辐射诱发淋巴细胞凋亡生成与抑制作用研究

研究了辐射诱发的人外周血淋巴细胞凋亡生成，以及水溶性维生素Ｅ类似物－Ｔｒｏｌｏｘ对辐射诱导人外周血淋巴细胞凋亡的抑制作用。照后３０分钟内Ｔｒｏｌｏｘ能有效地阻抑ＤＮＡ片段形成，而在照前或受照中加入Ｔｒｏｌｏｘ均不能抑制ＤＮＡ片段形成，揭示Ｔｒｏｌｏｘ并不是通过清除照射过程中产生的自由基而起作用。照后３０分钟内加Ｔｒｏｌｏｘ，２小时后撤去，同样能抑制ＤＮＡ片段形成，表明Ｔｒｏｌｏｘ能不可逆地阻抑细胞凋亡早期的＂关键＂事件。     

Inhibition of Poly(ADP-ribose) Polymerase as a Possible Reason for Activation of Ca2+/Mg2+-dependent Endonuclease in Thymocytes of Irradiated Rats

Summary

The molecular mechanism of activation of Ca²⁺/Mg²⁺-dependent endonuclease in thymocytes of irradiated rats was studied. Thymocyte nuclei of control and irradiated rats were pre-incubated with NAD under conditions favourable for poly ADP-ribosylation. Pre-incubation results in a decrease in the rate of autolytic DNA digestion by Ca²⁺/Mg²⁺-dependent endonuclease of 6–7- and 2–3-fold for control and irradiated animals, respectively. The activity of Ca²⁺/Mg²⁺-nuclease extracted from the nuclei pre-incubated with NAD is also considerably decreased. The presence of nicotinamide and thymidine in the preincubation medium prevents the suppression of Ca²⁺/Mg²⁺-nuclease activity. In the experiments performed with isolated nuclei and permeabilized thymocytes the synthesis of poly(ADP-ribose) does not significantly change within 1 h after irradiation at a dose of 10 Gy, whereas 2 and 3 h after the exposure it decreases by 35–40 and 45–55 per cent, respectively. The activity of poly(ADP-ribose) glycohydrolase in this period is similar to that in the controls. The average size of the de novo synthesized chains of poly(ADP-ribose) increases from 11 to 17 ADP-ribose units by the second hour after irradiation. Inhibition of poly(ADP-ribose) polymerase in the postirradiation period preceded the internucleosomal fragmentation of chromatin. The results suggest that activation of Ca²⁺/Mg²⁺-nuclease in irradiated thymocytes is accounted for by the disturbance of its poly ADP-ribosylation     

A role for calcium in regulating apoptosis in rat thymocytes irradiated in vitro.

Thymus-derived lymphocytes undergo death after gamma-irradiation via a pathway termed apoptosis, or programmed cell death. An early step in this pathway is the production of nucleosome-sized fragments of DNA. DNA fragmentation was used as the endpoint in these investigations to examine apoptosis in lymphocytes extracted from the rat thymus and irradiated in vitro. In unirradiated thymocytes the level of DNA fragmentation rose to 15% by the first hour of culture, where it remained approximately constant until the fifth hour. In contrast, thymocytes irradiated with a dose of 2.5 Gy exhibited a large and dramatic increase in DNA fragmentation beginning 2 h postirradiation. DNA fragmentation measured 6 h after irradiation was detected after as little as 0.25 Gy and reached a maximum of 90% with 10 Gy. Metabolic control of DNA fragmentation after irradiation was evidenced by the suppression of DNA fragmentation when thymocytes were incubated with cyclohexamide or actinomycin D. When gamma-irradiated thymocytes were incubated with the Ca2+ chelator EGTA, DNA fragmentation was reduced significantly. BAPTA-AM, a highly specific intracellular Ca2+ chelator, essentially eliminated DNA fragmentation in cells irradiated with 2.5 Gy and, unlike EGTA, eliminated the background level of fragmentation in unirradiated samples. Therefore, our data are consistent with the possibility that Ca2+ serves as a second messenger to induce DNA fragmentation in irradiated thymocytes, suggesting a common pathway for cells prompted to enter apoptosis from seemingly dissimilar interval events.     

Attenuation of caspase-3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells

PURPOSE: The relationship between post-irradiation treatment with Trolox, an antioxidant that inhibits lipid peroxidation, and X-ray-induced apoptosis, with regard to signal transduction pathways, was examined in MOLT-4, a human leukaemia cell line. MATERIALS AND METHODS: In MOLT-4 cells treated with Trolox after X-irradiation, viability, DNA fragmentation, expression of p53, BCL-2, BAX, active SAPK/JNK, active caspase-3 and the cleavage of PARP were measured by the trypan blue exclusion test, agarose gel electrophoresis and Western blotting. RESULTS: Stained cells and ladder-like DNA cleavage were observed after X-irradiation. Cell death and DNA fragmentation were significantly inhibited by the post-irradiation treatment with Trolox. The expression of p53 and active SAPK/JNK was increased after X-irradiation, and fragments of PARP and the activated fragment of caspase-3 were produced. Post-irradiation treatment with Trolox attenuated the X-irradiation-induced expression, fragmentation or activation of these apoptosis-related biomolecules. The expression of BCL-2 and BAX, which would occur downstream from p53, was not changed by irradiation and Trolox treatment. Furthermore, cell death was associated with caspase-3 because the ladder-like DNA cleavage was completely inhibited by Ac-DEVD-CHO but not Ac-YVAD-CHO, TLCK and PMSF. CONCLUSION: Post-irradiation events such as membrane damage induce caspase-3-dependent apoptosis, which might be mediated by the activation of SAPK/JNK and be independent of p53.     

Accumulation of cAMP in gamma-irradiated thymocytes and internucleosomal DNA fragmentation.

Ionizing radiation, glucocorticosteroids and chemical inducers of differentiation (CID) are cytotoxic to thymocytes, and induce internucleosomal DNA fragmentation. Tissue cAMP levels in thymi of irradiated mice were significantly elevated as early as 30 min post-irradiation. In contrast, cAMP content in the liver was not changed significantly up to 1 h post-irradiation, and then some decrease occurred. Irradiation of isolated thymocytes gave essentially the same results as after irradiation of animals, and the elevation in cAMP 30 min after the irradiation, DNA fragmentation and cell death were linearly related to the dose up to 2.5 Gy. The maximal induction of cAMP level occurs in the fractions of radiosensitive cortical thymocytes. In thymocytes all CID tested also induced the increase in cAMP level with concomitant DNA fragmentation. Unlike ionizing radiation, UVC light did not induce cAMP accumulation and DNA fragmentation in thymocytes. Treatment of UV-irradiated cells with But2 cAMP did not result in an increase in DNA fragmentation. Ionizing radiation induced DNA fragmentation and cell death can be prevented by adding the protein kinases inhibitor H-7. Theophylline was shown to reduce the cAMP response, DNA fragmentation and cell death in gamma-irradiated thymocytes, suggesting that the accumulation of cAMP may be partly related to adenosine receptor sites.     

Ultraviolet exposure of thymocytes: selective inhibition of apoptosis

Purpose: To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes.

Materials and methods: Cells were exposed to UV radiation with emission peaks of 365?nm (UVA) exposures of 1620–10?200?J?m^?2, of 312?nm (UVB) exposures of 34–1620?J?m^?2 or of 254?nm (UVC) exposures of 1.5–1620?J?m^?2, and incubated for 5.5?h with or without hydrocortisone, phorbol‐12‐myristate‐13‐acetate or anti‐Fas antibody. Additionally, cells were irradiated with gamma‐rays (5?Gy) before UVB exposure (408?J?m^?2) at different times. Apoptosis was quantified by DNA fragmentation.

Results: Up to an irradiation of 5000?J?m^?2, UVA exposure did not show any effect on thymocyte apoptosis, while at 10?200?J?m^?2 irradiation, considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibited natural and cortisone‐induced apoptosis. Moreover, UVB inhibited apoptosis triggered by phorbol‐12‐myristate‐13‐acetate and gamma‐irradiation, but not by anti‐Fas antibody.

Conclusions: The response of mouse thymocytes in culture to UV irradiation strongly depends on the wavelength used. It is suggested that either a survival or an apoptotic pathway occurs depending on the physiological state of the cell, spectral composition of the UV light and cell type. The possible involvement of extracellular signal‐regulated kinase and stress‐activated protein kinase/c‐Jun N‐terminal kinase in the apoptotic pathway is discussed.     

Role of Ca2+ in radiation‐induced damage in murine splenocytes

Purpose: To address the links between calcium, peroxidation, cell damage and death and the response of the enzymes involved in free radical metabolism, in splenocytes of mice irradiated with gamma‐rays.

Materials and methods: Splenocytes of Swiss albino mice were irradiated with various doses (0–7?Gy) of gamma‐rays (⁶⁰Co) at a dose‐rate of 0.0575?Gy?s^?1. Membrane peroxidation and fluidity were determined by the thiobarbituric acid‐reactive substances (TBARS) method, and fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), respectively. Apoptosis was analysed by nucleosomal ladder formation and activity of NF‐κB by electrophoretic mobility shift assay (EMSA). The specific activities of the antioxidant enzymes, lactate dehydrogenase (LDH), levels of nitric oxide (NO^?) and glutathione were determined spectrophotometrically. Modulatory effects of Ca²⁺ were examined at 3?Gy using different concentrations (1, 3 and 5?mM) in the presence or absence of the ionophore A23187.

Results: Irradiation of splenocytes resulted in enhanced peroxidative damage, membrane fluidity, apoptosis and DNA binding activity of NF‐κB. The specific activities of LDH and antioxidant enzymes superoxide dismutase (SOD), DT‐diaphorase (DTD), glutathione S‐transferase (GST) and levels of glutathione (GSH) and NO^? were increased with radiation dose up to 4?Gy. Ca²⁺ augmented the radiation‐induced responses. The presence of ionophore A23187 potentiated the modulatory effects of Ca²⁺.

Conclusions: These findings show that Ca²⁺ augments radiation damage and is more effective intracellularly. Ca²⁺, peroxidation, cellular damage and apoptosis are possibly interlinked through signals, as is evident from the increased activity of NF‐κB and generation of NO^?. The enhanced antioxidant status suggests an attempt made by the irradiated cells to maintain their normal functions.     

Antioxidant administration inhibits exercise-induced thymocyte apoptosis in rats

PURPOSE: The purpose of this study was to investigate the effect of antioxidant on exercise-induced apoptosis in rat thymocytes. METHODS: After exercise at 13.8 m x min(-1) for 60-90 min x d(-1) on a motor-driven drum exerciser for 2 consecutive days, rat thymocyte apoptosis was monitored by the feature of DNA fragmentation. To study the effect of antioxidant, rats were administered with butylated hydroxyanisole (BHA) for 7 d before exercise. RESULTS: Exercise could induce thymocyte DNA fragmentation as detected on electrophoretic gel and by cell death detection ELISA kit. Further studies indicated that pretreatment with antioxidant BHA to rats resulted in a blockage of exercise-induced DNA fragmentation. The concentrations of glutathione (GSH) were not significantly changed in rat thymocytes after exercise with or without BHA treatment. CONCLUSION: These results suggest that reactive oxygen species may play a role in thymocyte apoptosis induced by exercise. However, changes in GSH levels were not observed in this exercise model.     

The effect of chronic and acute exercise on thymocyte apoptosis and necrosis in ovariectomized mice given dietary genistein

AIM: At menopause, many women consume phytoestrogens instead of beginning hormone replacement therapy. Many also start exercise programs for health benefits. Genistein, a soy isoflavone with estrogen-like properties, induces lymphocyte apoptosis in vitro. Aerobic exercise also induces apoptosis in lymphoid cells. The present study was designed to determine the effect of chronic (wheel running, WR) and acute (treadmill) exercise on in vivo apoptosis of thymocytes using an animal model of menopause with supplementation from dietary genistein. METHODS: Using a randomized design, 99 ovariectomized B6D2F1 mice were fed 250 (GEN) or 0 (C) ppm genistein and given concurrent exercise (voluntary wheel running-WR; or WR followed by a bout of high intensity treadmill running-WR+TREAD) or remained sedentary (SED). After 21 days, mice were sacrificed for measurements of body weights, tissue weights, thymocyte apoptosis and necrosis by annexin-V FITC and propidium iodide staining and flow cytometry, DNA fragmentation by ELISA, and plasma estrogen concentrations by RIA. RESULTS: WR+TREAD mice had lower percentages of viable and higher percentages of apoptotic and necrotic cells from thymus compared with SED or WR conditions (p<0.001). WR resulted in greater DNA fragmentation in thymus cell lysates than in samples from SED mice (p<0.005). There were no differences in thymocyte apoptosis or DNA fragmentation between GEN and C mice, either independently or interactively with exercise. GEN mice tended to have greater wheel running activity than C mice (0.05相似文献   

12C重离子束对人淋巴细胞增殖、周期和凋亡的影响

目的 探讨¹²C重离子束对人淋巴细胞增殖以及周期、凋亡的影响.方法 ¹²C重离子束照射人淋巴细胞Peng-EBV,吸收剂量分别为0(对照组)、0.5、2.0 Gy.照射后用MTS法检测细胞增殖活力,流式细胞仪检测细胞周期和细胞凋亡.结果 与对照组相比,0.5 Gy照射可以增加细胞增殖活力(t=2.66~14.45, P<0.05),而2.0 Gy照射降低了细胞活力(t=7.65~64.45, P<0.05).受照射细胞的活力存在一个恢复和下降过程,受照后48 h内,细胞数量呈增加趋势,但72 h时细胞数量下降.照射后48 h,两组细胞G₂/M期呈明显上升趋势,高于对照组(t=2.01~99.80,P<0.05),且2.0 Gy组的周期阻滞较0.5 Gy组严重;照射后30 d,细胞周期阻滞恢复到正常水平.照射后12、24、48 h,两照射组与对照组相比,细胞凋亡率差异有统计学意义(t=-3.05~-1.05,P<0.05),在照后24 h最高,48 h明显下降,30 d恢复到对照水平.结论 ¹²C离子束辐射影响人淋巴细胞增殖,诱导人淋巴细胞发生明显的G₂/M期阻滞,并且明显地促进细胞凋亡.     

The Effect of Irradiation on Sub-cellular Components Metal Ion Transport in Mitochondria

Summary

Mitochondria from rat liver irradiated with electron doses within the range 2–100 kilorads readily lose potassium ions when incubated in media containing low concentrations of potassium (10 mM) for short periods at 25° or 37°. When incubated in media containing 0·1 M potassium, irradiated mitochondria take up more potassium than do control mitochondria. Active uptake of calcium from a Ca²⁺-containing medium is also inhibited by irradiation and Ca²⁺ ions are lost if irradiated mitochondria are incubated in a Ca²⁺-free medium.

Effects on the transport of both K⁺ and Ca²⁺ are observable after doses of 5 kilorads, and are believed to be a direct result of damage to the mitochondrial membrane and not to inhibition of mitochondrial ATP-ase or to inhibition of oxidative phosphorylation.     

Ferulic acid protects lymphocytes from radiation-predisposed oxidative stress through extracellular regulated kinase

Purpose:?The objective of this study was to investigate the radioprotective effect of ferulic acid (FA) on irradiated lymphocytes and discover the possible mechanisms of protection.

Materials and methods:?Lymphocytes were pretreated for 12?h with FA (0.001–0.1?μM) and then exposed to 3 Gy radiation. Cell apoptosis, intracellular reactive oxygen species (ROS), and signal pathway was analysed.

Results:?Irradiation increased cell death, DNA fragmentation and intracellular ROS. Pretreatment with FA significantly reversed this tendency and attenuated the irradiation-induced ROS generation. Furthermore, several anti-apoptotic characteristics of FA were determined, including the ability to diminish cytosolic Ca²⁺ concentration, inhibit caspase-3 activation and cytochrome c translocation, upregulate B-cell lymphoma 2 (Bcl-2) and downregulate Bcl-2-associated X protein (Bax) in 3 Gy-irradiated lymphocytes. Signal pathway analysis showed FA decreased the activation of extracellular regulated kinase (ERK), which had been activated by radiation.

Conclusion:?The results suggest that FA had a radioprotective effect through the ERK pathway to inhibit apoptosis and oxidation, and it may be an effective candidate for treating radiation diseases associated with oxidative stress.     

Role of Protein Kinase-C in Thymocyte Apoptosis Induced by Irradiation

The role of protein kinase C in radiation-induced death of thymocytes was studied. For this purpose murine thymocytes were irradiated and incubated for 6 h at 37°C and afterwards the fraction of fragmented DNA was measured. Results indicate that radiation-induced DNA fragmentation can be prevented by adding the protein kinase C inhibitor H-7 or staurosporine to the thymocytes during incubation time. Incubation of irradiated cells with HA-1004, an inhibitor of cAMP-dependent protein kinase, with a minor effect on protein kinase C did not affect the DNA fragmentation induced by irradiation. Incubation of cells with phorboldibutyrate gave a dose-dependent induction of DNA fragmentation. This effect can be inhibited by staurosporine. These results suggest that radiation-induced DNA fragmentation is an active cellular process in which protein kinase C plays an important role.     

Effect of the Inducers of Cellular Differentiation and of Ionizing Radiation of Thymus Lymphocytes: Chromatin Degradation and Programmed Cell Death

Summary

The cytotoxic response of thymocytes to chemical inducers of differentiation does not represent a non-specific toxic action of these drugs. The death of thymocytes treated with the inducers or with γ-rays is associated with internucleosomal chromatin fragmentation. All treatments are more effective on the most radiosensitive sub-population of these cells. This subpopulation is characterized by the maximal level of spontaneous DNA lesions. Incubation of thymocytes with the inducers of differentiation raises the level of these lesions. It is suggested that the processes of thymocyte death after the inducer treatment or irradiation and of cellular differentiation have features in common, and the capacity of thymocytes to limit or reverse the potentially lethal effects of these treatments is determined by the level of pre-existing spontaneous DNA lesions.     

Role of protein kinase-C in thymocyte apoptosis induced by irradiation.

The role of protein kinase C in radiation-induced death of thymocytes was studied. For this purpose murine thymocytes were irradiated and incubated for 6 h at 37 degrees C and afterwards the fraction of fragmented DNA was measured. Results indicate that radiation-induced DNA fragmentation can be prevented by adding the protein kinase C inhibitor H-7 or staurosporine to the thymocytes during incubation time. Incubation of irradiated cells with HA-1004, an inhibitor of cAMP-dependent protein kinase, with a minor effect on protein kinase C did not affect the DNA fragmentation induced by irradiation. Incubation of cells with phorboldibutyrate gave a dose-dependent induction of DNA fragmentation. This effect can be inhibited by staurosporine. These results suggest that radiation-induced DNA fragmentation is an active cellular process in which protein kinase C plays an important role.     

Early postirradiation chromatin degradation in thymocytes

The decrease in the average DNA size in thymocytes starts soon after in vivo irradiation and at approximately 45 min reaches a plateau, thereafter showing only minor changes up to 3 h. This fall in extent of chromatin cleavage coincides with the accumulation of 1.0-1.5 kb DNA fragments. Double-strand breaks generated by endonucleases are not randomly distributed along DNA but clustered in such a way that they give rise to fragments of 1-5 nucleosomes in size. Cycloheximide treatment partially inhibits nuclease activity in nuclear extracts isolated from thymus of irradiated mice. This suggest that DNA fragmentation is an early event in programmed death of thymocytes mediated by irradiation. The data indicate that it requires protein synthesis and that it precedes release of polydeoxyribonucleotides.     

New insight into mitochondrial changes in vascular endothelial cells irradiated by gamma ray

Purpose: To investigate alterations of mitochondria in irradiated endothelial cells to further elucidate the mechanism underlying radiation-induced heart disease.

Materials and methods: Experiments were performed using human umbilical vein endothelial cells (HUVECs). HUVECs were irradiated with single gamma ray dose of 0, 5, 10 and 20?Gy, respectively. Apoptosis was assessed by flow cytometry at 24, 48 and 72?h post-irradiation, respectively. The intracellular reactive oxygen species (ROS) was measured with 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) at 24?h post-irradiation. Mitochondrial membrane potential (ΔΨm) by JC-1 and the opening of mitochondrial permeability transition pore (mPTP) by a calcein-cobalt quenching method were detected at 24?h post-irradiation in order to measure changes of mitochondria induced by gamma ray irradiation.

Results: Gamma ray irradiation increased HUVECs apoptosis in a dose-dependent and time-dependent manner. Irradiation also promoted ROS production in HUVECs in a dose-dependent manner. At 24?h post-irradiation, the results showed that irradiation decreases ΔΨm, however, paradoxically, flow cytometry showed green fluorescence instensity higher in irradiated HUVECs than in control HUVECs in an irradiation dose-dependent manner which indicated gamma ray irradiation inhibited mPTP opening in HUVECs.

Conclusions: Gamma ray irradiation induces apoptosis and ROS production of endothelial cells, and decreases ΔΨm meanwhile contradictorily inhibiting the opening of mPTP.     

Reply to Letter by C. B. Seymour and C. Mothersill

Summary

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.     

Magnitude of radiation-induced DNA damage in peripheral blood leukocytes and its correlation with aggressiveness of thymic lymphoma in Swiss mice

Abstract

Purpose: The present study is aimed to investigate the magnitude and kinetics of DNA damage in peripheral blood leukocytes of mice exposed to whole body gamma irradiation (WBI; 3 Gy) and its correlation with aggressiveness of thymic lymphoma (TL).

Materials and methods: DNA damage was monitored in peripheral blood cells of individual mice by comet assay at different intervals of post-irradiation, which were correlated with weight of TL in respective mice at 120th day. To further study genomic radiosensitivity in TL development, peripheral blood samples collected at the 15th and 90th day of post-irradiation from control and WBI animals were irradiated (0.5 Gy) ex vivo followed by assessment of DNA damage by comet assay.

Results: The maximum DNA damage (tail moment) was observed at 5 min after WBI, which decreased at longer period, and was minimum at the 7th day after WBI. However, residual damage was observed in comparison to control and it persisted up to 90 days of irradiation. Tail moment values observed at an early time (5 min) of post-irradiation was better correlated (correlation coefficient, r = 0.84) with weight of TL than at longer time period (60 days; r = 0.21). Our results showed that in ex vivo irradiated (0.5 Gy) peripheral blood, the magnitude of DNA damage was higher in samples obtained from WBI mice than sham-irradiated controls suggesting enhanced genomic radiosensitivity in WBI mice. Genomic susceptibility to radiation observed in peripheral blood from WBI animals showed better correlation with weight of TL at the 15th day (r = 0.9) post-irradiation period than at the 90th day (r = 0.44).

Conclusion: These results suggest that the magnitude of radiation-induced initial DNA damage in peripheral blood leukocytes and genomic radiosensitivity could be an indicator of TL aggressiveness in mice.