* Department of Biomedical Sciences, University of Teramo, Italy;
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy;
Department of Physiological, Biochemical and Cellular Sciences, University of Sassari, Italy; and
Space Biology, ETH, Zürich, Switzerland
Correspondence: Mauro Maccarrone, Ph.D., Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. E-mail: Maccarrone{at}vet.unite.it
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Key Words: arachidonate cascade • calcium • caspase • cyclooxygenase • mitochondrial membrane potential
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10-2 g) directly enhances the catalytic efficiency of pure LOX up to fourfold over the ground (1 g) controls [18
]. Here, we created conditions similar to those that occur during exposure of cells to microgravity with the random positioning machine (RPM) [11
] and with the rotary cell culture system (RCCS) [9
] to ascertain whether these conditions might induce apoptosis in human lymphocytes. The possible involvement of the arachidonate cascade and mitochondrial alterations in the apoptotic program were also investigated. Taken together, the data indicate that creating conditions similar to those that occur during exposure of cells to microgravity induces lymphocyte apoptosis and that 5-LOX is a "gravity responder" that executes this process. The ability of 5-LOX to uncouple mitochondria, leading to cytochrome c release and caspase activation, seems to be the critical event along the apoptotic pathway, suggesting that creating conditions similar to those that occur during exposure of cells to microgravity triggers a signaling cascade common to other unrelated proapoptotic stimuli. |
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Creating conditions similar to those that occur during exposure of cells to microgravity
Conditions similar to those that occur during exposure of cells to microgravity were created by the three-dimensional clinostat developed by Fokker Space (Leiden, The Netherlands), also called RPM. The RPM consists of a frame that rotates within a second rotating frame, and each frame is driven by a separate motor. Rotation of each frame is random, autonomous, and regulated by computer software. Each frame rotates at a speed of 60° · s-1, which corresponds to a force of gravity of 2 x 10-3 g at the center of the frame. The RPM was located in a room at 37°C, and a box containing the cell cultures, sealed in 2 ml Eppendorf tubes, was placed at the center of the inner frame. Control (static) cultures were placed at the basement of the RPM. To avoid the presence of air bubbles, which could lead to shear force damage of the cells on the RPM, the Eppendorf tubes were completely filled [11
]. In the experiments on DNA fragmentation in human lymphocytes and in those with isolated mitochondria, the same level of force of gravity (2x10-3g) was obtained by means of the RCCS, developed by the National Aeronautics and Space Administration (Washington, DC) and manufactured by Synthecon (Houston, TX) [9
]. In this case, cell cultures and mitochondria suspensions were placed in 50 ml vessels and rotated at a speed of 7.2 rpm, according to the manufacturer’s instructions.
Lymphocyte isolation, culture, and treatment
Blood samples (20 ml per donor) were drawn from the antecubital vein of healthy donors (age range 28–35 years), who gave informed consent to the study, and were collected into heparinized sterile tubes. Peripheral lymphocytes were purified by gradient centrifugation using the density separation medium Lymphoprep (Nycomed Pharma, Oslo, Norway) and consisted of T lymphocytes (85%), B cells (10%), and monocytes (5%) [21
]. Isolated lymphocytes were resuspended in RPMI-1640 medium (Gibco, Paisley, UK), supplemented with 25 mM Hepes, 2.5 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) at a density of 1.5 x 106 cells/ml [21
] and were stored at 37°C for at least 4 h before exposure to conditions similar to those that occur during exposure of cells to microgravity to allow them to recover from the potential stress of the centrifugation steps during isolation [11
]. Incubation of lymphocytes with different inhibitors was performed at 37°C at the indicated concentrations and for the indicated periods of time. Controls were incubated with vehicles alone. Human lymphoma U937 cells were cultured and treated as the lymphocytes [21
]. The U937 cell line was obtained from an individual with histiocytic lymphoma and has monocyte-like characteristics; therefore, it cannot be considered a pure model for lymphocytes. However, it is widely used as a model for immune cells [17
, 22
, 23
].
Electrotransfer of anti-LOX mAb (200 µg/test) into lymphocytes (106 cells/test) was performed with a Gene Pulser II Plus apparatus (Bio-Rad). Exponentially decaying pulses were generated and delivered to cells suspended in phosphate-buffered saline (PBS; 0.7 ml/test) in sterile, disposable electroporation cuvettes (Bio-Rad) of 0.4 cm path length [22
]. Lymphocytes were electroporated at a capacitance of 125 µF and a field strength of 800 V/cm with a time constant of 1.5 ± 0.2 ms. Control cells were electroporated under the same experimental conditions in the presence of nonimmune mouse serum (200 µg/test) as reported [22
]. After electroporation, cells were kept for 5 min at 4°C, and then they were washed twice in PBS and were subjected to conditions similar to those that occur during exposure of cells to microgravity, as described for the nonelectroporated cells. Under these experimental conditions, 1.0 pg/cell (2.5 µg/mg protein) mAb was transferred inside the cells, where they could inhibit their intracellular target [22
].
Evaluation of cell death
After incubation for the indicated periods of time in culture medium, apoptosis was estimated in all experiments by cytofluorimetric analysis in a FACScalibur flow cytometer (Becton Dickinson, Bedford, MA), which quantified apoptotic body formation in dead cells by staining with propidium iodide (50 µg/ml, also pretreated with 15,000 U/ml RNase to reduce noise), as reported [24
]. Cells were fixed with cold methanol:acetone (4:1 v/v) diluted 1:1 in PBS and were stored at 4°C. Cells were excited at 488 nm using a 15 mW Argon laser, and the fluorescence was monitored at 570 nm. Events were triggered by the forward scattering signal and gated for FL2-A/FL2-W to skip aggregates. Ten-thousand events were evaluated using the Cell Quest Program. Controls of different batches of cells contained less than 4.0 ± 1.0 apoptotic bodies every 100 cells analyzed. Apoptosis was also quantified by the cell-death detection enzyme-linked immunosorbent assay (ELISA) kit (Boehringer Mannheim, Mannheim, Germany), based on the evaluation of DNA fragmentation by an immunoassay for histone-associated DNA fragments in the cell cytoplasm [22
].
Assay of arachidonate-degrading enzymes and of their metabolites
Arachidonic acid is converted into bioactive compounds such as PGs and LTs through the "cyclooxygenase branch" and the "lipoxygenase branch" of the arachidonate cascade, respectively [25
]. The activity of cyclooxygenase (COX; PG-H synthase; E.C. 1.14.99.1) was measured polarographically at 30°C in 0.1 M potassium phosphate, pH 7.2, 1 mM phenol, and 75 µM arachidonic acid [26
] and was expressed as nmol O2 consumed per min per µg protein. The activity of 5-LOX (arachidonate:oxygen 5-oxidoreductase; E.C. 1.13.11.34) was measured by incubating cell extracts for 10 min at 37°C in the presence of 1 mM adenosine 5'-triphosphate, 2 mM CaCl2, and 40 µM arachidonic acid [27
]. 5-LOX activity was expressed as nmol 5-HPETE formed per min per mg protein. The activity of 12- and 15-LOX in the cells was measured under the same experimental conditions in the presence of 40 µM arachidonic acid only [27
, 28
]. The activity of 12- and 15-LOX was expressed as nmol 12- or 15-HPETE formed per min per mg protein. The amount of proteins in cell extracts was determined according to Bradford [29
]. Arachidonate metabolites PGE2 and LTB4 were extracted from intact lymphocytes or from cell supernatants on octadecyl-solid phase extraction columns (Baker, Deventer, The Netherlands) and were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 3 x 3 CR column (SGE, Austin, TX), as reported [27
]. RP-HPLC was performed on a Perkin Elmer 1022 LC Plus liquid chromatograph at a flow rate of 1.2 ml/min, using methanol/water/trifluoroacetic acid (70:30:0.07 by vol) as mobile phase. Chromatograms were recorded at 270 nm, assessing peak identity by comparison with authentic standards. Quantitative determinations were performed by integrating peak areas of each compound. The effect of various inhibitors on cellular activity of 5-LOX, LTB4 content, cytochrome c release, and apoptotic body formation was evaluated by treating intact lymphocytes with each compound for the indicated periods of time and then performing the biochemical assays as detailed above. Inhibitors were used at concentrations found to be effective on their target enzymes in preliminary experiments and were in keeping with previous investigations [17
, 22
, 30
].
ELISA determined the content of 5-LOX protein [22 ] by coating each well overnight with cell extracts (25 µg/well) and reacting with rabbit anti-5-LOX (diluted 1:200) polyclonal antibodies as first antibody. GAR-AP conjugates were used as second antibody (diluted 1:2000), and color development of the alkaline phosphatase reaction was followed at 405 nm, using p-nitrophenylphosphate as substrate [22 ].
Isolation of mitochondria
Mitochondria were isolated from the liver of Wistar rats (weighing 350 g) by standard differential centrifugation, followed by Percoll purification [31
]. Mitochondria isolated by this procedure were 90% intact by cytofluorimetric analysis in a FACScalibur flow cytometer (Becton Dickinson), corroborating previous functional [31
] and morphological [32
] data on preparations obtained with the same method. Isolated mitochondria were resuspended in 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 0.1% bovine serum albumin, 15 mM Hepes (pH 7.4) buffer [17
], at a protein concentration of 10 mg/ml and were used as such for the treatment with pure 5-LOX, 5-HPETE, or 9-HPOD and subsequent biochemical analyses. In some experiments, mitochondria were incubated with 5-LOX (100 units/test) for 2 h in the RCCS to check the effect of conditions similar to those that occur during exposure of cells to microgravity.
Measurement of intracellular calcium, mitochondrial membrane potential, and cytochrome c release
Intracellular calcium concentration and mitochondrial membrane potential were evaluated by flow cytometric analysis in a FACScalibur flow cytometer (Becton Dickinson). Cytoplasmic-free calcium was measured using the fluorescent Ca2+ indicator Fluo-3 AM, as reported [33
]. Immediately before exposure to conditions similar to those that occur during microgravity, lymphocytes were collected by centrifugation and washed twice in Ca2+- and Mg2+-free PBS. Then, Fluo-3 AM [10 µM dissolved in dimethyl sulfoxide (DMSO)] was added, and cells were incubated 40 min at 37°C in the dark and frequently shaken manually and were collected by centrifugation and resuspended in culture medium without FCS. Control cells were treated with vehicle alone (1% of the final volume). After exposure to conditions similar to those that occur during microgravity, Fluo-3 AM fluorescence was recorded on a linear scale at 530 nm (bandwidth 30 nm) at a flow rate of 1000 cells/s. Mean fluorescence values for 3000 events were registered every 10 s. Changes in mean fluorescence were plotted versus time, and ionomycin (10 µg/ml) was used as positive control to set intracellular calcium to 100% [33
].
Mitochondrial membrane potential was measured using the fluorescent probe JC-1, as described [34 ]. JC-1 (dissolved in DMSO) was used at 20 µM final concentration, treating the controls with vehicle alone (1% of the final volume). After the treatment, human lymphocytes or isolated mitochondria were washed in PBS, incubated 20 min at 37°C in PBS, and then exposed to conditions similar to those that occur during microgravity. After this treatment, they were analyzed in a FL1/FL2 dot plot (530 nm/570 nm). CCCP (50 µM) was used as positive control, to set mitochondrial uncoupling to 100%, i.e., mitochondrial membrane potential to 0% [34 ].
The amount of cytochrome c released into the cytosol of human lymphocytes or into the medium by isolated mitochondria was quantified by ELISA, previously validated for both systems by Western blot analysis [22 , 32 ]. Cell extracts or mitochondria supernatants (25 µg protein/well) were prepared as reported [35 ] and were reacted with anticytochrome c mAb diluted 1:250. GAM-AP immunoglobulins were used as secondary antibody at 1:2000 dilution. Color development of the AP reaction was recorded at 405 nm, using p-nitrophenylphosphate as substrate [22 ]. The absorbance values of the unknown samples were within the linearity range of the ELISA test, assessed by calibration curves with known amounts of cytochrome c (in the range 0–500 ng/well).
Statistical analysis
Data reported in this paper are the mean (±SD) of at least three independent determinations, each in duplicate. Statistical analysis was performed by the nonparametric Mann-Whitney test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science, San Diego, CA).
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threefold over the control) and reached a level of five- to sixfold over the control 24–48 h later (Fig. 1A
and 1B)
. In the same time-window, we have previously observed a reduction of mitogenic activation of human lymphocytes exposed to microgravity [11
]. Conversely, lymphocytes incubated at 1 g under the same experimental conditions did not show any significant sign of apoptosis (Fig. 1A and 1B)
.
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Figure 1. Effect of conditions similar to those that occur during exposure of cells to microgravity on human lymphocyte apoptosis. Conditions similar to those that occur during exposure of cells to microgravity (µg) induced apoptotic body formation (A) and DNA fragmentation (B) time-dependently, whereas human lymphocytes kept at normal gravity (1 g) did not show indications of apoptosis under the same experimental conditions. Values were expressed as fold over controls (1=4±1% for apoptotic bodies and 0.300±0.030 A405 units for DNA fragmentation). *, P < 0.01 compared with 1 g controls.
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fourfold over 1 g controls) already 2 h after treatment, and it remained at least threefold higher than that of 1 g controls during the following 70 h (Fig. 2A
). The increase in 5-LOX activity under conditions similar to those that occur during exposure of cells to microgravity was paralleled by a 2.5-fold increase in the main product of enzyme activity, LTB4, intracellularly (Table 1)
and extracellularly (from 15±2 to 39±4 pmol · mg protein-1), whereas 5-LOX protein content was not affected (Table 1)
. Unlike 5-LOX, 12-LOX and 15-LOX were not affected by conditions similar to those that occur during exposure of cells to microgravity, and after 2 h or 48 h of treatment, their activity remained the same as in the untreated controls (320±30 pmol 12-HPETE · min-1 · mg protein-1 and 730±70 pmol 15-HPETE · min-1 · mg protein-1, respectively). Also, COX activity did not change under the same conditions (Fig. 2B)
; neither changed the level of its main product PGE2 (Table 1)
. Therefore, only the 5-LOX branch of the arachidonate cascade appears to be up-regulated in lymphocytes exposed to conditions similar to those that occur during exposure of cells to microgravity. |
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Table 1. Levels of Intracellular Calcium, 5-LOX Protein, LTB4, and PGE2 in Human Lymphocytes Subjected to Conditions Similar to Those that Occur during Exposure of Cells to Microgravity
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Figure 2. Effect of conditions similar to those that occur during exposure of cells to microgravity on 5-LOX and COX activity in human lymphocytes. The activity of 5-LOX (A) and COX (B) was measured in cells exposed to conditions similar to those that occur during exposure of cells to microgravity (µg) or normal gravity (1 g) for different periods of time. Values were expressed as fold over controls (1=1650±160 pmol · min-1 · mg protein-1 for 5-LOX and 800±75 nmol O2 · min-1 · mg protein-1 for COX). *, P < 0.01 compared with 1 g controls.
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1.3- and 1.6-fold the controls after 48 h (not shown). It is interesting that 5-LOX activity could not be detected in these lymphoma cells, corroborating a previous report [23
]. |
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Table 2. Effect of Various Compounds on 5-LOX Activity, Cytochrome c Release, and Apoptotic Body Formation in Human Lymphocytes Subjected to Conditions Similar to Those that Occur during Exposure of Cells to Microgravity
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5.5-fold over the controls after 4 h and remained 
fourfold over the controls in the following 68 h (Fig. 3A
). Conversely, 1 g controls did not show any significant change in mitochondrial membrane potential in the same time-window (Fig. 3A)
.
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Figure 3. Mitochondrial uncoupling and cytochrome c release in human lymphocytes subjected to conditions similar to those that occur during exposure of cells to microgravity. Cells exposed to conditions similar to those that occur during exposure of cells to microgravity (µg) showed a time-dependent, mitochondrial uncoupling (A) and cytochrome c release (B), whereas lymphocytes kept at normal gravity (1 g) under the same experimental conditions did not. Values were expressed as fold over controls (1=5±1% of the uncoupling induced by CCCP for mitochondrial uncoupling and 0.250±0.040 A405 units for cytochrome c release). *, P < 0.01 compared with 1 g controls.
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15 kDa in lymphocyte saps, corroborating our previous observations on other human cells [22
]. These antibodies were used to quantify cytochrome c release into cell cytosol by ELISA [22
]. Conditions similar to those that occur during exposure of cells to microgravity led to a fivefold increase in cytochrome c release after 8 h, and cytochrome release remained fourfold higher than that of 1 g controls in the following 64 h (Fig. 3B)
. The effect at 8 h was prevented by ETYA, CA, mAb, MK886, MAFP, EGTA-AM, SB203580, indomethacin, and the other compounds listed in Table 2
, in a way that fully resembled their ability to inhibit 5-LOX activity (Table 2)
. In particular, 10 µM cyclosporin A, a selective inhibitor of the mitochondrial permeability transition pore [42
], did not affect 5-LOX activity, cytochrome c release, or apoptosis (Table 2)
, suggesting that opening this pore did not contribute to lymphocyte death induced by conditions similar to those that occur during exposure of cells to microgravity. As the release of cytochrome c can trigger an apoptotic caspase cascade [35
, 43
], we tested the effect of inhibitors of caspase-3 and caspase-9 on apoptosis. Table 2
shows that the caspase-3 inhibitor Z-DEVD-FMK or the caspase-9 inhibitor Z-LEHD-FMK, each used at 50 µM, lowered, to 25–30% of the control cells, apoptosis induced in human lymphocytes by conditions similar to those that occur during exposure of cells to microgravity without affecting 5-LOX activity.
5-LOX and its products uncouple isolated mitochondria and lead to cytochrome c release
Treatment of mitochondrial suspensions with pure 5-LOX within a concentration range previously found to be physiological for the 15-isozyme [44
] dose-dependently increased the uncoupling of mitochondria (up to fourfold over the controls) and the release of cytochrome c (up to threefold), and ETYA and anti-LOX mAb fully prevented these effects of 5-LOX (Fig. 4A
). Conversely, cyclosporin A was ineffective (Fig. 4A)
, suggesting that the permeability transition pore did not contribute to mitochondrial membrane disruption by 5-LOX. Remarkably, conditions similar to those that occur during exposure of cells to microgravity enhanced the effect of 5-LOX on mitochondrial membrane potential and cytochrome c release (Fig. 4A)
. Time-course experiments showed that 5-LOX-induced mitochondrial uncoupling and cytochrome c release were significant (P<0.01) already after 30 min and remained threefold higher than in the untreated controls during the following 3.5 h (Fig. 4B)
. Moreover, 5-HPETE and 9-HPOD, the main hydroperoxides generated by 5-LOX from arachidonic [eicosatetraenoic (ETE)] and linoleic (octadecadienoic) acid, respectively [20
], dose-dependently increased cytochrome c release from mitochondria, reaching an threefold maximum at a concentration of 1 µM (Fig. 4C)
.
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Figure 4. Effect of 5-LOX and its products on isolated mitochondria. (A) Dose-dependence of mitochondrial uncoupling and cytochrome c release on the amount of pure barley 5-LOX after 2 h of incubation. Inhibitors of 5-LOX ETYA (10 µM), mAb (200 µg), and inhibitor of permeability transition pore cyclosporine A (CycA; 10 µM) were added to mitochondrial suspensions at the same time as 5-LOX. The effect of conditions similar to those that occur during exposure of cells to microgravity (µg) on the mitochondrial alterations induced by 5-LOX (100 units) was also checked. None of the inhibitors or conditions similar to those that occur during exposure of cells to microgravity affected mitochondrial parameters when used alone. (B) Time-dependence of mitochondrial uncoupling and cytochrome c release upon incubation with 5-LOX (100 units/test). (C) Effect of the 5-LOX products 5-HPETE and 9-HPOD on cytochrome c release after 2 h of incubation. *, P < 0.01; **, P < 0.05 compared with untreated controls; @, P < 0.01 compared with 5-LOX (100)-treated samples.
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Figure 5. Model of microgravity-induced apoptosis in human lymphocytes. Creating conditions similar to those that occur during exposure of cells to microgravity enhances, in a calcium (Ca2+)-dependent manner, 5-LOX activity, which is directly associated to decreased mitochondrial potential (  ), increased cytochrome c (cyt c) release, and caspase activation, ultimately leading to apoptosis. The conversion by 5-LOX of arachidonic (ETE) acid released from membranes through PLA2 into 5-HPETE can contribute to mitochondrial damage, and the FLAP is needed for this conversion. The model suggests a central role for 5-LOX in microgravity-induced apoptosis and also is based on the effect of specific inhibitors (EGTA-AM, ETYA, CA, MK886, and MAFP) and inhibitory mAb on their targets along the apoptotic pathway.
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30%) inhibited 5-LOX activity in lymphocytes exposed to conditions similar to those that occur during exposure of cells to microgravity, whereas 5-LOX inhibitors ETYA, CA, or mAb almost completely blocked it (Table 2)
. These findings suggest that most of 5-LOX activity (70%) in lymphocytes exposed to conditions similar to those that occur during exposure of cells to microgravity was not based on the oxygenation of membrane-derived ETE, and indeed, LTB4, the end-product of this pathway, was increased less than 5-LOX (2.5- vs. fourfold) in treated cells (Fig. 2A
and Table 1 ). Therefore, it can be suggested that fatty acids bound to mitochondrial membranes represent an alternate substrate of 5-LOX, in line with previous observations that 5-LOX oxygenates these organelles [17
]. Conversely, 12- and 15-LOX also can damage mitochondrial membranes [17
], and their products can induce apoptosis in human cells [30
]. However, creating conditions similar to those that occur during exposure of cells to microgravity does not affect 12- or 15-LOX activity in human lymphocytes, suggesting that these isozymes are not involved in the cellular sensitivity to weightlessness. Moreover, it should be recalled that each of the 5-LOX inhibitors used here, widely used to block the cellular activity of this enzyme [17
, 37
, 49
], also might have an alternate action: ETYA is an arachidonic acid analog that might affect multiple arachidonic acid-metabolizing enzymes, CA might inhibit glutathione S-transferase, and MK886 might inhibit PLA2 [17
, 37
, 50
]. These actions might contribute to the sensitivity of intact cells to conditions similar to those that occur during exposure of cells to microgravity and indirectly affect 5-LOX activity. Indeed, it should be recalled that MK886 is inactive in broken cells or with purified 5-LOX [37
]; therefore, the observation that intact cell treatment with this inhibitor led to reduced 5-LOX activity in a cell-free assay seems to suggest indirect effects of this compound. Overall, the mechanism by which conditions similar to those that occur during exposure of cells to microgravity enhance 5-LOX remains elusive, as investigations regarding the effect of microgravity on enzyme catalysis are also still very scant [18
, 51
]. However, it is noteworthy that in vitro the catalytic efficiency of soybean LOX-1, which is prototypal for structural and kinetic studies on LOXs from different species [37
], has demonstrated recently to be enhanced directly by microgravity [18
]. This effect might be a result of a better dispersion in water of the substrates of 5-LOX, leading to an easier formation of enzyme-substrate complexes [52
]. Also, the interaction of 5-LOX with cytoskeletal proteins [53
] might play a role in the formation of the enzyme-substrate complex under microgravity, based on reaction-diffusion processes such as those involved in microtubule self-organization [54
]. The availability of calcium ions might favor these interactions [48
, 53
]. Conversely, the possibility that the "tensegrity" paradigm [2
] might supplement reaction-diffusion processes [54
] in the sensitivity of enzyme reactions to microgravity remains to be explored. At any rate, our results clearly show that conditions similar to those that occur during exposure of cells to microgravity enhance 5-LOX activity in human lymphocytes.
5-LOX activation appears to be the critical event in apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity, as suggested by the effect of several compounds on 5-LOX and apoptosis (Table 2)
. In this context, it seems of particular interest that 5-LOX directly uncouples mitochondria leading to cytochrome c release and that 5-HPETE generated by 5-LOX through PLA2 and FLAP contributes to 30% of mitochondrial damage (Table 2)
. The effect of 5-LOX and its main products on isolated mitochondria (Fig. 4)
strongly supports this conclusion. These findings are in keeping with a function for LOXs in programmed organelle degradation, based on the ability of the 15-isozyme to make pore-like structures in the lipid bilayer [44
]. Calcium might favor the release of mitochondrial cytochrome c [55
], whereas opening the mitochondrial-permeability transition pore [42
] does not seem to contribute to 5-LOX-induced mitochondrial damage (Fig. 4A)
nor to apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity (Table 2)
, as suggested by the lack of effect of cyclosporin A. The release of cytochrome c is indeed a converging point in apoptotic death induced by unrelated stimuli in different cell types [31
, 43
, 55
]. Its release in the cytosol is usually followed by activation of a caspase cascade, initiated by caspase-3 and caspase-9, which are the most proximal members of the proteolytic chain [35
, 43
]. Caspases are thought to form a proteolytic machinery within the cell, resulting in the breakdown of key enzymes and cellular structures, and to activate DNases responsible for the chromatin degradation seen in apoptosis [35
, 43
]. Also, apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity appears to be executed through this series of events, as caspase-3 or caspase-9 inhibitors reduced apoptotic body formation to 25–30% of the controls (Table 2)
. In conclusion, we have demonstrated that human lymphocytes are forced to apoptosis by conditions similar to those that occur during exposure of cells to microgravity, through a pathway based on calcium-dependent 5-LOX activation, mitochondrial membrane disruption, and cytochrome c release, followed by caspase activation and cell death, as summarized in Figure 5
.
Received June 13, 2002; revised November 15, 2002; accepted December 24, 2002.
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