Ionizing radiation and the activation of caspase-8 in highly apoptosis-sensitive lymphoma cells

Purpose: Ionizing radiation induces apoptosis in human lymphoma cells. However, the participating molecules and their exact order is unknown. Caspases are reported as essential cysteine proteases required for the activation and execution of programmed cell death. The activator caspase-8 is a key component of the CD95/Fas/APO-1 death receptor-triggered apoptosis pathway. Since contributing molecules for radiation-induced apoptosis and their exact order have not been analyzed in greater detail, the involvement of caspase-8 for radiation-induced apoptosis in human lymphoma cell lines was studied. Materials and methods: Apoptosis induction by CD95 stimulation and ionizing radiation was analyzed in eight different cell lines. In parallel, Western blotting tested activation of caspase-8. Results: Activation of caspase-8 by ionizing radiation occurred in five cell lines and was associated with high apoptosis sensitivity. Caspase-8 activation and apoptosis was detectable in cells resistant to CD95 stimulation, so suggesting separate pathways for CD95 and radiation-induced caspase-8 activation. Conclusion: The activator caspase-8 is activated during radiation-induced cell death and, in some cases, ionizing radiation induces caspase-8 independently of the CD95 system.     

Ionizing radiation and the activation of caspase-8 in highly apoptosis-sensitive lymphoma cells.

PURPOSE: Ionizing radiation induces apoptosis in human lymphoma cells. However, the participating molecules and their exact order is unknown. Caspases are reported as essential cysteine proteases required for the activation and execution of programmed cell death. The activator caspase-8 is a key component of the CD95/Fas/APO-1 death receptor-triggered apoptosis pathway. Since contributing molecules for radiation-induced apoptosis and their exact order have not been analyzed in greater detail, the involvement of caspase-8 for radiation-induced apoptosis in human lymphoma cell lines was studied. MATERIALS AND METHODS: Apoptosis induction by CD95 stimulation and ionizing radiation was analyzed in eight different cell lines. In parallel, Western blotting tested activation of caspase-8. RESULTS: Activation of caspase-8 by ionizing radiation occurred in five cell lines and was associated with high apoptosis sensitivity. Caspase-8 activation and apoptosis was detectable in cells resistant to CD95 stimulation, so suggesting separate pathways for CD95 and radiation-induced caspase-8 activation. CONCLUSION: The activator caspase-8 is activated during radiation-induced cell death and, in some cases, ionizing radiation induces caspase-8 independently of the CD95 system.     

In vitro evaluation of 213Bi-rituximab versus external gamma irradiation for the treatment of B-CLL patients: relative biological efficacy with respect to apoptosis induction and chromosomal damage

External source radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues. We assessed the relative biological efficacy (RBE) of alpha RIT (in vitro) compared to external gamma irradiation with respect to induction of apoptosis in B chronic lymphocytic leukaemia (B-CLL) and induction of chromosomal damage in healthy donor B and T lymphocytes. The latter was measured by a micronucleus assay. ²¹³Bi was eluted from a ²²⁵Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A'-DTPA as a chelator. B-CLL cells from five patients were cultured for 24 h in RPMI/10% FCS while exposed to ²¹³Bi conjugated to CD20 antibody or after external ⁶⁰Co gamma irradiation. Binding assays were performed in samples of all patients to calculate the total absorbed dose. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V-FITC and 7-AAD. Apoptosis was expressed as % excess over spontaneous apoptosis in control. Full dose range experiments demonstrated ²¹³Bi-conjugated CD20 antibody to be more effective than equivalent doses of external gamma irradiation, but showed that similar plateau values were reached at 10 Gy. The RBE for induction of apoptosis in B-CLL was 2 between 1.5 and 7 Gy. The micronucleus yield in lymphocytes of healthy volunteers was measured to assess the late toxicity caused by induction of chromosomal instability. While gamma radiation induced a steady increase in micronucleus yields in B and T cells, the damage induced by ²¹³Bi was more dramatic, with RBE ranging from 5 to 2 between 0.1 Gy and 2 Gy respectively. In contrast to gamma irradiation, ²¹³Bi inhibited mitogen-stimulated mitosis almost completely at 2 Gy. In conclusion, high-LET targeted alpha particle exposure killed B-CLL cells more effectively than did external gamma irradiation at a low dose (RBE=2), while a plateau was reached at a high dose. Long-term toxicity on healthy B and T lymphocytes was systematically higher for the alpha emitter (RBE=5 to 2).     

Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin A

PURPOSE: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death. MATERIALS AND METHODS: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2x10(5) cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200-300 cGy/min. The cells were treated with 0.25 microM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins. RESULTS: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation. CONCLUSIONS: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.     

ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse

Purpose : To define the role of the ataxia telangiectasia (A-T) mutated gene (ATM) in activation and progress of apoptosis. Material and methods : Three normal and three A-T EBV-transformed cell lines were studied. Following irradiation (IR), Fas activation or ceramide exposure, viability and apoptosis were measured by trypan blue dye exclusion assay and as sub-G1 cell fraction by flow cytometric analysis of propidium iodide stained cultures, respectively. Activation of caspase-3 was evaluated by immunoblot and by an in vitro activity assay on cytosolic cell extracts. To assess changes in mitochondrial potential and reactive oxygen species, cells were stained by 3,3'-dihexyloxacarbocynine iodide or hydroethidine, respectively, and scored by flow cytometry. Results : The observations establish that A-T cells are equipped with a proficient apoptotic machinery, as demonstrated by their ability to undergo mitochondrial collapse and caspase-3 activation after Fas activation or ceramide treatment. Both treatments have a similar cytotoxic effect on normal and A-T cells. In contrast, in spite of the stronger cytotoxicity induced by IR exposure, irradiated A-T cells are unable to undergo mitochondrial collapse and caspase-3 activation. Conclusions : The data indicate that ATM is necessary in the initiation of molecular pathway(s) leading to IR-induced apoptosis, and suggest that increased radiosensitivity of A-T cells is more likely a direct consequence of necrotic cell death.     

Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin A

Purpose: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death.

Materials and methods: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2 × 10⁵ cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200 – 300 cGy/min. The cells were treated with 0.25 μM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins.

Results: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation.

Conclusions: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.     

ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse

PURPOSE: To define the role of the ataxia telangiectasia (A-T) mutated gene (ATM) in activation and progress of apoptosis. MATERIAL AND METHODS: Three normal and three A-T EBV-transformed cell lines were studied. Following irradiation (IR), Fas activation or ceramide exposure, viability and apoptosis were measured by trypan blue dye exclusion assay and as sub-G1 cell fraction by flow cytometric analysis of propidium iodide stained cultures, respectively. Activation of caspase-3 was evaluated by immunoblot and by an in vitro activity assay on cytosolic cell extracts. To assess changes in mitochondrial potential and reactive oxygen species, cells were stained by 3,3'-dihexyloxacarbocynine iodide or hydroethidine, respectively, and scored by flow cytometry. RESULTS: The observations establish that A-T cells are equipped with a proficient apoptotic machinery, as demonstrated by their ability to undergo mitochondrial collapse and caspase-3 activation after Fas activation or ceramide treatment. Both treatments have a similar cytotoxic effect on normal and A-T cells. In contrast, in spite of the stronger cytotoxicity induced by IR exposure, irradiated A-T cells are unable to undergo mitochondrial collapse and caspase-3 activation. CONCLUSIONS: The data indicate that ATM is necessary in the initiation of molecular pathway(s) leading to IR-induced apoptosis, and suggest that increased radiosensitivity of A-T cells is more likely a direct consequence of necrotic cell death.     

不同剂量γ射线照射后胎儿骨髓CD34~ 造血干细胞死亡规律

目的　探讨不同剂量γ射线照射后胎儿骨髓CD34 造血干细胞的死亡规律。方法　以流式细胞术PE -CD34 FITC AnnexinV 7AAD三色荧光法 ,多参数分析了受不同剂量γ射线照射后 ,不同时间点的胎儿骨髓CD34 造血干细胞凋亡、坏死和存活的时效和量效变化特点。结果　受大剂量率γ射线 1次 5Gy和 8Gy照射后 ,胎儿骨髓CD34 造血干细胞的死亡是在照射后为期 1周的连续性死亡 ,既有凋亡又有坏死 ,但是以凋亡为主 ;受大剂量率γ射线 1次 10Gy和 12Gy照射后 ,胎儿骨髓CD34 造血干细胞则在受照后当天即大量坏死 ,在持续 1周内全部死亡。结论　受大剂量率γ射线 1次照射后 ,造血干细胞在 1周内发生了增殖期死亡而连续性空出所占据的龛位     

Radiation sensitivity and apoptosis in human lymphoma cells

PURPOSE: The impact ofapoptosis on radiation-induced eradication of clonogenic tumour cells is uncertain. The aim was to analyse the relationship of different functional stages during the apoptotic process to cell death and clonogenic cell eradication. MATERIALS AND METHODS: Apoptosis in Jurkat T-cells was studied by morphology, light scatter and caspase activation. Mitochondrial integrity was determined by the mitochondrial membrane potential (delta(phi)m). Cell death was quantified using propidium iodide exclusion. Clonogenic cell death was determined using a dilution survival assay. The influence of Bcl-2 was tested using a Bcl-2 transfected Jurkat clone. RESULTS: Irradiation induced profound apoptosis within 48 h associated with caspase activation and breakdown of delta(phi)m. Inhibition of caspases abrogated the apoptotic morphology with no influence on breakdown of delta(phi)m and survival. Over-expression of Bcl-2 abrogated all hallmarks of apoptosis; delayed cell death, however, had no influence on clonogenic survival after irradiation. CONCLUSION: Based on Bcl-2 as a positional marker, radiation-induced apoptosis can be divided into two stages: the initiation/decision phase, characterized by a breakdown of the mitochondrial membrane potential, and the execution phase, characterized by caspase activation. The execution phase had no influence on survival, whereas the initiation/decision phase controls immediate survival. However, abrogation of both phases did not influence radiation sensitivity.     

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.     

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotease-mediated shedding

Abstract. Purpose : L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. Materials and methods : Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITClabelled annexin-V in the presence of propidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. Results : PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. Conclusion : UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte tra Ýcking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.     

Cell death by apoptosis following X-irradiation of the foetal and neonatal rat kidney

A light and electron microscopic study was undertaken to determine the type of cell death induced by X-irradiation in the developing kidney. Five-day-old Sprague-Dawley rats were exposed to a whole-body dose of either 2 or 5 Gy, and foetuses in the eighteenth day of development were exposed to a dose of 4 Gy. The kidneys were examined at 4, 8 and 24 h, and at 1 and 2 weeks post-irradiation. The dying cells from both control and treated kidneys showed the morphological features of apoptosis, a distinct form of cell death that has been identified in mammalian tissues under physiological as well as pathological conditions. Necrosis was not detected. Apoptosis was infrequent in control kidneys and insignificant in extent when compared with the proliferative activity of the cells of the superficial nephrons. There was a pronounced increase in apoptosis during the first day after irradiation. The findings are in agreement with recent ultrastructural studies which report the presence of apoptosis following irradiation of rapidly proliferating adult cell populations, and irradiation of other immature mammalian tissues. There is evidence that apoptosis involves active cellular self-destruction, and it has been suggested that activation of apoptosis might bring about selective elimination of cells with critical DNA damage in irradiated tissues, thus minimizing propagation of genetic abnormalities.     

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotese-mediated shedding

PURPOSE: L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITC-labelled annexin-V in the presence ofpropidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. RESULTS: PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. CONCLUSION: UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte trafficking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.     

Pulse mode of laser photodynamic treatment induced cell apoptosis

One of the factors limiting photodynamic therapy (PDT) is hypoxia in tumor cells during photodynamic action. PDT with pulse mode irradiation and appropriate irradiation parameters could be more effective in the singlet oxygen generation and tissue re-oxygenation than continuous wave (CW) mode. We theoretically demonstrate differences between the cumulative singlet oxygen concentration in PDT using pulse mode and CW mode of laser irradiation. In vitro experimental results show that photodynamic treatment with pulse mode irradiation has similar cytotoxicity to CW mode and induces mainly cell apoptosis, whereas CW mode induces necrotic cell death. We assume that the cumulative singlet oxygen concentration and the temporal distribution of singlet oxygen are important in photodynamic cytotoxicity and apoptosis initiation. We expect our research may improve irradiation protocols and photodynamic therapy efficiency.     

Protein synthesis-dependent apoptotic signalling pathway in X-irradiated MOLT-4 human leukaemia cell line.

PURPOSE: To demonstrate whether protein synthesis is required for ionizing radiation-induced apoptosis through activation of caspases in human leukaemia cell line MOLT-4, the effects of a protein synthesis inhibitor, cycloheximide, on the apoptotic signalling pathway including the activation of caspase family and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and the expression of Fas/CD95/APO-1 (Fas) were examined in X-irradiated MOLT-4 cells. MATERIALS AND METHODS: MOLT-4 cells pretreated with 0.5 microg/ml cycloheximide for 1h were exposed to 7.5Gy of X-rays. The appearance of apoptosis, expression of Fas, activation of caspases-3, -8, -9, SAPK/JNK and AP-1, the release of mitochondrial cytochrome-C and the formation of death-induced signalling complex (DISC) between Fas and the Fas-associated death domain (FADD) were measured by fluorescence microscopy, Western blotting, flow cytometry, gel shift assay and immunoprecipitation, respectively. RESULTS: Nuclear fragmentation and chromatin condensation were observed at 6 h after X-irradiation and gradually increased up to 12 h. These phenomena were significantly attenuated by cycloheximide. Cycloheximide also inhibited the activation of caspases and AP-1, the expression of Fas, the formation of DISC and the release of cytochrome-C, but not the activation of SAPK/JNK in X-irradiated MOLT-4 cells. CONCLUSION: These results indicate that apoptosis of X-ray-induced MOLT-4 cells is dependent on the activation of caspases regulated by de novo protein synthesis through SAPK/JNK activation.     

Gamma irradiation of human leukaemic cells HL-60 and MOLT-4 induces decrease in Mcl-1 and Bid, release of cytochrome c, and activation of caspase-8 and caspase-9

PURPOSE: Apoptosis is significantly controlled by proteins of Bcl-2 (B-cell lymphoma 2) family promoting cell death or maintaining cell survival. We selected two representatives of Bcl-2 family (anti-apoptotic Mcl-1 - myeloid cell line-1 and pro-apoptotic Bid - Bcl-2 homology domain 3 interacting death agonist), cytochrome c (cyt-c), and two initial caspases (-8 and -9) to evaluate their function in ionizing radiation (IR)-induced apoptosis in human leukaemic cell lines diverging in p53 (TP53 tumor suppressor gene) status. MATERIALS AND METHODS: A total of 30 microg of proteins of whole-cell lysates or 10 microg of mitochondrial protein fractions were electrophoretically separated and analyzed by Western-blotting. RESULTS: Here we show that in both HL-60 (p53 null) and MOLT-4 (p53 wild type) leukaemic cells the amount of Mcl-1 initially increased after irradiation by sublethal but not by lethal dose and later (when apoptosis occurred) it decreased in a dose-dependent manner. Caspase-8 was cleaved and afterwards the amount of Bid decreased as it was truncated. We also found cyt-c release from the inner mitochondrial membrane space into cytoplasm to be dose-dependent and it was followed by induction of apoptosis. In the p53-null cells caspase-8 was activated prior caspase-9, whereas the cells harboring p53 exhibited a simultaneous activation of both initial caspases. CONCLUSION: IR induced a decrease in Mcl-1, activation of Bid, caspase-8, and -9, and release of cyt-c. Presented data indicate that both extrinsic and intrinsic apoptosis signalling pathways were activated in HL-60 and MOLT-4 cells upon exposure to IR regardless to the p53 status.     

淫羊藿甙对辐照小鼠的抗凋亡作用与半胱天冬酶活性的相关性研究

目的：观察淫羊藿甙抗辐射损伤作用，并探讨其抗辐射损伤机制。方法：KM种小鼠于60Coγ射线照射前48，24h及照后即刻灌胃淫羊藿甙，剂量分别为1，10，100mg/kg，照射剂量8．0Gy，观察受照射小鼠30d存活率和死亡动物平均存活时间。另外观察淫羊藿甙对l锄种小鼠受5．5Gy 60Coγ射线照射后小鼠胸腺、脾脏和骨髓细胞凋亡率、半胱天冬酶-3(caspase-3)和半胱天冬酶-8(caspase-8))活性的影响。结果：中剂量淫羊藿甙组30d存活率较照射对照组提高50％，死亡动物平均存活时间延长2．5d。照射对照组胸腺、脾脏和骨髓细胞在照射后6，12和24h均有凋亡。胸腺和脾脏于照射后12h凋亡率最高，骨髓于照后6h凋亡率最高。照射24h后，3种组织的凋亡串均明显降低。中剂量淫羊藿甙可降低照射后6，12h胸腺、脾脏、骨髓细胞凋亡率和照射后24h脾脏、骨髓细胞凋亡率，抑制照射后6hcaspase-3活性，对半胱天冬酶-8活性无影响。结论：淫羊藿甙具有抗辐射损伤作用，其机制之一可能是通过抑制caspase-3活性降低细胞凋亡率。     

Radiation-induced apoptosis in vivo: therapeutic significance of apoptosis in radiation therapy

Apoptotic cell death is frequently found in certain tumor cells after irradiation; however, the incidence is not always high in vivo. Seven tumors were transplanted to nude mice, and their organs were histologically examined after irradiation to study the therapeutic significance of apoptosis in radiation therapy. A high incidence of apoptosis was found only in radiosensitive tumors or normal cells with wild-type p53, but the peak incidence in most cells was only a few percent or less. However, the calculated total incidence of apoptotic cell death was much higher than the actual peak incidence, because the half-life of apoptosis is very short. Even in radioresistant tumors, total radiation-induced apoptosis was estimated to be about 10 percent. These results suggest that apoptotic cell death in radiotherapy may be more important in vivo than previously estimated.     

Gamma irradiation of human leukaemic cells HL-60 and MOLT-4 induces decrease in Mcl-1 and Bid,release of cytochrome c,and activation of caspase-8 and caspase-9

Purpose:?Apoptosis is significantly controlled by proteins of Bcl-2 (B-cell lymphoma 2) family promoting cell death or maintaining cell survival. We selected two representatives of Bcl-2 family (anti-apoptotic Mcl-1 – myeloid cell line-1 and pro-apoptotic Bid – Bcl-2 homology domain 3 interacting death agonist), cytochrome c (cyt-c), and two initial caspases (-8 and -9) to evaluate their function in ionizing radiation (IR)-induced apoptosis in human leukaemic cell lines diverging in p53 (TP53 tumor suppressor gene) status.

Materials and methods:?A total of 30 μg of proteins of whole-cell lysates or 10 μg of mitochondrial protein fractions were electrophoretically separated and analyzed by Western-blotting.

Results:?Here we show that in both HL-60 (p53 null) and MOLT-4 (p53 wild type) leukaemic cells the amount of Mcl-1 initially increased after irradiation by sublethal but not by lethal dose and later (when apoptosis occurred) it decreased in a dose-dependent manner. Caspase-8 was cleaved and afterwards the amount of Bid decreased as it was truncated. We also found cyt-c release from the inner mitochondrial membrane space into cytoplasm to be dose-dependent and it was followed by induction of apoptosis. In the p53-null cells caspase-8 was activated prior caspase-9, whereas the cells harboring p53 exhibited a simultaneous activation of both initial caspases.

Conclusion:?IR induced a decrease in Mcl-1, activation of Bid, caspase-8, and -9, and release of cyt-c. Presented data indicate that both extrinsic and intrinsic apoptosis signalling pathways were activated in HL-60 and MOLT-4 cells upon exposure to IR regardless to the p53 status.     

低剂量辐射对小鼠胸腺细胞成熟、分化、凋亡和激活的影响

目的研究低剂量辐射对胸腺细胞成熟、分化、激活和细胞凋亡及有关基因蛋白表达的影响，以揭示低剂量辐射免疫增强效应的发生机理。方法采用双参数直接免疫荧光和间接免疫荧光流式细胞术，检测了低剂量辐射小鼠全身照射后胸腺细胞ＣＤ４、ＣＤ８、ＴＣＲ、ＣＤ３、ＩＬ－２Ｒ、［Ｃａ２＋］ｉ、Ｂｃ１－２、Ｂａｘ的表达和细胞凋亡。结果实验结果表明：低剂量辐射全身照射未引起ＴＳ减少和ＴＨ／ＴＳ比值上调；低剂量辐射未增加胸腺细胞凋亡，这与Ｂｃｌ－２／Ｂａｘ比值上调有关；低剂量辐射促进了胸腺细胞的成熟分化和信息传递过程。结论以上结果提示：低剂量辐射促进胸腺细胞的成熟、分化和激活，使胸腺向外周输送成熟的具有功能活性的效应性Ｔ细胞的储备增强，可能是低剂量辐射免疫增强效应的重要机理。