Micro-organisation of the membrane after radiation-induced apoptosis: a flow cytometry study

The aim of this study was to detect membrane fluidity modifications in blood lymphocytes that had been exposed to γ-radiation, at a graded series of depths from the surface to the centre of the membrane bilayer and as a function of cell viability. A time course was performed to verify the contribution of the membrane to radiation-induced apoptosis. In comparison with spectrofluorimetry, flow cytometry proved to be a reliable method for measuring radiation-induced membrane alterations. Late apoptotic lymphocytes were characterised by a significant decrease of the 3-SA, 6-SA and 9-SA fluorescence anisotropy values, compared to viable lymphocytes. Moreover, a highly significant difference was observed in the early apoptotic lymphocyte subpopulation between the fluorescence anisotropy values measured 24 h (radiation-induced apoptosis) and those measured 1 h (spontaneous apoptosis) after irradiation. The simultaneous assessment of cellular viability and membrane fluidity using n-(9-anthroyloxy) fatty acid probes, may be relevant for the investigation of interactions which may exist between membrane modifications and the apoptotic process. Our observations support the specificity of radiation-induced apoptosis compared to spontaneous apoptosis in terms of biophysical modifications of membrane properties. Received: 15 January 2001 / Accepted: 1 July 2001     

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.     

Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip

INTRODUCTION: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. METHODS: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. RESULTS: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. CONCLUSIONS: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Human lymphocyte apoptosis after exposure to influenza A virus

Infection of humans with influenza A virus (IAV) results in a severe transient leukopenia. The goal of these studies was to analyze possible mechanisms behind this IAV-induced leukopenia with emphasis on the potential induction of apoptosis of lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure to IAV showed that a portion of CD3(+), CD4(+), CD8(+), and CD19(+) lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response but could be a possible mechanism of disease pathogenesis.     

Radiation-induced DNA double-stranded breaks and the dynamics of apoptotic death of human peripheral lymphocytes

DNA double-stranded breaks and their association with the development of radiation-induced peripheral lymphocyte apoptosis were studied in healthy donors exposed to in vitro gamma-irradiation in a dose of 1 Gy. It was shown that irradiation in 1-Gy dose caused a significant (p < 0.05) increase in the frequency of cells in late apoptosis 4 hours after irradiation and a rise in their frequency in early apoptosis 24 hours following this procedure. A significant correlation (r = 0.52, p < 0.05) was recorded between the primary level of radiation-induced DNA double-stranded breaks and the frequency of cells in late apoptosis following 4 hours, which suggests that DNA double-stranded breaks as a signal to trigger cell apoptotic death are of great importance.     

Apoptosis induced by membrane damage in human lymphocytes; effects of arachidonic acid and its photoproducts

The effect of arachidonic acid (AA) combined with UVA irradiation was studied in a model system mimicking phototherapy PUVA (psoralen+UVA) ex vivo in vitro. The contribution of damage to the plasma membrane by PUVA was tested on human lymphocytes derived from healthy donors. The effect of arachidonic acid (AA) combined with UVA irradiation was compared with that of a psoralen photoadduct to AA added to the culture. The adduct, obtained photochemically and purified, was characterized by NMR and MS spectrometry as a cycloadduct of psoralen to the vinylene bond of the acid (AA<>PSO). The reactions of cultured cells, manifested 20 h after treatment by changes in apoptosis and mitochondrial depolarization, were monitored by flow cytometry by tagging lymphocytes with appropriate fluorescent probes. Treatment of lymphocyte suspension within AA doses from 40 to 100 microM gradually induced a shift from Anx-V(+) (single positive cells) to late apoptotic, Anx-V(+)PI(+) (double positive cells) in a dose dependent manner. The adduct, AAPSO, induced apoptotic changes at a concentration 2-3 times higher than free AA. Combination of psoralen (1 microM ) or arachidonic acid (20-120 microM) with UVA irradiation (2-6 J/cm(2)) accelerated the plasma membrane changes in a synergic way. Preliminary studies indicated that changes in the transmembrane potential of mitochondria paralleled the apoptosis when cells were treated by AA alone. Our findings showed that UVA radiation of lymphocytes in the presence of arachidonic acid, as in the presence of psoralen, enhanced apoptosis of cells in a synergic manner. Thus, PUVA-induced apoptosis may proceed in part by a still undefined signaling pathway(s) triggered in lymphocyte membranes.     

Two different types of apoptosis in thymocytes

Thymocyte cell death was investigated after UVA-irradiation (365 nm) of the primary thymocyte culture in vitro. To determine the mode of cell death two fluorescent DNA-binding dyes (Acridine Orange and Ethidium Bromide) were used along with flow cytometry. Thymocytes undergo apoptosis spontaneously, but a percentage of apoptotic cells was seen to increase in a dose dependent manner after thymocytes exposition to 1-100 J/m2 UVA. These doses are lower than those commonly used for apoptosis induction in other lymphoid cells. Using flow cytometry, we demonstrated that UVA-irradiation induced two different types of apoptosis. The one referred to as the "fast" apoptosis was recorded within 1-4 h following irradiation, whereas the other one, called the "delayed" apoptosis occurred within 3-24 h after irradiation. After UVA-irradiation, the activation of the former prevented the development of the latter; whereas the inhibition of delayed apoptosis brought about the induction of the fast apoptosis in thymocytes. The interrelation between the fast and delayed types of apoptosis in thymocytes can be modulated by cycloheximide, an inhibitor of protein synthesis.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Radiation-induced apoptosis: a new approach using infrared microspectroscopy

PURPOSE: to characterize radiation-induced apoptosis in human cells using Fourier transform infrared microspectroscopy (FT-IRM) as a new analytical tool. MATERIAL AND METHODS: Normal human circulating lymphocytes were given a gamma ray dose of 6 Gy, or treated with t-butyl hydroperoxide (t-BuOH). HaCaT keratinocytes were given a dose of 20 Gy. Cells were deposited on ZnS windows for infrared spectral acquisition 2 days and 2 h after irradiation and 2 h after t-BuOH treatment. Apoptosis was simultaneously assessed by flow cytometry analysis of cells displaying annexin-V-positive staining. RESULTS: The flow cytometry study showed that about 90% of sham and irradiated cells were annexin-V negative 2 h after irradiation. Two days after irradiation, 68% of lymphocytes and 76% of HaCaT cells were apoptotic, as well as 43% of lymphocytes treated with t-BuOH. In infrared spectra of these apoptotic cells, qualitative and quantitative changes were observed. In the 960-1245 and 1690-1720 cm-1 ranges, mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structure, in particular an increase in the amount of beta structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids in apoptotic HaCaT cells only. Two hours after irradiation, depending on the dose and (or) the cell type, qualitative and quantitative changes were observed in the IR spectra in the amide I and amide II bands, mainly attributed to proteins. These changes were associated with a significant decrease in the 1700-1750 cm-1 range, mainly attributed to the -C=O ester groups of DNA and phospholipids, in the irradiated HaCaT cells only. CONCLUSION: Our results are in agreement with biochemical published data on radiation-induced apoptosis, and show that DNA is the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. They also demonstrate that FT-IRM may be useful for assessing the early radiation damage at the molecular level in human cells.     

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.     

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.     

A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells

BACKGROUND: Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V-FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells. METHODS: Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus-containing cells are selected using CD45-phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy-coupled dye (ECD) labeling. Apoptosis is detected using Annexin V-FITC with 7-amino-Actinomycin-D (7-AAD) to distinguish early apoptotic from late apoptotic lymphocytes. RESULTS: We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%. CONCLUSIONS: We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies.     

N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24 h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.     

Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.     

UV light killing efficacy of fluorescent protein‐expressing cancer cells in vitro and in vivo

We investigated the cell‐killing efficacy of UV light on cancer cells expressing GFP in the nucleus and RFP in the cytoplasm (dual‐color cells). After exposure to various doses of UVA, UVB, or UVC, apoptotic and viable cells were quantitated under fluorescence microscopy using dual‐color 143B human osteosarcoma cells, HT‐1080 human fibrosarcoma cells, Lewis lung carcinoma (LLC), and XPA‐1 human pancreatic cancer cells in vitro. UV‐induced cancer cell death was wave‐length and dose dependent, as well as cell‐line dependent. After UVA exposure, most cells were viable even when the UV dose was increased up to 200 J/m². With UVB irradiation, cell death was observed with irradiation at 50 J/m². For UVC, as little as 25 J/m² UVC irradiation killed approximately 70% of the 143B dual‐color cells. This dose of UVB or UVA had almost no effect on the cancer cells. UV‐induced cancer cell death varied among the cell lines. Cell death began about 4 h after irradiation and continued until 10 h after irradiation. UVC exposure also suppressed cancer cell growth in nude mice in a model of minimal residual cancer (MRC). No apparent side effects of UVC exposure were observed. This study opens up the possibility of UVC treatment for MRC after surgical resection. J. Cell. Biochem. 110: 1439–1446, 2010. © 2010 Wiley‐Liss, Inc.     

Modality of Cell Death Induced by Foscan®-Based Photodynamic Treatment in Human Colon Adenocarcinoma Cell Line HT29

Apoptosis induced by photodynamic therapy (PDT) is considered to be an important factor defining the treatment outcome. Nevertheless, the relevance of apoptotic events in overall cell death should be established for every given photosensitizer. The present study addresses the contribution of Foscan-(meta-tetra(hydroxyphenyl)chlorine; mTHPC) photosensitized apoptosis in overall cell death in a model of cultured HT29 adenocarcinoma cells. Early events of cell death were assessed by the evaluation of mitochondrial response to mTHPC-mediated PDT, cytochrome c release and membrane depolarization. Apoptosis was measured through the activity of caspase-3 and the binding of the fluorescent conjugate Ca2+-dependent protein Annexin-V on membrane externalized phosphatidylserine at 2, 4, and 24 h post-PDT. Immediately after mTHPC-PDT, from 28 to 57% cells exhibited cytochrome c release concomitantly with mitochondrial membrane depolarization for light doses inducing more than 90% overall cell death. The maximum of caspase-3 activation (12-fold more than control) was reached 24 h after irradiation at fluence inducing 90% cell death (LD(90)). The corresponding measurement of apoptotic cells (12% of Annexin-V bound cells) confirmed the mild and delayed apoptotic response of HT29 cells to mTHPC-PDT.     

CD8+ natural killer cells have a potential of a sensitive and reliable biodosimetric marker in vitro

The aim of our work was to evaluate peripheral blood lymphocyte subsets as in vitro indicators of the received dose of ionizing radiation (biodosimetric markers) in the range of 3-20 Gy and to determine the appropriate time interval, during which a dose-dependent induction of apoptosis occurs upon gamma irradiation. In lymphocyte subsets characterized by double color surface immunophenotyping, four-color flow cytometry was used for visualizing cell death-associated increase in superficial phosphatidylserine exposure and cytoplasmic membrane permeability by fluorinated Annexin V and propidium iodide, respectively. No differences between sham-treated and lethal dose (7 Gy)-irradiated samples were observed upon 6 h cultivation in vitro. Ten and 18 h later, about 50 % of lymphocytes were apoptotic, but only the minority of them was in the late apoptotic phase. The only difference in radioresistance of the CD4(+)CD8(-) and CD4(-)CD8(+) lymphocyte subsets was seen upon 2-day cultivation when huge depletion of intact cells and prevalence of the late apoptotic population became obvious. A dose-dependence study in 16 and 48 h cultures confirmed the effectiveness of major T cell subsets as biodosimetric indicators. On the other hand, the minor CD8(+) subset of natural killer (NK) cells has been identified as a radiosensitive lymphocyte population the disappearance of which correlated with the received dose. We demonstrated that the CD3(-)CD8(+)NK subset can be used as a lethal/sublethal dose discriminator to 16 h cultivation. In addition, our data indicate that two-day cultivation followed by CD3/CD8 expression analysis in an intact lymphocyte population may provide a clue for low dosage biodosimetry.     

Danthron Triggers ROS and Mitochondria-Mediated Apoptotic Death in C6 Rat Glioma Cells Through Caspase Cascades, Apoptosis-Inducing Factor and Endonuclease G Multiple Signaling

This research focused on the induction of cytotoxic effects by danthron, a natural anthraquinone derivative on C6 rat glioma cells through exploring the means of cell death and the effects on mitochondrial function. We found that danthron decreased the percentage of viable C6 cells and induced cell morphological changes in a dose-and time-dependent manner. The morphological and nuclei changes (DAPI staining) in C6 cells were observed using a contrast-microscope and fluorescence microscopy, respectively. The results suggest that cell death of C6 cells which are induced by danthron is closely related to apoptotic death. Danthron decreased the level of mitochondrial membrane potential (ΔΨ( m )), stimulated the release of cytochrome c from mitochondria to cytosol and promoted the levels of caspase-9 and caspase-3, or induced the release of AIF and Endo G from mitochondria. Based on both observations, we suggest that the danthron-provoked apoptotic death of C6 cells is mediated through the mitochondria-dependent pathway. Furthermore, our results also indicated that danthron triggered apoptosis through reactive oxygen species (ROS) production which were increased after 1 h exposure of danthron, which was reversed by the ROS scavenger N-acetyl-L: -cysteine (NAC). As a consequence, danthron-mediated cell death of C6 cells via ROS production, mitochondrial transmembrane potential collapse and releases of cytochrome c, AIF and Endo G. Taken together, danthron was demonstrated to be effective in killing C6 rat glioma cells via the ROS-promoted and mitochondria-dependent apoptotic pathways.     

Dose- and time-dependent effects of TNFalpha and actinomycin D on cell death incidence and embryo growth in mouse blastocysts

This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors--TNFalpha and actinomycin D--at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFalpha. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFalpha and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.