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.     

存活素与辐射诱导的凋亡

存活素(survivin)是一种新发现的细胞内蛋白,属于抗细胞凋亡蛋白家族,在胚胎和各类肿瘤中均表达,但在除胸腺和睾丸以外的成人组织中未被发现。阐明存活素在细胞凋亡和增生中的分子机制及其作用,在抗癌治疗和抑制对机体有害的细胞凋亡方面具有重要意义。     

Cleavage of ATM during radiation-induced apoptosis: caspase-3-like apoptotic protease as a candidate

Purpose : To study the relationship of ionizing radiation-induced apoptosis with the integrity of ATM (mutated in ataxia telangiectasia) that has a critical role in DNA damage sensing and repair, cell-cycle checkpoint controls and maintenance of genomic stability. Materials and methods : U937 cells were treated with gamma-radiation. Sub-G1 DNA content, DNA fragmentation, cleavage of PARP, active caspase-3, cleavage of ATM in vivo and in vitro were measured by flow cytometry, agarose gel electrophoresis, cleaving of colorimetric caspase-3 substrate and Western blotting. Results : ATM is specifically cleaved in cells during the induction of apoptosis by ionizing radiation exposure. The time-course of cleavage coincided with the appearance of cells with a sub-G1 DNA content and activation of caspase-3. ATM was cleaved with similar kinetics as PARP and DEVD-FMK could abolish the cleavage. In vitro studies showed that ATM was cleaved by caspase-3 or related subfamily members at a DIVD/G site. Conclusion : ATM belongs to a group of repair proteins, including PARP, DNA-PK and HsRad51, which are specifically cleaved during apoptosis. These findings support the idea that repair mechanisms need to be inactivated for apoptosis to proceed effciently.     

Cleavage of ATM during radiation-induced apoptosis: caspase-3-like apoptotic protease as a candidate

PURPOSE: To study the relationship of ionizing radiation-induced apoptosis with the integrity of ATM (mutated in ataxia telangiectasia) that has a critical role in DNA damage sensing and repair, cell-cycle checkpoint controls and maintenance of genomic stability. MATERIALS AND METHODS: U937 cells were treated with gamma-radiation. Sub-G1 DNA content, DNA fragmentation, cleavage of PARP, active caspase-3, cleavage of ATM in vivo and in vitro were measured by flow cytometry, agarose gel electrophoresis, cleaving of colorimetric caspase-3 substrate and Western blotting. RESULTS: ATM is specifically cleaved in cells during the induction of apoptosis by ionizing radiation exposure. The time-course of cleavage coincided with the appearance of cells with a sub-G1 DNA content and activation of caspase-3. ATM was cleaved with similar kinetics as PARP and DEVD-FMK could abolish the cleavage. In vitro studies showed that ATM was cleaved by caspase-3 or related subfamily members at a DIVD/G site. CONCLUSION: ATM belongs to a group of repair proteins, including PARP, DNA-PK and HsRad51, which are specifically cleaved during apoptosis. These findings support the idea that repair mechanisms need to be inactivated for apoptosis to proceed efficiently.     

Staurosporine modulates radiosensitivity and radiation-induced apoptosis in U937 cells

PURPOSE: The present study aims at investigating the involvement of several genes in the cell cycle distribution and apoptosis in U937 cells, a cell line lacking functional p53 protein, after combined treatment with staurosporine and irradiation. MATERIALS AND METHODS: Using a DNA fragmentation assay, flow cytometry and western blot analysis, the molecular basis for the effects of staurosporine in combination with the irradiation of leukemia cells was investigated. RESULTS: Our results indicated that combined treatment led to an increased apoptotic cell death in U937 cells, which is correlated with the phosphorylation of the V-Jun sarcoma virus 17 oncogene homolog (c-JUN) NH(2)-terminal kinase protein (JNK), the activation of caspases, the increase in B cell leukemia/lymphoma 2 (Bcl-2) associated X protein (Bax), the decrease in Bcl xL protein (Bcl-XL) levels, the loss of mitochondria membrane potential and the release of cytochrome c. CONCLUSIONS: Abrogation of the G2 checkpoint should be an effective strategy against p53-deficient leukemia cells to irradiation-induced cell killing.     

Cellular origin of ionizing radiation-induced NF-kappaB activation in vivo and role of NF-kappaB in ionizing radiation-induced lymphocyte apoptosis

PURPOSE: To investigate the cellular origin of ionizing radiation (IR)-induced NF-kappaB activation in vivo and the role of NF-kappaB in IR-induced lymphocyte apoptosis. MATERIALS AND METHODS: NF-kappaB activities were analysed by gel shift/supershift assay in isolated murine T- and B-cells, macrophages (MPhi) and tissues from normal and T- and B-cell-deficient Rag1 mice with or without exposure to IR. IR-induced lymphocyte apoptosis was determined by analysis of 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)) uptake, annexin-V staining and the sub-G0/1 population, or by TUNEL assay. RESULTS: The results showed that IR activated NF-kappaB in lymphocytes, including both T- and B-cells, but failed to do so in MPhi. Furthermore, T- and B-cell-deficient Rag1 mice exposed to IR exhibited a significant reduction in NF-kappaB activation as compared with normal mice. Although NF-kappaB1 (p50) gene knockout or NF-kappaB decoy oligonucleotide treatment specifically inhibited IR-induced lymphocyte NF-kappaB activation, they had no significant effect on IR-induced lymphocyte apoptosis. CONCLUSIONS: This finding suggests that lymphocytes are the main cellular origin of IR-induced NF-kappaB activation in vivo. However, NF-kappaB activation has no significant effect on IR-induced lymphocyte apoptosis.     

Dose-response for radiation-induced apoptosis, residual 53BP1 foci and DNA-loop relaxation in human lymphocytes

The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53 (TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear dose dependence for all apoptotic end-points up to 1-2 Gy followed by a plateau at higher doses. Immediately after irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose-response with saturation at 2-3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to the average size of DNA-loops in resting lymphocytes. Similar saturations in dose-responses for apoptosis and DNA-loop relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one residual foci per cell was calculated. Radiation at 2-3 Gy induced at least one residual 53BP1 focus per cell. The dose-responses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2-3 Gy. The correlation between dose-responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may be linked to induction of radiation-induced apoptosis in lymphocytes.     

Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells.

PURPOSE: To investigate the mechanisms and pathways of X-ray apoptosis in Molt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+]i) regulation. MATERIALS AND METHODS: X-irradiated Molt-4 cells and cell extract (CE) were used to analyse: (1) induced apoptosis (Giemsa stain), (2) p53, Bcl-2 and Bax expressions (immunoblot), (3) mitochondrial potential deltapsi(m) and (4) [Ca2+]i (flow cytometry), (5) caspase-3 activity, and (6) roles of [Ca2+]- and caspase-3-mediated pathways by inhibiting either or both pathways for induced apoptosis. RESULTS: Molt-4 cells were sensitive to apoptosis since 5 Gy induced 57 and 94% apoptosis at 6 and 24 h. After 5Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bax reduced deltapsi(m), and low-deltapsi(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 was activated in apoptotic CE. The caspase-3 inhibitor Ac-DEVD-CHO inhibited apoptosis and DNA-ladder formation by approximately 50%, suggesting a approximately 50% role of caspase-3-activated DNase (CAD). [Ca2+]i was increased after 5 Gy. [Ca2+]i-chelating BAPTA-AM (5 microM) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited approximately 50% of induced apoptosis and DNA-laddering, indicating a 50% participation of Ca2+/Mg2+-dependent DNase gamma. CONCLUSIONS: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca+2]i-mediated DNase gamma pathway were involved in the regulation of X-ray apoptosis in sensitive Molt-4 cells.     

Enhanced scintigraphic protocol required for optimal preoperative localization before targeted minimally invasive parathyroidectomy

At our tertiary care institution, a targeted minimally invasive parathyroidectomy (MIP) is the preferred surgical procedure for primary hyperparathyroidism. Similar to unilateral neck exploration (UNE), preoperative scintigraphic localization of the adenoma in relation to the midline is required. However, in contrast to the abbreviated standard incision for UNE, 2 distinct incision sites, 1 medial and 1 lateral, are available on each side with MIP. The incision site is ultimately chosen based on scintigraphic determination of the adenoma's vascular origin to facilitate ligation and removal. Unfortunately, the scintigraphic location of a parathyroid adenoma does not necessarily reflect the site of its vascular origin. We reviewed our database to identify factors that accurately predict the site of vascular origin of parathyroid adenomas. A retrospective chart review was performed on 125 patients who underwent Tc-99m sestamibi scintigraphy and parathyroidectomy. Scintigraphic localization, surgical findings, and histopathology were recorded. Preoperative image interpretations that were discordant with operative findings were independently reviewed. Scintigraphy identified the presence of an adenoma in 105 of 118 patients (89%) with primary hyperparathyroidism. In 17 of the 105 cases (16%), the scintigraphic interpretation did not accurately reflect the site of superior or inferior vascular origin seen at surgery. In many discordant cases, anterior images were insufficient for determining the vascular origin. The posterior displacement of an adenoma in relation to the thyroid on early lateral images was often critical in determining the superior or inferior vascular origin. Scintigraphic determination of the superior or inferior vascular origin of a parathyroid adenoma directs incision placement for MIP. Imaging protocols should include early lateral images when localizing parathyroid adenomas before minimally invasive parathyroidectomy.     

Model for the initiation of ionizing radiation-induced apoptosis in lymphoid cells by complex DNA double-strand breaks

PURPOSE: To present a model for the molecular events that lead to the induction of apoptosis in irradiated lymphoid cells based on the assumption that the process is triggered by complex DNA double-strand breaks (DSB). OUTLINE OF THE MODEL: * Cellular DNA repair mechanisms have difficulty rejoining complex DSB because of the nature of the end groups on such breaks. * Association between p53 and DNA topoisomerase I (topo I) can occur at complex DSB in open regions of the genome and the enzymic activity of such associations is not suppressed by polyADP-ribosylation. * Binding of p53 and topo I at a complex DSB results in the transient trapping of a DNA-topo I cleavage complex. * Transiently trapped DNA-topo I cleavage complexes at complex DSB are reversed following association with topo I bound elsewhere in the genome, thus initiating a misrejoining event. * Topo I-mediated DNA misrejoining creates a structure that activates p53. Initiation of rapid interphase apoptosis requires that the inducing signal from activated p53 exceeds a threshold level. * Initiation of rapid interphase apoptosis is regulated by poly(ADP-ribose) polymerase.     

In utero radiation-induced apoptosis and p53 gene expression in the developing rat brain

Purpose : This study addressed the question of the role of the p53 gene in prenatal low-dose radiation-induced apoptosis in the neuroepithelium, in an effort to elucidate molecular mechanisms involved in the extreme radiosensitivity of the developing brain. Materials and methods : Pregnant Wistar rats were exposed to a single dose of 10, 20 or 40 cGy of X-rays on day 15 or 17 of gestation. Animals were sacrificed 4 or 24 h after exposure. Apoptosis was studied by gel electrophoresis of isolated DNA and in situ by the TUNEL reaction. Expression of the p53 gene was studied by immunocytochemistry and Western analysis, as well as Northern analysis, for the detection of the protein and mRNA respectively. Results : In utero low-dose irradiation led to apoptosis and an increase of p53 gene expression in the developing rat brain. Apoptotic as well as p53 immunopositive cells were detected among proliferating, migratory and post-mitotic neurones in the developing neuroepithelium following prenatal irradiation, even after only 10 cGy. In addition to the p53 protein, p53 mRNA brain levels were also increased following prenatal irradiation. Conclusions : Low-dose prenatal irradiation of the developing brain led to p53 induction and cell death by apoptosis.     

Sensitivity to radiation-induced apoptosis and neuron loss declines rapidly in the postnatal mouse neocortex

Therapeutic brain irradiation can cause progressive decline in cognitive function, particularly in children, but the reason for this effect is unclear. The study explored whether age-related differences in apoptotic sensitivity might contribute to the increased vulnerability of the young brain to radiation. Postnatal day 1 (P1) to P30 mice were treated with 0-16 Gy whole-body X-irradiation. Apoptotic cells were identified and quantified up to 48 h later using the TdT-UTP nick end-labelling method (TUNEL) and immunohistochemistry for activated caspase-3. The number of neuron-specific nuclear protein (NeuN)-positive and -negative cells were also counted to measure neuronal and non-neuronal cell loss. Significantly greater TUNEL labelling occurred in the cortex of irradiated P1 animals relative to the other age groups, but there was no difference among the P7, P14 and P30 groups. Irradiation decreased the %NeuN-positive cells in the mice irradiated on P1, whereas in P14 animals, irradiation led to an increase in the %NeuN-positive cells. These data demonstrate that neocortical neurons of very young mice are more susceptible to radiation-induced apoptosis. However, this sensitivity decreases rapidly after birth. By P14, acute cell loss due to radiation occurs primarily in non-neuronal populations.     

In utero radiation-induced apoptosis and p53 gene expression in the developing rat brain

PURPOSE: This study addressed the question of the role of the p53 gene in prenatal low-dose radiation-induced apoptosis in the neuroepithelium, in an effort to elucidate molecular mechanisms involved in the extreme radiosensitivity of the developing brain. MATERIALS AND METHODS: Pregnant Wistar rats were exposed to a single dose of 10, 20 or 40 cGy of X-rays on day 15 or 17 of gestation. Animals were sacrificed 4 or 24h after exposure. Apoptosis was studied by gel electrophoresis of isolated DNA and in situ by the TUNEL reaction. Expression of the p53 gene was studied by immunocytochemistry and Western analysis, as well as Northern analysis, for the detection of the protein and mRNA respectively. RESULTS: In utero low-dose irradiation led to apoptosis and an increase of p53 gene expression in the developing rat brain. Apoptotic as well as p53 immunopositive cells were detected among proliferating, migratory and post-mitotic neurones in the developing neuroepithelium following prenatal irradiation, even after only l0 cGy. In addition to the p53 protein, p53 mRNA brain levels were also increased following prenatal irradiation. CONCLUSIONS: Low-dose prenatal irradiation of the developing brain led to p53 induction and cell death by apoptosis.     

The circadian rhythm for the number and sensitivity of radiation-induced apoptosis in the crypts of mouse small intestine

Apoptosis is a mode of cell death involving nuclear pycnosis, cytoplasmic condensation and karyorrhexis. Changes in the number of apoptotic cells at various times (3-12 h) after a single dose of either 0.5 or 9.0 Gy given at 09.00, 21.00 or 03.00 h were studied in histological sections of small intestinal crypts of mice. The incidences of apoptosis were examined 3 or 6 h after irradiation at different times of day with different doses of gamma-rays ranging from 0.15 to 9.0 Gy. Survival curves were constructed from the dose-incidence curves for apoptosis, using the number of apoptotic cells after high doses (NM) as the maximum cell population size. The mean lethal doses (Do) for the dose range 0-0.5 Gy were calculated for each time of day. A circadian rhythm in both Do and NM values was detected, indicating that both the number and sensitivity of radiation-induced apoptosis were changing throughout the day. A possible explanation based on the cell-cycle states of the target cell population for apoptosis (presumably functional stem cells) was drawn. Most of the target cells were assumed to be in an extended G1 phase. Around 21.00 h a transition from G1 to S phase takes place in some of these cells (approximately seven or eight cells per whole crypt). The S phase then lasts till around 06.00 h. They may be at G2 and M around 06.00-09.00 h, and then they re-enter G1. The circadian rhythm for the number and sensitivity of the cells susceptible to apoptosis obtained in the present report agrees well with this pattern of cell-cycle phases of target cells.     

Sensitivity to radiation-induced apoptosis and neuron loss declines rapidly in the postnatal mouse neocortex

Therapeutic brain irradiation can cause progressive decline in cognitive function, particularly in children, but the reason for this effect is unclear. The study explored whether age-related differences in apoptotic sensitivity might contribute to the increased vulnerability of the young brain to radiation. Postnatal day 1 (P1) to P30 mice were treated with 0 – 16 Gy whole-body X-irradiation. Apoptotic cells were identified and quantified up to 48 h later using the TdT-UTP nick end-labelling method (TUNEL) and immunohistochemistry for activated caspase-3. The number of neuron-specific nuclear protein (NeuN)-positive and -negative cells were also counted to measure neuronal and non-neuronal cell loss. Significantly greater TUNEL labelling occurred in the cortex of irradiated P1 animals relative to the other age groups, but there was no difference among the P7, P14 and P30 groups. Irradiation decreased the %NeuN-positive cells in the mice irradiated on P1, whereas in P14 animals, irradiation led to an increase in the %NeuN-positive cells. These data demonstrate that neocortical neurons of very young mice are more susceptible to radiation-induced apoptosis. However, this sensitivity decreases rapidly after birth. By P14, acute cell loss due to radiation occurs primarily in non-neuronal populations.     

Model for the initiation of ionizing radiation-induced apoptosis in lymphoid cells by complex DNA double-strand breaks

Purpose : To present a model for the molecular events that lead to the induction of apoptosis in irradiated lymphoid cells based on the assumption that the process is triggered by complex DNA double-strand breaks (DSB). Outline of the model : ?Cellular DNA repair mechanisms have difficulty rejoining complex DSB because of the nature of the end groups on such breaks. ?Association between p53 and DNA topoisomerase I (topo I) can occur at complex DSB in open regions of the genome and the enzymic activity of such associations is not suppressed by polyADP-ribosylation. ?Binding of p53 and topo I at a complex DSB results in the transient trapping of a DNA-topo I cleavage complex. ?Transiently trapped DNA-topo I cleavage complexes at complex DSB are reversed following association with topo I bound elsewhere in the genome, thus initiating a misrejoining event. ?Topo I-mediated DNA misrejoining creates a structure that activates p53. Initiation of rapid interphase apoptosis requires that the inducing signal from activated p53 exceeds a threshold level. ?Initiation of rapid interphase apoptosis is regulated by poly(ADP-ribose) polymerase.     

Low and high LET radiation-induced apoptosis in M059J and M059K cells

PURPOSE: To investigate and compare the ability of DNA-dependent protein kinase (DNA-PK)-deficient and -proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation. MATERIALS AND METHODS: A human glioma cell line M059J lacking the catalytic subunit of DNA-PK (DNA-PKcs) and its DNA-PKcs-proficient counterpart, M059K, were exposed to 1 and 4 Gy of accelerated nitrogen ions (14N, 140 eV nm(-1), 8-12 Gy min(-1)) or 60Co gamma-rays (0.2 eV nm(-1), 0.7 Gy min(-1)). The induction of apoptosis was studied up to 144 h post-irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA-PKcs and poly(ADP-ribose) polymerase (PARP) as well as DNA-PK and caspase-3 activity were investigated. RESULTS: Low and high LET radiations (4 Gy) induced a time-dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144 h post-irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase-3 activation. CONCLUSIONS: M059J and M059K cells both display radiation-induced apoptosis, which occur independently of caspase-3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.     

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

Purpose : To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. Materials and methods : Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. Results : In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. Conclusion : These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.