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Cell Biology International (2007) 31, 13531358 (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
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 6
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.
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
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, 100
2.3 Cell viability analysis
Immediately after radiation, cells were seeded at the density of 5
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
2.6 Measurement of MMP
Changes in MMP of irradiated cells were measured by uptake of lipophilic cation Rhodamine 123 into mitochondria. About 4
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 (10
2.8 Immunoblot analysis
Cells were collected, washed twice with cold PBS, and pelleted. The cell pellet was resuspended in 100
2.9 Statistical analysis
Unless otherwise indicated, data are presented as means
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 6
Radiation inhibits AHH-1 cells proliferation. Cells were exposed to 60Co γ rays at a dose of 6
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 12
(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 6
Flow cytometric analysis of phosphatidylserine externalization and cell membrane integrity in AHH-1 cells undergoing apoptosis. AHH-1 cells were treated with radiation (6
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 3
Detection of MMP with Rhodamine 123. AHH-1 cells were treated with 6
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 0.5
ROS production in radiation-induced apoptosis in AHH-1 cells. AHH-1 cells were treated with 6
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 12
Effect of radiation on caspase-3 processing in AHH-1 cells. Cells were untreated or exposed to 6
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 12
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 0.5
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.
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.
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Received 25 February 2006/19 October 2006; accepted 12 May 2007doi:10.1016/j.cellbi.2007.05.009