988) J. Biol. Chem, 263, 18493-18499). In addition, morphological changes and DNA fragmentation characteristic of apoptosis are induced in 48 h concanavalin A-stimulated T blasts by gliotoxin and these changes are inhibited by Zn2+ (Waring, P., Egan, M., Braithwaite, A., Mullbacher, A., and Sjaarda, A. (1990) Int. J. Immunopharmacol., in press). We have studied the effects of actinomycin D and the protein synthesis inhibitor cycloheximide on apoptosis induced by gliotoxin in these cells, and these studies demonstrate no effect on apoptosis induced by gliotoxin. Cycloheximide and actinomycin D alone induce DNA fragmentation in these cells. Gliotoxin itself proved to be a potent inhibitor of protein synthesis. The fragmentation caused by cycloheximide correlated with the extent of protein synthesis inhibition. The toxin ricin also induced DNA fragmentation in T blasts characteristic of apoptosis. These results indicate that protein synthesis is not required for induction of apoptosis in macrophages or T blasts by gliotoxin. Gliotoxin caused elevated levels of inositol triphosphate in treated macrophages which may be related to mobilization of Ca2+ levels during apoptosis.     

Activation of poly(ADP-ribose)-polymerase inhibits apoptosis of thymocytes, caused by short-wave ultraviolet radiation

The effect of (i) aphidicolin, a specific inhibitor of delta- and epsilon-polymerases, and nucleotide excision repair; (ii) 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase and base excision repair; and (iii) actinomycin D and cycloheximide, inhibitors of protein and RNA synthesis, respectively, on the induction of suppression of apoptosis of rat thymocytes by different doses of short-wavelength ultraviolet radiation was studied by flow cytometry. 3-Aminobenzamide suppressed the inhibition of apoptosis induced by the doses of short-wavelength ultraviolet radiation higher than 20 J/m2, increasing the cell death to a maximum. Thus, the inhibition of apoptosis by high short-wavelength ultraviolet radiation doses depends on the status of poly(ADP-ribose) polymerase and is prevented by 3-aminobenzamide. As opposed to 3-aminobenzamide, aphidicolin did not affect the cell death at short-wavelength radiation doses higher than 10 J/m2 but induced the apoptosis of unirradiated cells and cells irradiated with short-wavelength ultraviolet radiation doses lower than 10 J/m2. The inhibitors of protein and RNA synthesis cycloheximide and actinomycin D prevented the induction of apoptosis caused by low and medium doses but did not abolish the apoptosis-inhibiting activity of high doses of short-wavelength ultraviolet radiation.     

Partially purified bacteriocin kills malignant cells by apoptosis: programmed cell death.

Bacterial proteins, partially purified bacteriocin (PPB), were investigated for their selective killing of malignant cells. It is shown here that upon PPB-cell interaction DNA fragmentation starts within one hour and peaks at 6 hrs. This process requires an on-going cellular metabolism. It is prevented by both actinomycin D, a DNA dependent RNA synthesis inhibitor, and cycloheximide, a protein synthesis inhibitor. We show here that the DNA fragmentation is triggered by PPB-cell membrane-receptor interaction which signals the activation of endogenous cellular endonucleases rather than actually penetrating the cell and interacting directly with the DNA, or serving as a nuclease itself. Thus, it is suggested that the cell death initiated by the lethal bacterial proteins, PPB, is a programmed, step-wise cell death involving apoptosis.     

Negative selection of thymocytes. A novel polymerase chain reaction-based molecular analysis detects requirements for macromolecular synthesis.

Self-tolerance is mainly established through clonal deletion of autoreactive T cells during thymic differentiation. The mechanisms by which deletion is achieved are poorly understood. Here we use a specific polymerase chain reaction-based system to characterize DNA fragmentation and show that after in vivo treatment of neonatal mice with staphylococcus enterotoxin B, selective apoptosis of V beta 8+ thymocytes occurs. This process precedes detectable deletion of V beta 8+ cells as determined by phenotypic analysis. Moreover, in vivo administration of cycloheximide and, to a lesser extent, actinomycin D, inhibits apoptosis of staphylococcus enterotoxin B specific thymocytes. Thus, macromolecular synthesis is a requirement for negative selection.     

Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation.

Some T cell hybridomas, upon activation via the TCR, rapidly undergo cell death. In this paper, we demonstrate that this activation-induced cell death (AICD) is accompanied by morphologic changes seen at the electron and light microscopy levels. The most striking changes are an extensive condensation of the chromatin and formation of membrane blebs. In addition to the morphologic changes, a significant portion of genomic DNA is broken at an interval of approximately 200 bp, producing a ladder of oligonucleosome-sized fragments after gel electrophoresis. Taken together, these observations indicate that AICD proceeds via apoptosis, or programmed cell death. This is additionally supported by the observation that AICD-associated phenomena are at least partially inhibited by cycloheximide or actinomycin D. Curiously, AICD and its associated DNA fragmentation are completely inhibited by aurintricarboxylic acid, a known nuclease inhibitor. The possible relationship between AICD in vitro, and the negative selection process (wherein selection may proceed via AICD of developing, autoreactive thymocytes) is discussed.     

Effect of gamma radiation on resting B lymphocytes. I. Oxygen-dependent damage to the plasma membrane results in increased permeability and cell enlargement

Although the susceptibility of resting B lymphocytes to radiation-induced interphase death is well known, the mechanism by which this occurs is not understood. In this report, we use three measures of plasma membrane integrity (increase in cell volume, uptake of trypan blue, and release of 51Cr) to assess the effect of radiation on the resting B cell plasma membrane. The delivery of 500 to 1000 rad caused the majority of resting B cells to enlarge slightly, whereas 3000 rad caused virtually all of the cells to approximately double in size within 3 to 4 hr. Measurement of the release of 51Cr from resting B cells revealed a similar relationship between the dose of radiation and the loss of radioactive label. Trypan blue exclusion was also found to diminish as a function of radiation dose. An analysis of a variety of lymphoid cells suggested that sensitivity to the membrane damaging effects of gamma radiation was in the order of resting B cells greater than resting T cells greater than a long-term L3T4+ T cell clone greater than a B cell lymphoma. LPS-induced B cell blasts treated with 3000 rad were equivalent to 1000 rad-treated resting B cells. The effects of the gamma radiation could be ameliorated by excluding oxygen (a diradical molecule that can potentially enhance the generation and propagation of highly reactive free radicals) at the time of irradiation, or by adding the free radical scavenging agent cysteamine. These data are compatible with the hypothesis that gamma radiation results in damage to the plasma membrane of resting lymphocytes via the generation of highly reactive free radical species. This damage is reflected in a rapid increase in plasma membrane permeability and swelling of the cells, and may play a major role in causing interphase death.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.     

Apoptosis: mode of cell death induced in T cell leukemia lines by dexamethasone and other agents

Glucocorticoids can mediate the destruction of thymocytes and T cell-derived leukemia cells through a mechanism known as apoptosis. The characteristic feature of apoptosis is fragmentation of DNA at internucleosomal linkers through the activity of a specific endonuclease. In this study, an attempt was made to compare dexamethasone-induced apoptosis in two T cell-derived human leukemia lines (CEM-C1 and CEM-C7) to the cell killing brought about by selected cytotoxic agents. In the CEM-C7 cell line (dexamethasone-sensitive), apoptosis was induced not only by dexamethasone but by actinomycin D, cycloheximide, and 25-OH cholesterol. In the CEM-C1 cell line (dexamethasone-resistant) cycloheximide, 25-OH cholesterol, or cell starvation could induce apoptosis. It appears that in leukemic cells apoptosis may be induced by a variety of unrelated toxic agents and is not limited to glucocorticoids.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

T cell receptor-mediated DNA fragmentation and cell death in T cell hybridomas

Activation of Ag-specific T cell hybridomas with a high density of immobilized anti-CD3 antibody resulted in not only secretion of IL-2 but also cell death of up to 60 to 80% in selected hybridomas after 14 h. Similar results were obtained with V beta 8+ T cell hybridomas stimulated with cross-linked F23.1 antibody. In these activated hybridomas, we found that DNA was fragmented into 180- to 200-bp multiples. DNA fragmentation was not observed when T cells were maintained after killing with anti-Thy-1 plus C or with heat treatment at 45 degrees C, nor when T cells were incubated with fixed anti-CD4 antibody. Furthermore, fragmentation was detectable at 6 h after incubation when almost all of the cells were still viable as evaluated by trypan blue dye exclusion test. Cell death was prevented by addition of EGTA, cycloheximide, actinomycin D, and zinc, suggesting that the induction of cell death requires Ca2+ influx, newly synthesized protein(s), and involvement of endonuclease.     

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A₂, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A₂ can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.     

Control of mitogen-induced transformation: characterization of a splenic suppressor cell and its mode of action.

The present study demonstrates that mouse spleen cells contain a population of glass wool adherent T lymphocytes which exhibit the capacity to suppress non-glass adherent lymphocyte responses to mitogens. These suppressor cells are stimulated by both low and high doses of PHA¹ and high doses of con A. The suppressor cell effect is observed when UNA, but not RNA or protein synthesis, is studied. This glass-adherent suppressor cell population is characterized as being the primary DNA synthesizing cells during the early (0–8 hr) stages of culture. Suppression still occurs when the suppressor cells are treated with mitomycin C, actinomycin D or cycloheximide. This implies that new macromolecular synthesis may not be required for suppression to occur. Suppression is blocked by inhibiting synthesis of prostaglandin and is mimicked by Bu₂cAMP. This suggests that mitogen activated suppressor cells regulate T cell responses via production of prostaglandin which modulates the concentration of intracellular cyclic nucleotide levels.