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Cell Biology International (2007) 31, 1353–1358 (Printed in Great Britain)
Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells
Zi‑Bing Wanga1, Yu‑Qing Liub, Ying Zhanga, Yan Lia, Xiao‑Xia Ana, Han Xua, Ying Guoa, Wei Jina, Zhu‑Jun Jianga and Yu‑Fang Cuia*
aDepartment of Immunology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, P.R. China
bDepartment of Oncology, Third Affiliated Hospital of Xinxiang Medical College, Henan Province, P.R. China


Abstract

The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6Gy of γ radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12h postirradiation and fraction of apoptotic cells was highest at 24h. Intracellular ROS generation measured with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30min postirradiation and resumed to normal level at 6h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3h after radiation and exhibited an aberrant increase at 6h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.


Keywords: Ionizing radiation, Apoptosis, Mitochondrial membrane potential, Reactive oxygen species, Caspase-3.

1Present address: Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, P.R. China.

*Corresponding author. Tel.: +86 10 6693 1353.


1 Introduction

Apoptosis, or programmed cell death, is a highly regulated process that involves activation of a series of molecular events. It is characterized by cell shrinkage, plasma membrane blebbing, and chromatin condensation (Gupta, 2001; Chen and Wang, 2002). Various extracellular stresses, including reactive oxygen species, ultraviolet radiation, viral infection, and anti-cancer agents are known to induce apoptosis in many cell systems (Henseleit et al., 1996; Mathiasen and Jaattela, 2002). Signaling for apoptosis occurs through multiple independent pathways, but several of them converge on mitochondria, which constitute a critical event in the apoptotic process (Henkart and Grinstein, 1996; Curtin et al., 2002). In this regard, many studies have shown that opening of the mitochondrial permeability transition (MPT) pore results in a disruption of mitochondrial membrane integrity and the loss of MMP. The mitochondrial changes promote the release of apoptogenic proteins such as cytochrome c and apoptosis inducing factor (AIF) from the mitochondrial intermembrane space into the cytosol, which generally lead to caspase activation (Zou et al., 1999). Recent studies have shown that the oxidative stress induced by ROS plays a role as a common mediator of apoptosis (Jing et al., 2002; Hou et al., 2003). Many stimuli such as tumor necrosis factor-α (TNFα), anti-cancer drugs, and chemopreventive agents stimulate cells to produce ROS (Hasnain et al., 2003). Generated ROS can cause MMP loss by activating MPT, and induce apoptosis (Zou et al., 1999). In several models of apoptosis, increased formation of ROS has been noted as an early event in apoptosis and as a main cause of chromosomal aberrations in survived cells (Petit et al., 1995; Zamzami et al., 1995). However, whether ROS is the primary trigger of radiation-induced apoptotic pathway has not been determined.

T lymphocyte seems to be one of the most radiosensitive cells in the body (Cui et al., 1999, 2002). However, few studies have been reported about the sensitivity of human AHH-1 lymphoblastoid cells to radiation. In this study, we used AHH-1 cells to examine the sensitivity to radiation-induced apoptosis and determine its relevance to MMP and ROS.

2 Materials and methods

2.1 Materials

Standard fetal bovine serum (FBS) was purchased from Gibco; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Amresco; RPMI 1640, dimethyl sulfoxide (DMSO), 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI), Rhodamine 123, and DCFH-DA were all purchased from Sigma.

2.2 Cell culture and radiation treatment

AHH-1 cells, a kind gift from Dr Ping-Kun Zhou (Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine), were routinely cultured in medium RPMI 1640 supplemented with 10% heat-inactivated FBS, 100U/ml penicillin, and 100μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C with a 100% humidity atmosphere. Cells were maintained at a density of 2–5×105 cells/ml and the medium was replenished at 48–72h intervals. Cells were harvested during exponential growth and mortality never exceeded 10% as assessed by Trypan blue exclusion test. The cells were irradiated at room temperature with a 60Co γ rays resource at a dose rate of approximately 250cGy/min.

2.3 Cell viability analysis

Immediately after radiation, cells were seeded at the density of 5×103/100μl per well in 96-well cell culture plates. 20μl MTT solution (5mg/ml) was added to the culture medium for a further 4h incubation at 37°C, 5% CO2. Then cells were collected and dissolved in dimethyl sulfoxide. Colorimetric analysis at 570nm was measured.

2.4 Analysis for apoptosis

At the defined times after radiation, cells were fixed with methanol, stained by DAPI solution, and examined under a fluorescent microscope. The apoptotic cells exhibiting morphological features of apoptosis including chromatin condensation and nuclear fragmentation were counted in 5–10 randomly selected fields. Approximately 300–1000 nuclei were examined for each sample, and the results were expressed as the number of apoptotic nuclei divided by the total number of nuclei counted.

2.5 Annexin V–FITC and propidium iodide simultaneous staining

Phosphatidylserine externalization (PS) was assessed by measuring annexin V–FITC binding using a kit from Jingmei and according to the manufacturer's instructions with slight modifications. Approximately 4×105 cells were collected and washed in 1ml cold PBS (pH 7.4) twice. Then the cells were resuspended in 200μl Binding Buffer (pH 7.4) and added to 10μl annexin V–FITC and 5μl PI. After 10min of incubation in the dark at room temperature, each sample was added 300μl Binding Buffer and then immediately (within 1h) analyzed using a flow cytometer (Becton Dickinson).

2.6 Measurement of MMP

Changes in MMP of irradiated cells were measured by uptake of lipophilic cation Rhodamine 123 into mitochondria. About 4×105 cells were collected at the confined time points after radiation and incubated with Rhodamine 123 (10μg/ml) at 37°C for 30min. Then the cells were washed twice with PBS and resuspended in 500μl PBS. The samples were analyzed for fluorescence using a flow cytometer.

2.7 Measurement of intracellular ROS generation

Intracellular ROS production was measured by using a fluorescent dye, DCFH-DA, which can be converted to DCFH by esterases when taken up. DCFH reacts with ROS to generate a new highly fluorescent compound, dichlorofluorescein, which can be analyzed with flow cytometry (FACS). The treated cells were incubated by DCFH-DA (10μM) at 37°C for 30min, washed twice with PBS, and then measured with FACS.

2.8 Immunoblot analysis

Cells were collected, washed twice with cold PBS, and pelleted. The cell pellet was resuspended in 100ml RIPA buffer containing 1% NP-40, 0.5% sodium deoxycholate, 1% SDS, 50mg/ml aprotinin, 100mg/ml PMSF, 1mM dithiothreitol (DTT), and PBS (pH 7.4). After incubation on ice for 20min, the cell lysate concentration was measured with a BioRad protein assay kit, and then by spectrophotometry. Proteins were normalized to 20mg/lane resolved on 10% SDS–PAGE and blotted onto a nitrocellulose membrane with an electroblot apparatus. The membrane was blocked for 2h at room temperature with a blocking reagent (5% [w/v] defatted dry milk), incubated overnight at 4°C with an rabbit anti-Caspase-3 antibody (1:1000), washed three times, and then incubated with a horseradish peroxidase-conjugated secondary antibody in PBS and 0.5% (v/v) Tween-20 for another 30min with gentle shaking. After final washing, the proteins were visualized by using the Enhanced Chemiluminescence (ECL) system.

2.9 Statistical analysis

Unless otherwise indicated, data are presented as means±SD of three or more experiments. Student's t-test was used to compare two means. Statistical significance was defined at the level of P<0.05.

3 Results

3.1 Radiation inhibits proliferation of AHH-1 cells

To examine the effect of radiation on the proliferation of AHH-1 cells, they were treated with 6Gy of γ radiation, and then the cell growth was studied 6, 12, 24, 48, and 72h postirradiation. The result showed that radiation treatment started to significantly inhibit the proliferation of AHH-1 cells after 12h and enhanced its effect in a time-dependent manner (Fig. 1).


Fig. 1

Radiation inhibits AHH-1 cells proliferation. Cells were exposed to 60Co γ rays at a dose of 6Gy. MTT assay was performed at different times after irradiation (6 to 72h). All values are means of triplicate determinations±SD from representative experiments performed at least 4 times. **P<0.01 vs. control.


3.2 Radiation induces apoptosis in AHH-1 cells

To elucidate whether radiation decreases cell survival by the induction of apoptosis, we confirmed the apoptotic characterizations in radiation-treated AHH-1 cells by several approaches. Fig. 2A illustrates the morphological features of apoptosis including chromatin condensation and DNA fragmentation. Fig. 2B shows the kinetics of apoptotic rate by radiation. An increase in the apoptotic index was seen as early as 12h after radiation treatment and became more remarkable at 24h postirradiation, followed by a decrease after that, but still significant statistically at 48h. To confirm this, we further performed annexin V–FITC binding assay. We found that 6h and 24h after radiation, the apoptotic cells respectively reached 10.79% and 23.61%, whereas necrotic cells accounted for only 8.64% and 12.18%, respectively (Fig. 3). Taken together, we conclude that radiation decreases cell survival primarily by apoptosis within 24h, and after that, by necrosis might be the dominant manner of cell death.


Fig. 2

(A) Representative photomicrographs showing the morphological features of radiation induced apoptosis of AHH-1 cells. (B) Radiation-induced apoptosis in AHH-1 cells. Cells were exposed to 6Gy of γ radiation and processed at different time points for apoptosis detection as described in Section 2. Data are means±SD of triplicate experiments. **P<0.01 vs. control.


Fig. 3

Flow cytometric analysis of phosphatidylserine externalization and cell membrane integrity in AHH-1 cells undergoing apoptosis. AHH-1 cells were treated with radiation (6Gy) and detected at 0, 6, and 24h time points. The dual parametric dot plots combining annexin V–FITC and PI fluorescence show the viable cell population in the lower left quadrant (annexin V PI), the early apoptotic cells in the lower right quadrant (annexin V+ PI), and the necrotic cells in the upper right quadrant (annexin V+ PI+).



3.3 Radiation-induced apoptosis does not involve loss of MMP detected by Rhodamine 123

One of the early events in apoptotic signaling appears to be the alteration of mitochondrial membrane integrity. We next examined the effect of radiation on the MMP by using the mitochondria-specific dye Rhodamine 123. Unexpectedly, the fluorescence intensity of Rhodamine 123 did not change at all during the first 3h postirradiation, and exhibited an aberrant increase at 6h (Fig. 4). No loss of MMP was detected with the fluorochrome.


Fig. 4

Detection of MMP with Rhodamine 123. AHH-1 cells were treated with 6Gy of radiation and then stained with Rhodamine 123 at 37°C for 30min at different time points postirradiation. (A) Mean Rhodamine 123 fluorescence intensity detected by flow. (B) The results were expressed as the relative fluorescence intensity (%) with respect to untreated cells. Data are means±SD from representative experiments performed at least 3 times. *P<0.05 vs. control.


3.4 Radiation induces immediate increase of ROS

Studies in a variety of cell types have suggested that cancer chemotherapy drugs induce tumor cell apoptosis, in part, by inducing the formation of ROS. To investigate whether ROS is involved in radiation-induced apoptosis in AHH-1 cells, we examined ROS generation at indicated time points after radiation. As shown in Fig. 5, ROS generation increased quickly and reached maximum at 0.5h after radiation, but decreased gradually after that, and resumed to normal level at 6h. These results indicate that ROS generation is an early phase in apoptosis induced by radiation.


Fig. 5

ROS production in radiation-induced apoptosis in AHH-1 cells. AHH-1 cells were treated with 6Gy of radiation and then stained with DCFH-DA at 37°C for 30min. At each time point, the fluorescence intensity was measured by FACS. (A) Mean DCF fluorescence intensity detected by flow. (B) The results were expressed as the relative fluorescence intensity (%) with respect to untreated cells. Data are means±SD from representative experiments performed at least 3 times. *P<0.05 vs. control. **P<0.01 vs. control.


3.5 Caspase-3 activation is observed in radiation-induced apoptosis

Caspase-3 is a zymogen that is cleaved to a 2-chain polypeptide during apoptosis, resulting in its functional activation. The cleaved form of Caspase-3 was undetectable in nonirradiated cells, while activation and processing of caspase-3 could be detected in AHH-1 cells 12h after 6Gy of radiation (Fig. 6).


Fig. 6

Effect of radiation on caspase-3 processing in AHH-1 cells. Cells were untreated or exposed to 6Gy of radiation, incubated for approximately 12h, and then processed for Western blot analysis using an anti-caspase-3 polyclonal antibody.


4 Discussion

In our previous work, we found that the serious injuries of immune tissues and the inhibition of immune function in irradiated mice were closely related to the apoptosis of the lymphocytes (Cui et al., 1999, 2002). In the present study, we found that AHH-1 cell, which is a valid model for in vitro study, was sensitive to radiation-induced apoptosis. As shown in Fig. 1, radiation treatment caused the significant decrease of cell survival in a time-response manner. To elucidate the causes for the decreased cell viability, we determined the cell apoptotic index by using DAPI staining technique. The results showed that apoptotic cells began to increase dramatically 12h postirradiation, with a maximal effect at 24h, and a decrease after that, but still significant statistically at 48h (Fig. 2). To confirm this, we further detected apoptosis by flow cytometry. The results showed that both apoptotic and necrotic cells increased in a time-dependent manner (Fig. 3). These results made us to think that the primary manner of cell death was by apoptosis within 24h postirradiation, and after that, by necrosis might be the dominant manner of cell death.

Some studies have reported that cancer chemopreventive agents induce apoptosis in part with ROS generation and disruption of redox homeostasis (Jing et al., 2002). On the other hand, some studies reported that apoptosis induced by some anti-cancer agents is independent of ROS generation (Bratton et al., 2000; Chang et al., 2003; Lin et al., 2003). In the present study, we found that radiation induced immediate generation of ROS, with a maximal effect at 0.5h after radiation and resuming to normal level at 6h, indicating that ROS generation might be an early phase in apoptosis process.

Mitochondria have been reported to play a pivotal role in the regulation of apoptosis (Herr and Debatin, 2001; Grebenova et al., 2003). In most, if not in all, models of apoptosis, cells manifest a disruption of the MMP that often precedes nuclear DNA degradation (Marchetti et al., 1996). Changes in the mitochondrial inner membrane function are accompanied by an increase in outer membrane permeability, leading to the release of apoptogenic factors such as cytochrome c and AIF from mitochondria into cytosol (Kim et al., 2002). Methods have been proposed to estimate MMP modifications, notably by flow cytometry with cationic lipophilic dyes (Darzynkiewicz et al., 1982), which are according to the Nernst equation sequestered in the mitochondrial matrix. Among these dyes, Rhodamine 123 is popular for its low cytotoxicity and insignificant fluorescence quenching at high concentration characteristics (Lopez-Mediavilla et al., 1995). However, in the present study we found that after inducing apoptosis, AHH-1 cells did not change or tended to exhibit an increase in fluorescence obtained with Rhodamine 123. The finding is consistent with one of our reports (Wang et al., 2006) in which we suggested three possibilities leading to these contradictory results. From these results, the authors conclude that analyzing MMP might not be a good measure to assess apoptosis. This view, although contrary to many previous studies, is in good agreement with a recent report stating that cytochrome c release proceeds in a single step that is independent of changes in MMP (Goldstein et al., 2005).

In short, we concluded first time from our data that AHH-1 human lymphoblastoid cells are sensitive to radiation-induced apoptosis. ROS generation is an early phase in the apoptosis process. Moreover, the findings that irradiated cells do not change or tend to exhibit an aberrant increase in the fluorescence obtained with Rhodamine 123 might cast doubts on those studies which, using Rhodamine 123, hypothesized that the fall of MMP is one of the early events of apoptosis.

Acknowledgements

The authors would like to acknowledge Dr Pingkun Zhou for the generous gift of AHH-1 cells. In addition, the authors would like to acknowledge Dr Qihong Sun, Dr Jianping Mao, Dr Jianen Gao, Xiaolan Liu and Bo Dong for many helpful comments and suggestions during the course of these experiments.

References

Bratton SB, Lau, SS, Monks, TJ. The putative benzene metabolite 2,3,5-tris(glutathion-S-yl)hydroquinone depletes glutathione, stimulates sphingomyelin turnover, and induces apoptosis in HL-60 cells. Chem Res Toxicol 2000:13:550-6
Crossref   Medline   1st Citation  

Chang H, Oehrl, W, Elsner, P, Thiele, JJ. The role of H2O2 as a mediator of UVB-induced apoptosis in keratinocytes. Free Radic Res 2003:37:655-63
Crossref   Medline   1st Citation  

Chen M, Wang, J. Initiator caspases in apoptosis signaling pathways. Apoptosis 2002:7:313-9
Crossref   Medline   1st Citation  

Cui YF, Gao, YB, Yang, H, Xiong, CQ, Xia, GW, Wang, DW. Apoptosis of circulating lymphocytes induced by whole body gamma-irradiation and its mechanism. J Environ Pathol Toxicol Oncol 1999:18:185-9
Medline   1st Citation   2nd  

Cui Y, Yang, H, Wu, S, Gao, L, Gao, Y, Peng, R. Molecular mechanism of damage and repair of mouse thymus lymphocytes induced by radiation. Chin Med J 2002:115:1070-3
Medline   1st Citation   2nd  

Curtin JF, Donovan, M, Cotter, TG. Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 2002:265:49-72
Crossref   Medline   1st Citation  

Darzynkiewicz Z, Traganos, F, Staiano-Coico, L, Kapuscinski, J, Melamed, MR. Interaction of rhodamine 123 with living cells studied by flow cytometry. Cancer Res 1982:42:799-806
Medline   1st Citation  

Goldstein JC, Munoz-Pinedo, C, Ricci, JE, Adams, SR, Kelekar, A, Schuler, M. Cytochrome c is released in a single step during apoptosis. Cell Death Differ 2005:12:453-62
Crossref   Medline   1st Citation  

Grebenova D, Kuzelova, K, Smetana, K, Pluskalova, M, Cajthamlova, H, Marinov, I. Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells. J Photochem Photobiol B 2003:69:71-85
Crossref   Medline   1st Citation  

Gupta S. Molecular steps of death receptor and mitochondrial pathways of apoptosis. Life Sci 2001:69:2957-64
Crossref   Medline   1st Citation  

Hasnain SE, Begum, R, Ramaiah, KV, Sahdev, S, Shajil, EM, Taneja, TK. Host-pathogen interactions during apoptosis. J Biosci 2003:28:349-58
Crossref   Medline   1st Citation  

Henkart PA, Grinstein, S. Apoptosis: mitochondria resurrected? J Exp Med 1996:183:1293-5
Crossref   Medline   1st Citation  

Henseleit U, Rosenbach, T, Kolde, G. Induction of apoptosis in human HaCaT keratinocytes. Arch Dermatol Res 1996:288:676-83
Crossref   Medline   1st Citation  

Herr I, Debatin, KM. Cellular stress response and apoptosis in cancer therapy. Blood 2001:98:2603-14
Crossref   Medline   1st Citation  

Hou DX, Ose, T, Lin, S, Harazoro, K, Imamura, I, Kubo, M. Anthocyanidins induce apoptosis in human promyelocytic leukemia cells: structure-activity relationship and mechanisms involved. Int J Oncol 2003:23:705-12
Medline   1st Citation  

Jing X, Ueki, N, Cheng, J, Imanishi, H, Hada, T. Induction of apoptosis in hepatocellular carcinoma cell lines by emodin. Jpn J Cancer Res 2002:93:874-82
Medline   1st Citation   2nd  

Kim R, Tanabe, K, Uchida, Y, Emi, M, Inoue, H, Toge, T. Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy. Cancer Chemother Pharmacol 2002:50:343-52
Crossref   Medline   1st Citation  

Lin S, Fujii, M, Hou, DX. Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway. Arch Biochem Biophys 2003:418:99-107
Crossref   Medline   1st Citation  

Lopez-Mediavilla C, Orfao, A, Garcia, MV, Medina, JM. Changes in adult rat liver mitochondrial populations at different energy states analyzed by flow cytometry. Biochim Biophys Acta 1995:1232:27-32
Crossref   Medline   1st Citation  

Marchetti P, Susin, SA, Decaudin, D, Gamen, S, Castedo, M, Hirsch, T. Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA. Cancer Res 1996:56:2033-8
Medline   1st Citation  

Mathiasen IS, Jaattela, M. Triggering caspase-independent cell death to combat cancer. Trends Mol Med 2002:8:212-20
Crossref   Medline   1st Citation  

Petit PX, Lecoeur, H, Zorn, E, Dauguet, C, Mignotte, B, Gougeon, ML. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J Cell Biol 1995:130:157-67
Crossref   Medline   1st Citation  

Wang ZB, Zhang, Y, Liu, YQ, Guo, Y, Xu, H, Dong, B. Bcl-xL overexpression restricts gamma-radiation-induced apoptosis. Cell Biol Int 2006:30:15-20
Crossref   Medline   1st Citation  

Zamzami N, Marchetti, P, Castedo, M, Decaudin, D, Macho, A, Hirsch, T. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995:182:367-77
Crossref   Medline   1st Citation  

Zou H, Li, Y, Liu, X, Wang, X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999:274:11549-56
Crossref   Medline   1st Citation   2nd  


Received 25 February 2006/19 October 2006; accepted 12 May 2007

doi:10.1016/j.cellbi.2007.05.009


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)