Lymphoblasts were treated for 48 or 72 h with the Ca²⁺ ionophore A23187 at concentrations of 100, 150 and 200 nM (Figs. 1a and 1b). Control cells showed normal proportions of DNA PI staining, corresponding to normal 2N or 4N DNA content; A23187 even exerted a somewhat protective effect against basal apoptosis. At the opposite, in Scott B cells cytometric analysis revealed nuclei with low DNA staining (sub-G1 peak) corresponding to apoptotic cells. The percentage of apoptotic nuclei was A23187 concentration-dependent and at 200 nM, 17.6 ± 2.7 % of the cells have hypodiploid DNA after 72 h. The apoptotic character of A23187-treated lymphoblasts was confirmed by TUNEL assay (see below). A reduced capacitative Ca²⁺ entry has been shown to correlate with apoptosis 19. Recently, we have reported that Scott lymphoblasts show a defective store-operated Ca²⁺ entry 22. Therefore, it was interesting to assess whether inhibition of capacitative Ca²⁺ channels could induce apoptosis in control B lymphoblasts. The experiments were performed in the presence of 10 μM SKF 96365, a blocker of store-operated Ca²⁺ channels 25. At 10 μM, SKF 96365 reduced by approximately 30 % the level of capacitative Ca²⁺ entry induced by thapsigargin (1 μM) in either control or Scott cells (not shown). As shown in Fig. 1c, the same concentration of SKF 96365, was able to induce apoptosis in both control and Scott cells.

After 48 h, SKF 96365-induced apoptosis in control cells was significantly higher than in Scott cells (9.6 ± 0.9 % and 6.6 ± 0.6 % in control and Scott cells, respectively; P < 0.01), however, after 72 h of treatment, SKF 96365 induced apoptosis at comparable degrees in both types of cells (12.4 ± 0.8 % and 12.9 ± 2.4 % in control and Scott cells, respectively). Higher concentrations of SKF 96365 were responsible for necrosis in both types of cells. It should be noted that when cells were treated with the combination of Ca²⁺ ionophore A23187 + SKF 96365, the degrees of apoptosis were almost additive at 48 h, and ~40 % higher at 72 h in Scott cells with respect to values obtained in the presence of A23187 alone, whereas A23187 remained protective in control lymphoblasts (Figure 1d).

Some studies have shown that cytochrome P450 activity is critical with respect to the regulation of store-operated Ca²⁺ influx 2728. To verify whether cytochrome P450 pathway is implicated in Ca²⁺ entry induced by depletion of Ca²⁺ stores in EBV lymphoblasts, experiments were also performed in the presence of 10 μM SKF 525A, a cytochrome P450 inhibitor 28. As observed with SKF 96365, SKF 525A reduced capacitative Ca²⁺ entry induced by thapsigargin (1 μM) by approximately 30 % in either control or Scott cells (not shown). In addition, after 48 or 72 h of treatment, 10 μM SKF 525A induced apoptosis at comparable extents in control and Scott cells (Fig. 2a). When cells were treated with the combination of Ca²⁺ ionophore A23187 + SKF 525A, the degrees of apoptosis in Scott cells were not significantly different from those observed in the presence of A23187 alone (Fig. 2b). Again, A23187 exerted a protective effect against SKF 525A-induced apoptosis in control lymphoblasts, more pronounced at 72 h (Fig. 2b).

Experiments using the extracellular Ca²⁺ chelator EGTA (1–2 mM) were designed in order to evaluate the relationship between store-operated Ca²⁺ entry and apoptosis. At 1 mM, EGTA did not induce apoptosis nor affected Ca²⁺ ionophore-evoked apoptosis. However, higher concentrations of EGTA (1.5 and 2 mM) enhanced cell mortality by necrosis, making measurements of the degree of apoptosis almost impossible (not shown).

To determine whether caspases are involved in this apoptotic process, Ca²⁺ ionophore- or SKF 96365-treated cells were incubated in the absence or presence of the broad-spectrum caspase inhibitor z-VAD.fmk (50 μM) or the more specific caspase-3 inhibitor, Ac-DEVD-CHO (50 μM) 29. Apoptosis analysis, assessed by PI staining, showed that none of these inhibitors had significant effect on DNA fragmentation after 48 or 72 h of A23187 treatment of either control or Scott cells (Fig. 3a). However, z-VAD.fmk interfered in the effect of SKF 96365 on Scott cells, even exerting an apparent paradoxical potentiation of apoptosis, whereas it did not affect the fate of control cells to a significant extent (Fig. 3b).

In order to assess the effects of Ca²⁺ ionophore and channel blockers on apoptosis, Ca²⁺ signaling was monitored in viable cells using Fluo-3 fluorochrome and PI to define a gate excluding necrotic cells, i.e. PI positive cells representing less than 10 % of the whole population.

Under control condition (Fig. 4), approximately 95 % of viable cells presented "normal" [Ca²⁺]_i (68 ± 6 and 78 ± 14 fluorescence arbitrary units in control and Scott cells respectively) and only 3 ± 1 % of cells showed elevated [Ca²⁺]_i (Table 1). After A23187 (200 nM) treatment, the latter population with elevated [Ca²⁺]_i was significantly increased as well as [Ca²⁺]_i itself, but this occurred in control cells only, and the effect was not inhibited by the caspase-3 inhibitor Ac-DEVD-CHO (Table 1). In addition, SKF 96365 did not modify either [Ca²⁺]_i or number of events in both types of cells. Similar results were obtained with SKF 525A (not shown). However, it should be noted that when cells were treated with the combination of Ca²⁺ ionophore A23187 + SKF 96365, [Ca²⁺]_i was enhanced in control but not in Scott cells. Furthermore, in response to UV irradiation, both types of cells underwent apoptosis as determined by DNA fragmentation but [Ca²⁺]_i was not modified. For example, at 5 J/cm², 23 ± 3 % and 28 ± 5 % of the normal and Scott cells had hypodiploid DNA, respectively.

Apoptotic cells were assessed for phosphatidylserine exposure by functional procoagulant prothrombinase assay, and by annexin V labeling. After Ca²⁺ ionophore or SKF 96365 treatment, the degree of phosphatidylserine externalization remained weak at 48 h and was not affected by the caspase inhibitor z-VAD.fmk (Fig. 6a). At 72 h of A23187 or SKF 96365 treatment, the degree of phosphatidylserine exposure was enhanced in both types of cells, and more significantly in SKF 96365-treated cells (P < 0.05). Phosphatidylserine externalization was not affected by the caspase inhibitor z-VAD.fmk in control cells. In Scott cells, z-VAD.fmk however prevented the phosphatidylserine redistribution induced by Ca²⁺ ionophore (P < 0.05), but did not reduce the effect of SKF 96365, even exerting an apparent paradoxical potentiation of phosphatidylserine externalization probably due to a membrane destabilizing effect when used at 50 μM 32 (Fig. 6b).

In order to compare with more drastic and rapid stimulation, it should be noted that after 10 min of A23187 treatment at 1 μM, prothrombinase activity was enhanced in both type of cells, but as previously reported 1222 B lymphoblasts from the Scott patient displayed a ~60 % reduction of prothrombinase activity when compared with control cells (Fig. 6c). Consistent with the data obtained by the prothrombinase assay at 72 h of A23187 or SKF 96365 treatment (Fig. 6b) and [Ca²⁺]_i determinations (Fig. 4), the number of annexin V-positive cells increased in both types of cells (Fig. 7). Moreover, upon induction of apoptosis by UV irradiation, control and Scott cells showed approximately the same proportion of annexin V-positive cells (~31 ± 5 % and 34 ± 6 % of control and Scott cells, respectively). Ac-DEVD-CHO did not prevent annexin V labeling (not shown), suggesting that phosphatidylserine externalization is independent of caspase-3 activity.

X-VIVO 15 medium was from BioWhittaker (Walkersville, MD). PI, type I-A RNAse A and Phycoerythrin-conjugated streptavidin were from Sigma Chemical Co. (St Louis, MO). Ca²⁺ ionophore A23187, SKF 96365, SKF 525A, z-VAD.fmk and Ac-DEVD-CHO were from Calbiochem (La Jolla, CA). Fluo-3/AM and JC-1 were from Molecular Probes (Eugene, OR). TUNEL assay kit was from Roche Diagnostics (Mannheim, Germany). Biotin-labeled annexin V has been prepared and characterized as previously described 52.

The case of Scott syndrome has been detailed in another study where homozygous status has been suggested 12. Lymphocytes were isolated under sterile conditions and stored in liquid nitrogen. Infection of B lymphocytes by EBV (EBV B958, Marmouset) was performed in RPMI culture medium containing 20% FCS in the presence of 50 ng/ml cyclosporin and was achieved within 3 weeks 12. Three independent EBV infections could be performed. It should be noted that the Scott phenotype was observed in these cells after drastic activation with Ca²⁺ ionophore A23187, as previously described 1222. EBV-infected B cells were expanded in X-VIVO 15 culture medium (free Ca²⁺ concentration = 1.8 mM).

The cell viability was checked by Trypan blue exclusion. Cells were seeded at 5 × 10⁵ cells/ml in the presence or absence of A23187 (100–200 nM) for 48 or 72 h. In some experiments, cells were treated with 10 μM SKF 96365 or 10 μM SKF 525A in the absence or presence of Ca²⁺ ionophore A23187 (200 nM) for 48 or 72 h.

In another set of experiments, cells cultured in a standard 6-well plate were submitted to UV light by exposure to the germicidal lamp (peak sensitivity approximately 254 nm) in the tissue culture hood for 10, 20 et 30 s (5, 10 and 15 J/cm², respectively) and apoptosis was measured 18 h after irradiation.

After the different treatments, cells were harvested and numbered. Concentration was adjusted to 5 × 10⁵ cells/ml of 70% ethanol solution in H₂O, and fixation was allowed to proceed during at least 1 h at 4°C. Cells were washed once in HBSS before resuspension in a solution containing type I-A RNAse A (0.5 mg/ml) in HBSS and were incubated for 10 min at 37°C. PI was then added at a final concentration of 0.1 mg/ml. Samples were allowed to stand another 15 min in the dark at room temperature before flow cytometry analysis using CELLQuest software (Becton Dickinson, San Jose, CA).

Cells (10⁶ cells/ml) were loaded with 3 μM Fluo-3/AM for 30 min at room temperature. Cells were then washed twice in X-VIVO 15 medium. Then 10 μg/ml PI was added in order to determine the proportion of necrotic cells uptaking PI. Fluo-3 and PI fluorescences were recorded by flow cytometry. Fluo-3 fluorescence was plotted as FL-1 versus PI fluorescence as FL-2, and both were measured in fluorescence arbitrary intensity units.

In another set of experiments, phosphatidylserine externalization was assessed by monitoring annexin V-biotin binding, simultaneously with [Ca²⁺]_i measurement in viable cells only, i.e. in those excluding PI. Briefly, cells were loaded with fluo-3 as described above. After 20 min of Fluo-3 loading, 5 μl of annexin V-biotin at 0.5 mg/ml was added. After 10 min of incubation in the dark, at room temperature, cells were washed by one centrifugation-resuspension step in X-VIVO 15 medium. Labeling was achieved following another 10 min incubation at room temperature with phycoerythrin-conjugated streptavidin. Cells were washed by a centrifugation-resuspension step with X-VIVO 15.

To assess the occurrence of A23187- or SKF 96365-induced apoptosis in B cells, labeling of fragmented DNA was performed by TUNEL assay using a commercially available cell death detection kit according to the manufacturer's instruction. In brief, cells were fixed with 4% paraformaldehyde solution for 20 min at 4°C and then washed with PBS. Endogenous peroxidases were inactivated by 0.3% H₂O₂ in a 70% methanol solution for 20 min, then cells were washed with PBS and incubated with permeabilization solution (0.1% Triton X-100) for 2 min at 4°C. Apoptotic cells were incubated with 50 μl of TUNEL reaction mixture for 80 min at 37 °C. After substrate addition, stained cells were analyzed by flow cytometry. Negative control samples for TUNEL staining lacked terminal deoxynucleotidyl transferase.

Δψ was determined by monitoring the decrease in red fluorescence and the increase in green fluorescence by flow cytometry after labeling the cells with JC-1. Cells suspensions were ajusted to a density of 5 × 10⁵ cells/ml and incubated in complete medium with JC-1 (1 μg/ml) for 15 min at room temperature in the dark. At the end of the incubation period, the cells were washed twice in PBS. For each experiment, A23187 or SKF 96365-treated and control cells were analyzed for both red and green fluorescences after JC-1 labeling. The mean fluorescence ratio was calculated from the mean red and green fluorescence for the entire sample population 53.

Procoagulant phospholipid exposure in apoptotic cells was detected using a human prothrombinase assay in which phosphatidylserine promotes the activation of prothrombin by factor Xa in the presence of factor Va 54. Thrombin generated by assembled prothrombinase complex was measured using a chromogenic assay as described elsewhere 12. EBV-infected B lymphocytes were studied at 2 × 10⁵ cells/ml and the ability to externalize procoagulant phosphatidylserine was examined after treatment by A23187 (200 nM) or SKF 96365 (10 μM) in the absence or presence of z-VAD.fmk (50 μM) during 48 and 72 h, or 10 min only in case of more drastic stimulation by 1 μM A23187. The cells were separated from derived membrane microparticles by centrifugation at 12,000 g for 1 min before measurement. For each protocol, results were compared with the prothrombinase activity developed in samples from healthy volunteers. Linear absorbance changes were converted to concentration of generated thrombin by reference to a standard curve. Prothrombinase activity was normalized to 2 × 10⁵ cells/ml and was expressed as the increase in activity (□) with respect to baseline prothrombinase activity of untreated cells.

Experiments were performed at different culture stages. Unpaired Student's t-test was used for the statistical analysis. A P < 0.05 value was considered significant.