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。     

Post-irradiation Defects in Newly Synthesized DNA: Neutrons and Electrons Compared and the Effect of Misonidazole

The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentative in thymocytes exposed to γ-radiation. Trolox is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation, suggesting that it does not work by scavenging free radicals generated during radiation exposure. Incubation of the irradiated cell suspension with trolox for 2 h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells. This suggests that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca²⁺ into the cell. At 3 h post-irradiation the amount of Ca²⁺ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Membrane damage has been shown to affect the transport of Ca²⁺. Trolox treatment completely blocked the radiation-induced influx of Ca²⁺ into the thymocytes. These results suggest that membrane damage is a critical lesion that is involved in DNA fragmentation in thymocyte apoptosis.     

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.     

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.     

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.     

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.     

Ascorbic acid reduced mutagenicity at the HPRT locus in CHO cells against thermal neutron radiation.

We investigated the biological effects of the long-lived radicals induced following neutron irradiation. It has been reported that radiation-induced long-lived radicals were scavenged by post-irradiation treatment of ascorbic acid (Koyama, 1998). We studied the effects of ascorbic acid acting as a long-lived radical scavenger on cell killing and mutagenicity in Chinese hamster ovary cells against thermal neutrons produced at the Kyoto University Research reactor. Ascorbic acid was added to cells 30 min after neutron irradiation and removed 150 min after irradiation. The biological end point of cell survival was measured by colony formation assay. The mutagenicity was measured by the mutant frequency in the HPRT locus. The post-irradiation treatment of ascorbic acid did not alter the cell killing effect of neutron radiation. However, the mutagenicity was decreased, especially when the cells were irradiated with boron. Our results suggested that ascorbic acid scavenged long-lived radicals effectively and caused apparent protective effects against mutagenicity of boron neutron capture therapy.     

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.     

X射线对小鼠胸腺细胞凋亡的影响

采用荧光分光光度法对裂解ＤＮＡ进行定量，并用琼脂糖凝胶电泳法定性研究Ｘ射线全身照射后小鼠胸腺细胞凋亡。结果表明，４ＧｙＸ射线照后２小时胸腺细胞凋亡开始增加，于照后１４小时达峰值，尔后呈下降趋势。ＤＮＡ裂解率在照后２４小时降至１４小时的５０％，这可能是内环境中巨噬细胞吞噬凋亡小体所致。在照射后１４小时研究其剂量－效应关系发现，高剂量照射使ＤＮＡ裂解明显增多，形成１８０ｂｐ（ｂａｓｅｐａｉｒｓ）左右或其整倍数的ＤＮＡ断片，电泳呈现＂梯形图谱＂；而低剂量辐射可使ＤＮＡ裂解率降低。剂量－效应曲线呈Ｊ型。     

Is the NAD-poly (ADP-ribose) polymerase system the trigger in radiation-induced death of mouse thymocytes?

Agents that induce DNA strand breaks evoke a drop in the NAD content in mouse thymocytes. A decrease in the endogenous NAD content that occurs immediately after gamma-irradiation of thymocytes is entirely attributed to the activation of poly(ADP-ribosylation). The addition of 5 mM benzamide before irradiation prevents the postirradiation drop of the NAD level but has no effect on chromatin degradation and cell death. In contrast to liver nuclei, pre-incubation of mouse thymic nuclei with NAD had no effect on the subsequent chromatin endonucleolysis by Ca2+/Mg2+-dependent endonuclease. It is suggested that the NAD-poly(ADP-ribose) polymerase system is probably not the trigger in the radiation-induced programmed death of mouse thymocytes, but may merely be indicative of the radiation response of these cells.     

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 1620-10200 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 10200 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.     

放射性核素147Pm诱发Molt-4细胞和Ana-1细胞凋亡

对放射性核素１４７Ｐｍ内照射诱发Ｍｏｌｔ－４细胞和Ａｎａ－１细胞的死亡形式进行研究。方法通过形态学变化、琼脂糖电泳、ＭＴＴ和ＪＡＭ实验对核素诱发的细胞死亡形式进行了定性及定量分析。结果表明放射性核素１４７Ｐｍ诱发的细胞死亡具有典型的细胞凋亡形态学特征，且细胞凋亡的程度及ＤＮＡ链断裂量与１４７Ｐｍ作用的时间和内照射的累积吸收剂量有关。结论放射性核素１４７Ｐｍ内照射可诱发细胞凋亡。     

Effects of cycloheximide and actinomycin D on radiation-induced apoptotic cell death in the developing mouse cerebellum.

Effects of cycloheximide and actinomycin D on radiation-induced cell death in the external granular layer (EGL) of the cerebellum were studied in vivo. Newborn mice were exposed to 0.24 Gy gamma-radiation, and dying cells which exhibited pyknosis of nuclei in the EGL were examined at various post-irradiation periods. The number of pyknotic cells began to increase 3 h after irradiation, reached a peak incidence at 6 h, and then gradually fell to the sham-irradiated level by 18 h. When pups were injected with cycloheximide 1 h after irradiation, cell death was suppressed for 6 h, but a peak mortality as high as in the case of radiation alone was attained at 15 h after irradiation. When pups were treated with cycloheximide twice, at 1 and 6 h after irradiation, cell death did not occur for 15 h, but then the incidence rose to a level similar to that after irradiation alone. These findings showed that radiation-induced cell death in the EGL is suppressed by cycloheximide until the chemical is metabolized. Hence, death is by apoptosis which is known to require macromolecular synthesis, and the 'signal' for apoptosis in the cell persists for at least 15 h after irradiation. On the other hand, actinomycin D injected immediately before or after irradiation did not affect the initiation of cell death; actinomycin D alone induced cell death.     

Effects of Cycloheximide and Actinomycin D on Radiation-induced Apoptotic Cell Death in the Developing Mouse Cerebellum

Effects of cycloheximide and actinomycin D on radiation-induced cell death in the external granular layer (EGL) of the cerebellum were studied in vivo. Newborn mice were exposed to 0·24 Gy γ-radiation, and dying cells which exhibited pyknosis of nuclei in the EGL were examined at various post-irradiation periods. The number of pyknotic cells began to increase 3 h after irradiation, reached a peak incidence at 6 h, and then gradually fell to the sham-irradiated level by 18 h. When pups were injected with cycloheximide 1 h after irradiation, cell death was suppressed for 6 h, but a peak mortality as high as in the case of radiation alone was attained at 15 h after irradiation. When pups were treated with cycloheximide twice, at 1 and 6 h after irradiation, cell death did not occur for 15 h, but then the incidence rose to a level similar to that after irradiation alone. These findings showed that radiation-induced cell death in the EGL is suppressed by cycloheximide until the chemical is metabolized. Hence, death is by apoptosis which is known to require macromolecular synthesis, and the ‘signal’ for apoptosis in the cell persists for at least 15 h after irradiation. On the other hand, actinomycin D injected immediately before or after irradiation did not affect the initiation of cell death; actinomycin D alone induced cell death.     

60Coγ射线诱导的新生大鼠大脑皮质神经细胞凋亡

目的 采用电离辐射诱导的新生大鼠大脑皮质神经细胞损伤模型,探讨电离辐射诱导发育脑神经细胞死亡的特征和机制。方法 新生大鼠接受单次2.0 Gyγ射线照射后,采用DNA凝胶电泳、TUNEL和HE染色鉴定大脑皮质神经细胞死亡的类型和时程变化特点;应用免疫组织化学方法研究p53和iNOS在细胞死亡过程中的可能作用。结果 DNA凝胶电泳、TUNEL和HE染色表明,2.0 Gyγ射线照射可诱导新生大鼠大脑皮质神经细胞凋亡。大脑皮质各区域凋亡指数于照射后4 h开始增高,至12 h达到峰值;照射后相同时间点各皮质区域凋亡指数比较,新皮质最高,海马次之,旧皮质最低;P53和iNOS免疫阳性细胞计数定量结果显示,照射后相同时间点各皮质区域P53和iNOS免疫阳性细胞数差异无统计学意义。结论 2.0 Gyγ射线照射诱导了新生大鼠大脑皮质神经细胞凋亡;不同皮质区域的神经细胞对电离照射引起的损伤反应是相似的,但不同皮质区域的神经细胞凋亡存在差异。     

γ射线对培养的兔血管平滑肌细胞的抑制作用

目的 观察体外培养的兔动脉平滑肌细胞(SMCs）对7射线照射的剂量效应关系，探讨电离辐射抑制SMCs增殖的细胞生物学机理。方法 静止期SMCs分别给予γ射线2．5，5，10，20及30Gy一次性照射后继续培养4，24或72h。以^3H—TdR掺入法、流式细胞术分析、微核实验和电镜观察γ射线对SMCs的DNA合成、增殖周期和损伤的影响。结果 在2．5Gy以上剂量γ射线一次性照射时，SMCs增殖明显抑制，细胞G0／G1期阻滞，均呈剂量依赖性。2．5Gy以下剂量γ射线照射后24h左右可见细胞凋亡增加，2．5Gy以上剂量7射线照射可使SMCs发生坏死。结论 电离辐射能抑制SMCs增殖，使细胞产生G0／G1期阻滞，并呈剂量依赖关系。SMCs死亡方式与受照射剂量有关。     

DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and alpha-tocopherol

PURPOSE: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of alpha-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation. MATERIALS AND METHODS: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 - 21 days). RESULTS: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively. CONCLUSION: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.     

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.     

Radiation Studies on Sensitivity and Repair of Human Mammary Epithelial Cells

We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2–12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis.