We have previously reported that Scott cells present a defect of phosphatidylserine externalization in association with a reduced store-operated Ca2+ entry after stimulation . In hamster embryo cells, a reduced store-operated Ca2+ entry correlates with a dysregulation of intracellular vesicular traffic and apoptosis . Here, we provide direct evidence that impairment of store-operated Ca2+ entry can causally be implicated in the development of apoptosis since Ca2+ influx protects control cells against apoptosis. In addition, Scott cells are able to expose phosphatidylserine during apoptosis indicating that the mechanisms controlling membrane phosphatidylserine redistribution during apoptosis and procoagulant response are probably different.
Ca2+ ionophore has been reported to induce Ca2+ release from intracellular stores, and after sustained depletion, voltage-independent Ca2+ entry across the plasma membrane . In this study, A23187 treatment induced apoptosis only in Scott, but not in control cells, and an increase in [Ca2+]i only in the latter, indicating that Ca2+ exerts a protective effect against apoptosis. Because Ca2+ release from intracellular stores was similar in Scott and control cells , the present results suggest that reduced Ca2+ entry indeed correlates with apoptosis induction.
In T and B lymphocytes, it has been shown that stimulation of CD95, which leads to apoptosis, blocks store-operated Ca2+ channels and influx through the activation of acidic sphingomyelinase and ceramide release . In our study, if capacitative Ca2+ entry is involved in the induction of apoptosis, direct inhibition of this particular way of Ca2+ entry should trigger apoptosis in cells with a normal store-operated Ca2+ entry, as observed in other cells [19, 20]. SKF 96365, an inhibitor of store-operated Ca2+ channels , was able to induce apoptosis in both control and Scott cells in the same proportions. These results are consistent with the observations of Jayadev et al.  and indicate that after inhibition of store-operated Ca2+ channels, cells acquire a high propensity to undergo apoptosis. Also, the importance of store-operated Ca2+ entry in maintaining cell viability has been observed in HL60 cells after induction of apoptosis by SKF 96365 . Here, SKF 96365 did not affect [Ca2+]i in both cell types, and the data obtained with SKF 525A, an inhibitor of cytochrome P450 that also blocks store-operated Ca2+ influx , confirm the relationship between store-operated Ca2+ entry and apoptosis and suggest that, in B cells, Ca2+ entry is regulated, at least in part, by the cytochrome P450 enzymatic system.
It has been proposed that Ca2+ release from stores is by itself sufficient to induce apoptosis [34, 35]. Preston et al.  have shown that decreased Ca2+ in endoplasmic reticulum stores due to partial reduction of capacitative Ca2+ entry leads to poor refilling of these stores and to apoptosis. In the present study, combinations of SKF 96365 + A23187 or SKF 525A + A23187 resulted in a weak degree of apoptosis in control cells, which was lower than in cells treated with Ca2+ entry inhibitors alone (i.e. in the absence of A23187). [Ca2+]i measurements suggest that even when store-operated Ca2+ entry is inhibited, A23187-induced Ca2+ influx remains protective against apoptosis in control cells.
Scott cells show a lack of rapid procoagulant phosphatidylserine exposure after drastic ionophore stimulation [11, 12, 14]. Here, both control and more markedly Scott cells externalized phosphatidylserine after induction of apoptosis, indicating that this process when accounting for the procoagulant response after a swift elevation of intracellular Ca2+ depends on a different mechanism and/or involve different transporters. This does not rule out that some steps are however common. Our observations are consistent with those reported in Jurkat  or Raji cells , where it has been suggested that phosphatidylserine exposure is regulated by multiple pathways. Very recently, Williamson et al.  have shown that Scott EBV-lymphoblasts from another patient are able to externalize phosphatidylserine during apoptosis. In addition, it is well established that Ca2+ influx is necessary to promote the migration of phosphatidylserine in the membrane outer leaflet . Then, how phosphatidylserine can be externalized in Scott cells if Ca2+ entry is reduced? One possible explanation is that a ~30% reduction of Ca2+ influx is harmless for a normal exposure of phosphatidylserine during nucleated cell apoptosis, but not for the rapid exposure of phosphatidylserine by platelets necessary for the hemostatic response. Phosphatidylserine externalization is believed to result from the inhibition of aminophospholipid translocase and the activation of Ca2+-dependent outer transport of phospholipids possibly mediated by a nonspecific phospholipid scramblase [15, 39, 40] or a vectorial transporter such as ABCA1 . In this respect, phosphatidylserine exposure does not correlate with scramblase expression . Moreover, Scott cells show a normal scramblase expression [42–44] and, although ABCA1 has been reported to participate in the transbilayer movement of phosphatidylserine , the involvement of other transporters cannot be ruled out .
Finally, we have observed a dissociation of membrane changes of cells undergoing apoptosis from other features of programmed death, such as DNA fragmentation and decrease of mitochondrial Δψ. While the latter was caspase-dependent after 48 h treatment, phosphatidylserine exposure was not. However, after 72 h treatment, DNA fragmentation and changes of mitochondrial Δψ were caspase-independent in both control and Scott cells, whereas procoagulant phosphatidylserine exposure induced by A23187, during the same time period, was inhibited by the caspase inhibitor z-VAD.fmk in Scott but not in control cells. Nevertheless, a more specific caspase-3 inhibitor had no effect on DNA fragmentation, [Ca2+]i and phosphatidylserine exposure, suggesting that caspase-3 pathway is not involved in these apoptotic features. However, the participation of other caspases cannot be ruled out. The mechanisms controlling membrane phosphatidylserine redistribution during apoptosis are certainly complex, some studies pointing to the importance of caspase activation [5, 46, 47], whereas in other models, caspase are not involved [48, 49]. In B cells particularly, apoptotic pathways seem to be partitioned into caspase-independent and caspase-dependent steps, phosphatidylserine exposure following BCR activation being a caspase-dependent process, but not change of Δψ . Also, caspase inhibition after BCR stimulation blocks DNA fragmentation but does not prevent cytochrome c release and cell death . The present results are in favor of alternative pathways used by Scott cells in order to allow normal viability, apoptosis and perhaps phagocytosis, which may explain the absence of other apparent pathogenic consequences of the syndrome.