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Caspase activation is currently proposed as a common feature of apoptosis. However, although the apoptotic events triggered by Fas ligation are well documented, the terminal effectors of the ceramide-induced pathway are not completely identified. In this work, we found that C2-ceramide (Cer)-induced apoptosis was antagonised by leupeptin while Fastriggered cell death occurred in the presence of this protease inhibitor. Nevertheless, this Cer-induced apoptosis could not be attributed to chymotrypsin, calpain or proteasome activation. In addition, the caspase inhibitor Z-VAD-fmk suppressed Fas-triggered death but did not prevent ceramide-induced apoptosis. In MCF7, a caspase-3-deficient cell line, Cer has been found to induce cell death whereas an anti-Fas IgM (7C11) treatment was inefficient. Moreover, Cer induced apoptosis without DEVDase activation in U937 cell line. Finally, Cer induced an intracytoplasmic calcium release while Fas ligation remained without effect. These results are consistent with the notion that Cer acts, at least in part, independently of Fas signalling, and sheds light on a new caspase 3-free apoptotic pathway triggered by ceramide.

A b s t r a c tWe evaluated reticulocyte counting and measurement of immature reticulocyte fraction (IRF) We evaluated the ABX PENTRA 120 Retic (ABX, Montpellier, France) blood analyzer for reticulocyte counting (carryover, precision, linearity, stability) and IRF parameters. The results obtained with this analyzer were compared with those obtained with flow cytometry, Sysmex R-2000 (TOA, Kobe, Japan), and manual counting in 300 blood samples from healthy individuals or patients with blood disease. Materials and Methods PatientsReticulocyte counts and indices were measured within a maximum of 6 hours in 300 normal and pathologic samples from the blood collected daily for routine CBC counts.

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (<30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (>2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (<10 min) transitory increase in pHi. Both Bax immunoreactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.

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