Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro ˚ berg c , J.E. Larsson c , S. Ceccatelli a,⁎ R. Tofighi a , N. Tillmark b , E. Daré a , A.M. A a

Institute of Environmental Medicine, Division of Toxicology and Neurotoxicology, Karolinska Institutet, S-171 77 Stockholm, Sweden Division of Mechanics, Royal Institute of Technology, S-100 44 Stockholm, Sweden c Department of Surgery and Perioperative Sciences, Division of Anaesthesia and Intensive Care, Umeå University Hospital, S-901 85 Umeå, Sweden b

A R T I C LE I N FO

AB S T R A C T

Article history:

The neurotoxic effects of carbon monoxide (CO) are well known. Brain hypoxia due to the

Accepted 16 April 2006

binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct

Available online 13 June 2006

effect of CO on intracellular targets remains poorly understood. In the present study, we have investigated the pathways leading to neural cell death induced by in vitro exposure to

Keywords:

CO using a gas exposure chamber that we have developed. Mouse hippocampal neurons

Neural cells

(HT22) and human glial cells (D384) were exposed to concentrations of CO ranging from 300

Neurotoxicity

to 1000 ppm in the presence of 20% oxygen. Cytotoxicity was observed after 48 h exposure to

Cell death

1000 ppm, corresponding to approximately 1 μM CO in the cultured medium, as measured by

Caspases

gas chromatography. CO induced cell death with characteristic features of apoptosis.

Calpains

Exposed cells exhibited loss of mitochondrial membrane potential, release of cytochrome c

Oxidative stress

into the cytosol, nuclei with chromatin condensation, and exposure of phosphatidyl serine on the external leaflet of the plasma membrane. CO also triggered activation of caspase and calpain proteases. Pre-incubation with either the pancaspase inhibitor Z-VAD-fmk (20μM) or the calpain inhibitor E64d (25 μM) reduced by 50% the occurrence of apoptosis. When preincubating the cells with the two inhibitors together there was an additional reduction in the number of cells with apoptotic nuclei. These data suggest that CO causes apoptosis via activation of parallel proteolytic pathways involving both caspases and calpains. Furthermore, pre-treatment with the antioxidant MnTBAP (100 μM) significantly reduced the number of apoptotic nuclei, pointing to a critical role of oxidative stress in CO toxicity. © 2006 Published by Elsevier B.V.

1.

Introduction

Carbon monoxide (CO) is an endogenously produced gas generated by oxidation of organic molecules and degradation of heme. It has important physiological roles in modulation of certain neuronal processes, including intercellular signal transduction (Verma et al., 1993) and generation of long-

⁎ Corresponding author. Fax: +46 8 329041. E-mail address: [email protected] (S. Ceccatelli). 0006-8993/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.brainres.2006.04.095

term potentiation (LTP) in hippocampus (Alkadhi et al., 2001; Zhuo et al., 1993). However, poisoning from exposure to exogenous CO such as accidental poisoning from home heating, automobile exhausts and smoke, as well as intentional for suicidal purposes occurs frequently. Once CO enters the bloodstream through the lungs, it attaches to hemoglobin (Hb), forming carboxyhemoglobin (COHb) and thereby reduc-

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occurrence of cell death, HT22 and D384 cells were exposed to different concentrations of CO ranging from 300 to 1000 ppm for 24 to 48 h in both cell lines. Cytotoxic effects such as abnormal cell attachment and cell shrinkage were detected after exposure to 1000 ppm for 48 h (Figs. 2A, E and B, F). Nuclear staining of fixed cells showed characteristic alterations, i.e., condensed chromatin and intensively propidium iodiode (PI) stained nuclei (Figs. 2C, G and D, H), hallmarks of apoptosis (Kerr et al., 1972). Additional tests with Trypan blue showed that CO induced a significant decrease in the total cell number only after exposure to 1000 ppm for 48 h (Fig. 2I) with no alterations of cell membrane permeability (Fig. 2J), confirming that apoptosis and not necrosis was the type of cell death occurring in the exposed cells.

Fig. 1 – Front view of the test chamber.

ing oxygen (O2) transport with subsequent hypoxia. The acute symptoms of CO poisoning depend on severity of exposure and include headache, dizziness, nausea, vomiting, confusion, impaired language and cognition, collapse, and coma. At lower concentrations, CO affects visual perception, manual deftness, learning and attention level (see review Raub and Benignus, 2002; Gorman et al., 2003). Perhaps, the most insidious effects of CO are on the developing central nervous system. Children of women smoking during pregnancy have lowered intellectual development (Frydman, 1996), which could be a consequence of exposure to CO via smoke. Experimental studies have shown that prenatal exposure to low concentrations of CO lead to disrupted hippocampal long-term potentiation (LTP) (Alkadhi et al., 2001; Zhuo et al., 1993; Mereu et al., 2000), and altered habituation and working memory (Giustino et al., 1999; Mactutus and Fechter, 1984). Brain hypoxia has been considered the major cause of CO neurotoxicity, but in addition, CO can exert a direct damage to cells, possibly by binding directly to intracellular targets such as cytochrome P450 mono-oxygenase and cytochrome c oxidase (Uemura et al., 2001). However, it is still unclear to what extent cell death induced by CO may be due to a direct effect not involving hypoxia. In an attempt to characterize the mechanism(s) of CO toxicity, we have used an in vitro exposure system to investigate the intracellular pathways leading to neural cell death induced by CO. The mouse hippocampal cell line HT22 and the human astrocytoma cell line D384 were exposed to different CO concentrations under normoxic conditions using an exposure chamber that we have developed to expose cultured cells to volatile agents (Fig. 1).

2.

Results

2.1.

Cell damage induced by CO

We focused our studies on the neuronal HT22 and glial D384 cell lines because hippocampal and glial cells are known CO targets in vivo (Piantadosi et al., 1997). To investigate the

2.2. Phosphatidylserine (PS) exposure on the plasma membrane Translocation of PS to the outer leaflet of the plasma membrane plays a critical role in phagocytosis of apoptotic cells (Fadok et al., 1992). To characterize the effects of CO on PS exposure, HT22 and D384 cells were triple stained with Hoechst 33358, Annexin V and PI. Exposed HT22 and D384 cells exhibited nuclear condensation with PS translocation, which were visualized with the vital dye Hoechst 33358 and Annexin V (Figs. 3D, E and J, K). As expected, cells were not stained by the cell-impermeant dye PI, indicating that the plasma membrane was intact and cells were indeed undergoing apoptosis (Figs. 3F, L).

2.3. Loss of mitochondrial membrane potential and release of cytochrome c We used the vital dye tetramethylrhodamine ethyl ester (TMRE) to evaluate the mitochondrial potential. HT22 cells and D384 cells exposed to CO for 48 h showed loss of membrane potential, resulting in the lack of mitochondrial TMRE staining in cells with condensed nuclei (Figs. 4A–B and E–F). To determine further alterations in mitochondrial function, we used immunocytochemistry to look at cytochrome c (cyt c) release into cytosol. The release of this protein from mitochondria to the cytosol plays an essential role in the formation of the apoptosome complex with subsequent activation of the caspase cascade executing apoptosis (Li et al., 1997). In control HT22 and D384 cells cyt c was localized in the mitochondria, whereas in CO exposed cells mostly in the cytosol (Figs. 4C–D and G–H).

2.4.

Activation of caspases and calpains

Caspases, a family of cystein proteases cleaving after aspartate residues, play an important function in neuronal apoptosis (Gorman et al., 1998). Therefore, we investigated the role of caspases in CO-induced apoptosis by measuring their proteolytic activity. Exposure to CO induced a significant increase in caspase 3-like activity in whole cell extracts from both cell lines, approximately 5 folds in HT22, and 9 folds in D384 cells (Fig. 5A). Calpains are cysteine proteases activated by changes in intracellular calcium concentrations. Calpains can either inactivate caspases or participate together with caspases in

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Fig. 2 – (A–H) Morphological features of HT22 and D384 cells exposed to 1000 ppm CO for 48 h. Phase contrast pictures of HT22 (A, E), D384 (B, F) and confocal images showing nuclei stained with PI in HT22 (C, G) and D384 cells (D, H). A–D show control cells and E–H show CO-exposed cells. I, Number of cells after exposure to CO for 48 h, relative to the number of control cells at the same time point. Values are means ± SEM of 3 determinations. Statistical analysis was performed with two-tailed Student's t test (**P ≤ 0.01 and ***P ≤ 0.001). J, Alterations in cell membrane permeability after 48 h CO exposure were evaluated by staining HT22 and D384 cells with Trypan blue. Values are expressed as percentage of cells impermeable to Trypan blue. Scale bar = 20 μm in C–D and G–H.

the execution phase of apoptosis. The cytoskeletal protein αfodrin is a known substrate for both calpains and caspases. These two proteases cleave α-fodrin into distinct fragments of 150 kDa, and 120 kDa, respectively (Nath et al., 1996). Analysis by immunoblotting of HT22 and D384 cells (Fig. 5B) revealed that the 150 kDa breakdown product increased in CO exposed cells. In accordance with the presence of caspase activity, there was also an increase in the 120 kDa product (Fig. 5B).

2.5. Protection with pan-caspase inhibitor, calpain inhibitor and antioxidant Our results pointed to a concomitant activation of both caspases and calpains in cells exposed to CO. In agreement, the percentage of condensed nuclei was significantly decreased when HT22 and D384 cells were pre-incubated with the pan-caspase inhibitor z-VAD-fmk (20 μM) or the calpain inhibitor E64d (25 μM), 30 min prior the exposure to CO (Figs. 6A, B). When cells were pre-incubated with both inhibitors together, the number of apoptotic nuclei was additionally decreased (Figs. 6A, B).

To evaluate the role of oxidative stress in CO toxicity in our experimental models, we pre-incubated cells with the antioxidant MnTBAP, a superoxide dismutase mimetic, 30 min before exposure to CO. Addition of the antioxidant led to a significant decrease in the percentage of nuclei with condensed chromatin (Fig. 6C).

2.6.

CO-concentration in medium

In our experimental design cells attached to plastic vessels and covered by a thin layer of culture medium were kept in the exposure chamber with constant air CO concentration (ranging from 300 to 1000 ppm). Only a modest fraction of CO present in the chamber air dissolves in the medium, resulting in micromolar concentrations, as measured by gas chromatography with flame ionization detection. The actual concentration in the exposed samples was found to be 1.3 ± 0.16 μM (mean ± SEM, n = 3). The concentrations in the control samples were below detection levels. The equilibrium CO concentration in the fluid was also calculated by using Henry's law (XCO = pCO/H) (Moore, 1972), where XCO is the mole fraction of

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Fig. 3 – HT22 and D384 cells were triple stained with Hoechst 33358 (A, D and G, J), Annexin V (B, E and H, K) and PI (C, F, and I, L). Cells exposed to 1000 ppm CO for 48 h displayed bright and fragmented apoptotic chromatin (D and J) and exposed PS (E and K). A, B, C and G, H, I show control cells. Scale bar = 20 μm.

CO dissolved in the liquid phase, pCO the partial pressure in the gas and H is Henry's constant. According to the manufacturer 95% of the medium (w/w) is water and hence the physical constants of water were used for the determination of CO concentration and diffusion in the medium. For water COsystem at 310 K, H is 6.6 × 104 bar, (Battino, 1999), which gave

an estimated CO concentration in the medium in the order of 10−8. At a CO partial pressure of 1000 ppm and a total pressure of 1 bar, the equilibrium CO concentration is 1 μM. This is in accordance with our results obtained with gas chromatography and also with a publication by Thom et al. (Thom et al., 1997), where they reported a CO concentration of 0.01–0.11 μM

Fig. 4 – The mitochondrial membrane potential was evaluated by TMRE staining in control HT22 (A), control D384 (E) and cells exposed to 1000 ppm CO for 48 h (B and F). Co-staining with Hoechst 33358 visualized the nuclei. The drop of mitochondrial potential is indicated by loss of dot-like structures in cells with condensed nuclei. Control and treated samples at the same time point were immunostained with a monoclonal cytochrome c antibody. Control HT22 and D384 cells showed a mitochondrial dot-like pattern (C and G), while the exposed cells exhibited a diffuse cytosolic staining (D and H). Scale bar = 20 μm.

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Fig. 5 – (A) Measurement of caspase 3-like activity in HT22 and D384 cells exposed to 1000 ppm CO for 48 h. The activity was measured in whole cell extracts using the fluorogenic DEVDase assay. Values are means ± SEM of 3 determinations. Statistical analysis was performed with two-tailed Student's t test (*P ≤ 0.05 and ***P ≤ 0.001). (B) Western blot analysis of cleavage of α-fodrin in HT22 and D384 cells exposed to 1000 ppm CO for 48 h. Total proteins (50 μg) were separated by SDS-PAGE and blotted on nitrocellouse membranes. The monoclonal antibody used is known to detect both a 150 kDa fragment produced by calpain cleavage and a 120 kDa fragment produced by caspase cleavage. The level of both cleavage products was increased in exposed cells (B). The blot is representative for 3 independent experiments.

in a cell culture medium subjected to 10–100 ppm CO at atmospheric pressure.

3.

Discussion

Carbon monoxide (CO), a public health concern polluting both outdoor and indoor environments, is believed to exert most of its neurotoxic action via hemoglobin-mediated oxygen deprivation that occurs in the brain of exposed organisms. In the present study, we have investigated the direct neurotoxic effect of CO, independent from hypoxia, by using an in vitro experimental system. CO intoxication leading to neuronal cell death with features of both necrosis and apoptosis has been reported in humans as well as in experimental animals (Piantadosi et al., 1997; Uemura et al., 2001). While necrosis is characterized by ATP-independent cell and organelle swelling, loss of plasma membrane integrity, and cell lysis, apoptosis is an ATP-driven process with cell shrinkage, chromatin condensation, plasma membrane blebbing, and activation of specific proteases (e.g., caspases and calpains) (Nagata, 1997). There are two distinct signaling pathways that can lead to apoptosis. The extrinsic pathway requires the binding of death receptors with their associated ligands (Nagata, 1997) resulting in the activation of

Fig. 6 – Preincubation with z-VAD-fmk (20 μM), E64d (25 μM), or MnTBAP (100 μM) prevented CO-induced nuclear condensation in HT22 (A, C) and D384 cells (B, C). In some experiments the pan-caspase inhibitor z-VAD-fmk and the calpain inhibitor E64d were added together (A, B). Fixed cells were stained with PI and the nuclei were scored at the fluorescence microscope. Apoptotic nuclei are characterized by smaller size and brighter chromatin. Values are means ± SEM of 8 determinations. Statistical analysis was carried out with the one-way analysis of variance ANOVA followed by Fisher's protected least significant difference (PLSD) test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001).

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procaspase-8 which then sequentially activates procaspase-3. The intrinsic pathway involves the exertion of death signals directly or indirectly on the mitochondria (Liu et al., 1996; Bratton et al., 2000), resulting in the release of mitochondrial proteins such as cyt c into the cytosol. In the presence of ATP, cyt c can interact with apoptotic protease-activating factor-1 (Apaf-1) and recruit procaspase-9 in order to form the apoptosome complex. The formation of this complex then initiates the activation of procaspase-3, the main executioner caspase (Bratton et al., 2000). Our studies show that both hippocampal HT22 cells and glial D384 cells exposed to CO undergo apoptotic cell death. Exposed cells exhibited abnormal cell morphology with cell shrinkage and nuclear condensation. Externalization of phosphatidylserine on the outer leaflet of the plasma membrane, which in apoptotic cells is a signal for attracting the phagocytic cells, was also observed. In our experimental models, CO activates the intrinsic mitochondria-mediated cell death pathway as manifested by loss of mitochondrial potential, release of cyt c and activation of caspase 3. Besides caspases, other cysteine and serine proteases can participate in apoptosis. The role of the Ca2+-activated proteases calpains in apoptosis is more prominent in certain cell types including neuronal cells (Vanags et al., 1996) and there is growing evidence for a crosstalk between caspase and calpain pathways (see review Orrenius et al., 2003). Both proteases were concomitantly activated in our CO exposed cells as shown by the presences of the specific caspase (120 kDa) and the calpain (150 kDa) cleavage products of αfodrin. The observed increased protection against CO toxicity when cells were pre-incubated with both the pancaspase inhibitor z-VAD-fmk and the calpain inhibitor E64d, further indicates that these two proteases act in parallel. It has been shown that CO can bind to cyt c oxidase (Brown and Piantadosi, 1990; Thom, 1990), thus inhibition of its activity could lead to an increased reactive oxygen species (ROS) production in cells exposed to CO. ROS are also known to induce alterations of mitochondrial membrane potential with subsequent opening of mitochondrial permeability transition pores and release of cyt c into the cytosol (Ly et al., 2003; Kim et al., 2003). In our experiments, the loss of mitochondrial potential in cells with apoptotic nuclei, the release of cyt c into the cytosol and the protective effects exerted by the antioxidant MnTBAP point to the occurrence of oxidative stress in HT22 and D384 cells exposed to CO. This is in agreement with the study by Uemura et al. (2003), where COinhalation of rats led to generation of ROS and cell death in cortical neurons, independently of hypoxia. In vitro models are gaining increasing consensus as tools to study toxic processes. The complexity of the nervous system and the limited knowledge available about biochemical processes involved in neurotoxicity makes it difficult to reproduce the in vivo systems with cell cultures. However, in vitro models have proven to be well suited for investigations of the molecular, cellular and physiological mechanisms of toxicity in a controlled and isolated context. Furthermore, in vitro tests by providing information on basic mechanistic processes lead to the identification of mechanism-based end points that can then be evaluated in vivo by addressing specific experimental questions. Exposure systems like the one that

we have developed allowing exposure of cell cultures to volatile compounds in a controlled and safe manner are also valuable tools for in vitro research. These experimental approaches greatly contribute to the reduction of the use of experimental animals in toxicity studies. In conclusion, we have used an in vitro model system to investigate the mechanisms of CO neurotoxicity independent from the hemoglobin binding effects. Our results indicate that CO induces mitochondria-mediated apoptosis via a parallel activation of both caspases and calpains, and that oxidative stress plays a critical role in CO toxicity independently of hypoxia.

4.

Experimental procedures

4.1.

Exposure system components

Electrochemical sensor (CO LS-6809620), O2 measuring instrument (Pac III) and remote transmitter (Polytron 2) were purchased from Dräger (Dräger Sicherheitstechnik GmbH, Germany). Pressure regulator and flow controller were purchased from Brooks Instruments (Brooks Instrument, dev. of Emerson Process Management, USA). The Plexiglas chamber was made in collaboration with Eurometric Instruments (Hässelby, Sweden).

4.2.

Gas handling system and test chamber

The experimental device consisted of a gas handling system and a test chamber where the cell cultures were exposed to the toxic gas (Fig. 1). The gas concentration in the atmosphere inside the chamber was measured with an electrochemical sensor. The air came from a 7 bar in house air-supply system and the test gas from a high-pressure bottle (AGA, Stockholm, Sweden). The gas and the air flow branches merged in a tee connector and a mixing filter downstream the connector ensured that the gas mixture was homogenous before it reached the test chamber. The test chamber made of 10 mm thick acryl plastic (Plexiglas) was housed in a standard laboratory oven during the experiments keeping the cell cultures at a fixed stable temperature.

4.3.

CO transport and diffusion

The gas was first transported by the air flow to the inside of the multidishes and thereafter it diffused throughout the cell culture medium. Measurements showed an effective gas transport from the outside chamber atmosphere to inside the casted multidishes. The CO concentration inside the plate deviated less than 10% from the one outside within 15 min from the beginning of the exposure, independently of the culture plate position in the test chamber.

4.4.

Gas chromatography (GC)

The actual concentration of CO in the medium was measured by gas chromatography with flame ionization detection (Sundin and Larsson, 2002). To septum fitted gas tight tubes, 400 μl of the cell medium and a glass bead were added. Eight

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hundred microliter of a liberating solution (saponin 15 g/l and sulphuric acid 1 M) was added, using 2 hypodermic needles through the septum, and the samples were mixed for 40 min. An aliquot (200 μl) of the gas phase was injected manually with a gastight syringe on the GC column. The GC system was a HP 5790 with flame ionization detection, before the flame detector a nickel catalyst system was placed. The injector temperature was 40 °C, the oven temperature 35 °C, the nickel catalyst 375 °C and the detector 250 °C. The carrier gas was helium at a flow of 30 ml/min. The flow for the gases to the detector was 400 ml/min for air and 30 ml/min for hydrogen. Standard samples were made by mixing 400 μl water, 800 μl liberating solution and a glass bead before the tubes were capped. Different amounts of CO were added with gas tight syringes. The amounts used were 0, 2.5, 5, 10, 20 and 50 μl 5% CO in nitrogen. Triplicate samples were prepared. The zero calibration sample was obtained as a blank sample. The calibration samples were mixed for 40 min and 200 μl of the gas phase was injected manually with a gastight syringe on the GC. Calibration curves were calculated and concentrations of CO were expressed in micromolar.

4.5.

In vitro experiments

4.5.1.

Chemicals

floating cells were collected by centrifugation and then pooled together with the adherent cells detached by scraping or with trypsin.

4.5.3.

4.5.2.

Cell culture

The mouse hippocampal HT22 clonal cell line and the human astrocytoma D384 cell line were used as experimental models. HT22 and D384 cells were routinely seeded at a density of 3000 and 10 000 cells/cm2, respectively, in CO2-independent medium (LifeTechnologies, Gibco BRL, catalogue number: 18045054) supplemented with 10% fetal calf serum (FCS), 4 mM Lglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The cell culture flasks were locked and kept in the incubator at 37 °C with proper humidity for 24 h before exposure to the gas. Cells were then exposed to CO concentrations ranging from 300 to 1000 ppm inside the chamber. Control cells were kept in similar conditions in a separate oven. The percentage of O2 in the chamber was constant (20%) during the entire experimental procedure. During the exposure to CO, the bottles and dishes were no longer kept sealed. The caspase inhibitor zVAD-fmk, the calpain inhibitor E64d and the antioxidant MnTBAP were added 30 min before the exposure to CO and left in the culture for the entire exposure period. For harvesting,

Trypan blue exclusion test

Cells were detached from the flasks with trypsin and pooled with the cells floating in the medium. An aliquot of the cell suspension was mixed with an equal volume of 0.4% Trypan blue in PBS. Cells with damaged membrane stained blue (dead cells), while cells with intact plasma membrane remained unstained (healthy and apoptotic cells). Cells were scored at the phase contrast microscope using a Neubauer improved counting chamber.

4.5.4.

Vital triple staining

Annexin V is a phospholipid-binding protein with high affinity for phosphatidyl serine (PS). Cells grown on coverslips were incubated with a solution of Annexin V (0.5 μg/ml), cell impermeable PI (1 μg/ml) and cell permeable Hoechst 33358 (1 μg/ml) in a buffer containing 10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2. Stained cells were examined at the fluorescence microscope (Olympus BX60).

4.5.5. Trypan blue (TB), propidium iodiode (PI), fetal calf serum (FCS), the calpain inhibitor E64d and the antioxidant MnTBAP (Manganese (III) tetrakis-(4-benzoic acid)porphyrin) were purchased from Sigma (St. Louis, MO, USA). Annexin V-fluorescein isothiocyanate (FITC) was purchased from Pharmingen (Bio-Rad, Stockholm, Sweden). Hoechst 33358 and tetramethylrhodamine ethyl ester (TMRE) were obtained from Molecular Probes (Eugene, OR, USA). All other chemicals for cell culture were supplied by Life Technologies (Life Technologies, Gibco BRL, Grand Island, NY, USA). The caspase substrate DEVD-MCA [Ac-Asp-Glu-Val-Asp-α-(4-methyl-coumaryl-7-amide)] and the caspase inhibitor z-VAD-fmk [Z-ValAla-Asp(Ome)-FMK] were purchased from Peptide Institute (Osaka, Japan). The Micro BCA protein assay kit was purchased from Pierce (Boule Nordic AB, Stockholm, Sweden).

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Nuclear staining with propidium iodide

HT22 and D384 cells were grown on coverslips, fixed with icecold methanol/water (8/2 = v/v) at −20 °C for 30 min, washed in phosphate-buffered saline (PBS) and stained with PI (2.5 μg/ml) for 5 min. The coverslips were mounted onto glass slides with PBS/glycerol (1/9 = v/v) containing 0.1% (w/v) phenylendiamine. The coverslips were analyzed and photographed using a confocal microscope (Zeiss LSM 510 meta). The percentage of nuclei with chromatin condensation was determined by scoring at least 100 nuclei in four fields on each coverslip examined (n = 3), using a fluorescence microscope.

4.5.6.

Caspase 3-like activity

Caspase activity was measured by using a fluorogenic assay which evaluates the activity of class II caspases (caspase 2, 3 and 7), a method which has been previously described (Nicholson et al., 1995) with some modifications (Gorman et al., 2000). Substrate cleavage leading to the release of free 4-methyl-coumaryl-7 amide (excitation 355 nm, emission 460 nm) was monitored at 37 °C using a Fluoroscan II (Labsystem AB, Stockholm, Sweden). Fluorescence units were converted to pmoles of 4-methyl-coumaryl-7 amide release using a standard curve generated with 4-methyl-coumaryl-7 amide and subsequently related to protein content.

4.5.7. Measurements of the mitochondrial membrane potential (ΔΨ) TMRE is a dye that partitions to the negatively charged mitochondrial matrix according to the Nernst equation and acts as a voltage sensitive probe. Decreases in ΔΨ are paralleled by a reduction of the fluorescence emitted by TMRE (Daré et al., 2001; Ehrenberg et al., 1988). Cells grown on coverslips were exposed to CO, then incubated with 5 nM TMRE (in PBS) for 30 min at room temperature and co-stained with Hoechst (1 μg/ml) for 5 min. The mitochondria and nuclei were analyzed at the confocal microscope (Zeiss LSM 510 meta).

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4.5.8. Evaluation of cytochrome c release by immunocytochemistry Cells grown on coverslips were fixed with 4% paraformaldehyde (Sigma) for 1 h at 4 °C. Primary anti-cytochrome c antibody (1:100, BD PharMingen) was diluted in PBS supplemented with 0.3% Triton-X100 and 0.5% BSA (Boehringer Mannheim, Bromma, Sweden). Cells were incubated in a humid chamber at 4 °C overnight, rinsed with PBS and incubated with secondary FITC-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature (RT). After rinsing with PBS, coverslips were mounted in glycerol-PBS containing 0.1% phenylenediamine.

4.5.9.

Evaluation of α-fodrin cleavage by immunoblotting

Cells were harvested with tryspin, centrifuged and washed with PBS. The cells were sonicated in a solution containing 1 mM Pefablock (Boehringer Mannheim, Bromma, Sweden), 10 mM EDTA and 2 mM DTT in PBS. Protein content was determined using the Micro BCA kit. After adding the sample buffer (0.4% SDS, 4% glycerol, 1% β-mercaptoethanol, 12.5 mM Tris–HCL, pH 6.8), 50 μg total protein was boiled for 5 min and subsequently resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 6%) and immunoblotted according to methods previously described (Daré et al., 2002). Fodrin cleavage was detected using a mouse anti-spectrin monoclonal primary antibody (dilution 1:1000, Chemicon, California, USA) and a goat anti mouse secondary antibody, horseradish peroxidase conjugated (dilution 1: 20 000, Pierce Rockford, IL USA).

Acknowledgments This work was supported by grants from the European Commission (QLK-CT-1999-01356) and the Swedish Board for Laboratory Animals (CFN).

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