Apoptosis and cell cycle progression in an acidic environment after irradiation

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.     

The effects of ionizing radiation on stem cells and hematopoietic progenitors: the place of apoptosis and the therapeutic potential of anti-apoptosis treatments

Abstract: Bone marrow aplasia observed following ionizing radiation exposure (Total Body Irradiation; gamma dose range: 2-10 Gy) is a result, in particular, of the radiation-induced (RI) apoptosis in hematopoietic stem and progenitor cells (HSPC). We have previously shown in a baboon model of mobilized peripheral blood CD34+ cell irradiation in vitro that RI apoptosis in HSPC was an early event, mostly occurring within the first 24 hours, which involves the CD95 Fas pathway. Apoptosis may be significantly reduced with a combination of 4 cytokines (4F): Stem Cell Factor (SCF), FLT-3 Ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3), each at 50 ng x mL(-1) (15% survival versus <3% untreated cells, 24 h post-irradiation at 2.5 Gy). In this study we show that addition of TNF-alpha(800 IU/ml) induces an increase in 4F efficacy in terms of cell survival 24 h after incubation (26% survival after 24 h irradiation exposure at 2.5 Gy) and amplification (k) of CD34+ cells after 6 days in a serum free culture medium (SFM) (kCD34+ = 4.3 and 6.3 respectively for 4F and successive 4F + TNF-a/ 4F treatments). In addition, the 4F combination allows culture on pre-established allogenic irradiated stromal cells in vitro at 4 Gy (kCD34+ = 4.5). Overall this study suggests (i) the potential therapeutic interest for an early administration of anti-apoptotic cytokines with or without hematopoiesis inhibitors (emergency cytokine therapy) and (ii) the feasibility in the accidentally irradiated individual, of autologous cell therapy based on ex vivo expansion in order to perform autograft of residual HSPC collected after the accident.     

Effect of Epicatechin against Radiation-Induced Oral Mucositis: In Vitro and In Vivo Study

Purpose

Radiation-induced oral mucositis limits the delivery of high-dose radiation to head and neck cancer. This study investigated the effectiveness of epicatechin (EC), a component of green tea extracts, on radiation-induced oral mucositis in vitro and in vivo.

Experimental Design

The effect of EC on radiation-induced cytotoxicity was analyzed in the human keratinocyte line HaCaT. Radiation-induced apoptosis, change in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation and changes in the signaling pathway were investigated. In vivo therapeutic effects of EC for oral mucositis were explored in a rat model. Rats were monitored by daily inspections of the oral cavity, amount of oral intake, weight change and survival rate. For histopathologic evaluation, hematoxylin-eosin staining and TUNEL staining were performed.

Results

EC significantly inhibited radiation-induced apoptosis, change of MMP, and intracellular ROS generation in HaCaT cells. EC treatment markedly attenuated the expression of p-JNK, p-38, and cleaved caspase-3 after irradiation in the HaCaT cells. Rats with radiation-induced oral mucositis showed decreased oral intake, weight and survival rate, but oral administration of EC significantly restored all three parameters. Histopathologic changes were significantly decreased in the EC-treated irradiated rats. TUNEL staining of rat oral mucosa revealed that EC treatment significantly decreased radiation-induced apoptotic cells.

Conclusions

This study suggests that EC significantly inhibited radiation-induced apoptosis in keratinocytes and rat oral mucosa and may be a safe and effective candidate treatment for the prevention of radiation-induced mucositis.     

Differential gene expression in gamma-irradiated BALB/3T3 fibroblasts under the influence of 3-aminobenzamide,an inhibitior of parp enzyme

3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination with 3AB. A31 cells in exponential growth were pre-treated with 3AB 4mM 1h before gamma-irradiation (4Gy), remaining in culture during 6h until harvesting time. A31 cells treated with 3AB alone presented a down-regulation in genes involved in protein processing and cell cycle control, while an up-regulation of genes involved in apoptosis and related to DNA/RNA synthesis and repair was verified. A31 cells irradiated with 4Gy displayed 41 genes differentially expressed, being detected a down-regulation of genes involved in protein processing and apoptosis, and genes controlling the cell cycle. Concomitantly, another set of genes for protein processing and related to DNA/RNA synthesis and repair were found to be up-regulated. A positive or negative interaction effect between 3AB and radiation was verified for 29 known genes. While the combined treatment induced a synergistic effect on the expression of LCK proto-oncogene and several genes related to protein synthesis/processing, a negative interaction effect was found for the expression of genes related to cytoskeleton and extracellular matrix assembly (SATB1 and Anexin III), cell cycle control (tyrosine kinase), and genes participating in DNA/RNA synthesis and repair (RNA helicase, FLAP endonuclease-1, DNA-3 glycosylase methyladenine, splicing factor SC35 and Soh1). The present data open the possibility to investigate the direct participation of specific genes, or gene products acting in concert in the mechanism underlying the cell response to radiation-induced DNA damage under the influence of PARP inhibitor.     

Low-Dose X-Ray Irradiation Promotes Osteoblast Proliferation,Differentiation and Fracture Healing

Great controversy exists regarding the biologic responses of osteoblasts to X-ray irradiation, and the mechanisms are poorly understood. In this study, the biological effects of low-dose radiation on stimulating osteoblast proliferation, differentiation and fracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5 Gy) X-ray irradiation induced the cell viability and proliferation of MC3T3-E1 cells. However, high-dose (5 Gy) X-ray irradiation inhibited the viability and proliferation of osteoblasts. In addition, dynamic variations in osteoblast differentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterix and osteocalcin, were observed after both low-dose and high-dose irradiation by Western blot analysis. Second, fracture healing was evaluated via histology and gene expression after single-dose X-ray irradiation, and low-dose X-ray irradiation accelerates fracture healing of closed femoral fractures in rats. In low-dose X-ray irradiated fractures, an increase in proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation and fracture calluses was observed. In addition, we observed more rapid completion of endochondral and intramembranous ossification, which was accompanied by altered expression of genes involved in bone remodeling and fracture callus mineralization. Although the expression level of several osteoblast differentiation genes was increased in the fracture calluses of high-dose irradiated rats, the callus formation and fracture union were delayed compared with the control and low-dose irradiated fractures. These results reveal beneficial effects of low-dose irradiation, including the stimulation of osteoblast proliferation, differentiation and fracture healing, and highlight its potential translational application in novel therapies against bone-related diseases.     

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.     

Mouse embryonic stem cells that survive γ-rays exposure maintain pluripotent differentiation potential and genome stability

This study aimed to investigate the cell cycle, apoptosis, cytogenetics and differentiation capacity of mouse embryonic stem cells (mESCs) that survived a single dose of 2 or 5 Gy γ-rays during a period of up to 96 h of culture. After 2 Gy irradiation and 24 h culture, compared to control, a significant majority of cells was blocked at the G2/M phase and a massive apoptosis was recorded. Between 48 and 72 h post-irradiation, the parameters used to describe the cell cycle and apoptosis returned similar to those of control samples. When mESCs were irradiated with 5 Gy, a small fraction of cells, even after 96 h of culture, still presented clear evidences of a G2/M block and apoptosis. The cytogenetic analysis performed at 96 h showed that the structural stability of the aberrations did not change significantly when comparing control and 2 or 5 Gy-treated populations. However, the chromosomal damage observed in the progeny of the survived cells after 5 Gy exposure is significantly higher than that observed in control samples, although it is mostly of the stable and transmissible type. Ninety-six hours after irradiation, the survived mESCs maintained their undifferentiated status and capability to differentiate into the three germ layers. Overall, these results indicate a commitment of mESCs to maintain pluripotency and genome stability.     

The generation and characterization of a radiation-resistant model system to study radioresistance in human breast cancer cells.

To systematically study the selection of radioresistant cells in clinically advanced breast cancer, a model system was generated by treating MDA-MB231 breast cancer cells with fractionated gamma radiation. A clonogenic assay of the surviving cell populations showed that 2-6 Gy per fraction resulted in a rapid selection of radioresistant populations, within three to five fractions. Irradiation with additional fractions after this initial increase did not increase the radioresistance of the surviving population significantly. Doses of 0.5 and 8 Gy per fraction were not effective in selecting radioresistant cells. To further determine the cause of the changes in radiosensitivity, 15 clones were isolated from the cell populations treated with 40 or 60 Gy with 2 or 4 Gy per fraction, respectively, and were analyzed for radiosensitivity. The average D(10) for these clones was 6.75 +/- 0.36 Gy, which was higher than that for the parental cell population (D(10) = 6.0 +/- 0.2 Gy). The operation of cell cycle checkpoints and the doubling time were similar for both the nonirradiated parental population and the isolated radioresistant subclones. In contrast, a decrease in the apoptotic potential was correlated (r = 0.7, P < 0.01) with increased survival after irradiation, suggesting that apoptosis is an important factor in determining radioresistance under our experimental conditions. We also isolated several subclones from the nonirradiated parental cell population and analyzed them to determine their radiosensitivity after fractionated irradiation. Ten fractions of 4 Gy (40 Gy in total) did not result in a significant increase in the radioresistance of these subclones compared to the irradiated cell populations. The possible mechanisms of the increased radioresistance after fractionated irradiation are discussed.     

Monitoring of premitotic and postmitotic apoptosis in gamma-irradiated HL-60 cells by the mitochondrial membrane protein-specific monoclonal antibody APO2.7

BACKGROUND: Majority of hematopoietic cells die by apoptosis after irradiation with ionizing radiation. In present study it is shown that human promyelocytic leukemia HL-60 cells can undergo two different types of apoptosis, premitotic and postmitotic. METHODS: HL-60 cells were irradiated with doses 8 and 20 Gy. For apoptosis detection APO2.7 antigen (mitochondrial membrane specific protein) expression without and with permeabilization by digitonin was used. This method was compared with flow-cytometric analysis of cell light scattering properties and determination of subG1 DNA. RESULT: Cells irradiated with high dose (20 Gy) died rapidly by premitotic apoptosis (interphase death) from all phases of cell cycle. 2 hours after irradiation cells with subdiploid DNA content and cells stained by APO2.7 after digitonin permeabilization appeared. After 6 hours 40% of cells were apoptotic, nonapoptotic cells were mainly in G1-phase. Lower dose (8 Gy) after 6 hours of irradiation caused accumulation of cells in S-phase. After 24 hours majority of cells was in G2-phase and apoptotic cells appeared (subG1 peak, APO2.7 with permeabilization). CONCLUSION: Data presented herein indicate that mitochondrial membrane protein-specific antibody APO2.7 after permeabilization is a useful marker for detection of early apoptotic cells dying by premitotic and postmitotic apoptosis.     

Evaluation of delayed apoptotic response in lethally irradiated human melanoma cell lines

To assess the lethal doses of gamma radiation and corresponding apoptotic response in new established human melanoma cell lines we exposed exponentially growing cultures to 8-100 Gy gamma radiation. The apoptosis and cell survival were determined by trypan blue exclusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction, agarose gel electrophoresis, colony forming assay, and long-term survival assay. The maximal DNA fragmentation 3 days after irradiation was observed in cultures irradiated with 20 Gy (36.9% TUNEL positive cells). The cultures irradiated with 50 and 100 Gy contained 18.7% and 16.4% TUNEL positive cells, respectively. Cultures exposed to 8 and 20 Gy gamma radiation recovered by week 3-4. Lethally irradiated (50 and 100 Gy) cultures which contained less apoptotic cells by day 3 died by week 5. A detectable increase in melanoma cell pigmentation after irradiation was also observed. The survival of human melanoma cell cultures after exposure to gamma radiation does not correlate with the level of apoptotic cells by day 3. At high radiation doses (> 50 Gy) when the radiation induced cell pigmentation is not inhibited the processes of apoptotic DNA fragmentation might be preferentially inactivated.     

Application of autologous hematopoietic cell therapy to a nonhuman primate model of heterogeneous high-dose irradiation

We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.     

Modulation of ionizing radiation-induced apoptosis and cell cycle arrest by all-trans retinoic acid in promyelocytic leukemia cells (HL-60)

Acute promyelocytic leukemia is characterized by a block of myeloid differentiation. The incubation of cells with 1 micromol/l all-trans retinoic acid (ATRA) for 72 h induced differentiation of HL-60 cells and increased the number of CD11b positive cells. Morphological and functional changes were accompanied by a loss of proliferative capacity. Differentiation caused by preincubation of leukemic cells HL-60 with ATRA is accompanied by loss of clonogenicity (control cells: 870 colonies/10(3) cells, cells preincubated with ATRA: 150 colonies/10(3) cells). D0 for undifferentiated cells was 2.35 Gy, for ATRA differentiated cells 2.46 Gy. Statistical comparison of clonogenity curves indicated that in the whole range 0.5-10 Gy the curves are not significantly different, however, in the range 0.5-3 Gy ATRA differentiated cells were significantly more radioresistant than non-differentiated cells. When HL-60 cells preincubated with 1 micromol/l ATRA were irradiated by a sublethal dose of 6 Gy, more marked increase of apoptotic cells number was observed 24 h after irradiation and the surviving cells were mainly in the G1 phase of the cell cycle, while only irradiated cells were accumulated in G(2) phase. Our results imply that preincubation of cells with ATRA accelerates apoptosis occurrence (24 h) after irradiation by high sublethal dose of 6 Gy. Forty-eight hours after 6 Gy irradiation, late apoptotic cells were found in the group of ATRA pretreated cells, as determined by APO2.7 positivity. This test showed an increased effect (considering cell death induction) in comparison to ATRA or irradiation itself.     

Degradation of the nuclear matrix is a common element during radiation-induced apoptosis and necrosis

Human promyelocytic leukemia (HL60) cells were irradiated with 10 or 50 Gy of X rays and studied for up to 72 h postirradiation to determine the mode of death and assess changes in the nuclear matrix. After 50 Gy irradiation, cells were found to die early, primarily by apoptosis, while cells irradiated with 10 Gy died predominantly by necrosis. Disassembly of the nuclear lamina and degradation of the nuclear matrix protein lamin B occurred in cells undergoing radiation-induced apoptosis or necrosis. However, using Western blotting and a recently developed flow cytometry assay to detect changes in nuclear matrix protein content, we found that the kinetics and mechanisms of disassembly of the nuclear lamina are different for each mode of cell death. During radiation-induced apoptosis, cleavage and degradation of lamin B to a approximately 28-kDa fragment was detected in most cells within 4-12 h after irradiation. Measurements of dual-labeled apoptotic cells revealed that nonrandom DNA fragmentation was evident prior to or concomitant with breakdown of the nuclear lamina. Disassembly of the nuclear lamina during radiation-induced necrosis occurred much later (between 30-60 h after irradiation), and a different cleavage pattern of lamin B was observed. Degradation of the nuclear lamina was also inhibited in apoptosis-resistant BCL2-overexpressing HL60 cells exposed to 50 Gy until approximately 48 h after irradiation. These data indicate that breakdown of the nuclear matrix may be a common element in radiation-induced apoptosis and necrosis, but that the mechanisms and temporal patterns of breakdown of the nuclear lamina during apoptosis are distinct from those of necrosis.     

Alterations of DNA copy number and expression in genes involved in cell cycle regulation and apoptosis signal pathways in gamma-radiation-sensitive SX9 cells and -resistant SR-1 cells

In the present study, genomic differences related to sensitivity to radiation were examined by comparative genomic hybridization and GeneChip 45K microarray in SX9 cells (radiation-sensitive) and their parental line, SR-1 (radiation-resistant). SX9 cells have defective DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity. DNA-PKcs is a DNA double-strand break repair protein that maintains chromosomal stability through nonhomologous end joining. However, the molecular basis of the radiation sensitivity of SX9 cells is unclear. Flow cytometry analysis showed that SR-1 and SX9 cells had a larger G2/M-phase population at 12 h after 4 Gy gamma irradiation, while only SR-1 cells progressed to G1/S at 24-36 h. SX9 and SR-1 cells had similar patterns of DNA copy number alteration, but the gains were observed on chromosome 9 (cent-E2), 11 (cent-A3), and 12 (C1-E) only in SX9 cells. Expression of genes located on those regions is higher in SX-9 cells than in SR1 cells, and the regions include genes associated with apoptosis and cell cycle regulation. Time-course data for gene expression at 0, 1, 3, 6 and 12 h after 4 Gy gamma irradiation revealed that the genes whose expression was altered in SX9 cells but not in SR-1 cells are in 16 clusters. Three of these clusters included genes for cell cycle regulation: JNK, PKC (PRKC) and ceramide cascade protein. These results suggest that amplification and altered expression of genes associated with cell cycle and apoptosis regulators in DNA-PK-deficient SX9 cells affect the differences in response to gamma radiation between SX9 and SR-1 cells.     

PI3K激活NF-κB信号通路保护中子辐射致IEC-6细胞损伤

中子属于高传能线密度电离辐射,能产生比κ射线更为严重的放射损伤,肠上皮对中子辐射高度敏感,迄今未见有关中子辐射致肠上皮细胞损伤中PI3K对NF-κB信号通路调控的研究报道.本研究旨在探讨中子照射后肠上皮细胞中PI3K对NF-κB信号通路的调控及其在中子辐射致肠上皮细胞损伤中的作用.选取肠上皮细胞系-6（intestinal epithelial cell No.6,IEC-6）进行传代培养,随机分为对照组、4Gy中子照射组和4Gy中子照射＋LY294002处理组,照射组和LY294002处理组细胞采用4Gy中子均匀照射,LY294002处理组细胞在照前24h给予终浓度为10κmol/L的LY294002,各组于照射后6和24h采用MTT比色法、流式细胞术和免疫印迹（Western blot）方法检测IEC-6细胞增殖活力、凋亡与坏死率以及NF-κB信号通路相关分子NF-κB（p65）,IKKκ和IκBκ的表达变化.研究发现,4Gy中子照射后6和24h,IEC-6细胞增殖活力下降,凋亡和坏死率增加;应用LY294002后IEC-6细胞增殖活力较照射组明显下降,IEC-6细胞凋亡和坏死率较照射组增加.4Gy中子照射后6和24h,IEC-6细胞NF-κB（p65）和IKKκ表达升高,IκBκ表达降低;应用LY294002后NF-κB（p65）和IKKκ表达降低,IκBκ表达升高,表明4Gy中子照射可引起IEC-6细胞增殖活力下降,凋亡和坏死率增加;PI3K可激活NF-κB信号通路,对中子辐射IEC-6细胞损伤发挥保护作用.