Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis

Abstract

To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2 h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Delta(psi)m), between 4 and 48 h after treatment. Ceramide levels began to increase 2 h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48 h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations.

Overcoming resistance to γ-rays in squamous carcinoma cells by poly-drug elevation of ceramide levels

“…A sustained decrease in Dc m was also observed in response to the poly-drug treatment of SQ20B and SCC61 cells. Experiments conducted on different radiosensitive cell lines also confirm that exogenous (Decaudin et al, 1998;Ghafourifar et al, 1999) or endogenous ceramide (Tepper et al, 1999;Thomas et al, 1999;Rodriguez-Lafrasse et al, 2001;Alphonse et al, 2002) can directly trigger the loss of Dc m (Birbes et al, 2002). The present work confirms that endogenous ceramide can induce apoptosis via a caspasedependent transduction pathway, in agreement with previous reports (Ito et al, 1999;Tepper et al, 1999;Thomas et al, 1999;Alphonse et al, 2002).…”

“…In Jurkat leukaemia cells and SCC61 squamous carcinoma cells sensitive to anti-Fas and g-irradiation, we demonstrated that ceramide is a determining factor in the commitment phase of apoptosis (Rodriguez-Lafrasse et al, 2001;Alphonse et al, 2002). Ceramide acts upstream from and conveys the apoptotic signal to the mitochondria, leading to the subsequent activation of executive caspases.…”

“…After derivatization with o-phthalaldehyde, glutathione was quantified after separation by HPLC with fluorimetric detection at 340-nm excitation and 420-nm emission (Rodriguez-Lafrasse et al, 2001).…”

“…Ceramide, glucosylceramide, and free sphingoid bases were quantified by high-performance liquid chromatography (HPLC) with fluorimetric detection, as previously described (Rodriguez-Lafrasse et al, 2001). Briefly, total cellular lipid extracts were divided into three parts that were either hydrolysed for 1 h at 1001C with 1 M KOH in methanol (total sphingoid base content), or for 1.5 h at 371C in 0.1 M KOH in chloroform : methanol (2 : 1, v/v) (free sphingoid bases content).…”

Candida albicans Phospholipomannan Promotes Survival of Phagocytosed Yeasts through Modulation of Bad Phosphorylation and Macrophage Apoptosis

“…The glycosylinositol phospholipid from Trypanosoma cruzi, another member of the Trypanosomatideaea with an extracellular parasitic phase, induces host cell apoptosis (20). However, in contrast to lipophosphoglycan, the lipid core of glycosylinositol phospholipid belongs to a family of ceramides whose proapoptotic effects are well known (37,38).…”