Abstract
Ceramide synthases (CERS) produce ceramides which are key intermediators in the biosynthesis of complex sphingolipids and play an important role in cell proliferation, differentiation, apoptosis and senescence. CERS6 is an isoform of ceramide synthases known to generate ceramides with C16 acyl chain (C16-Cer). CERS6 and C16-Cer levels were significantly higher in acute lymphoblastic leukemia (ALL) cells in comparison to peripheral blood mononuclear cells and T lymphocytes derived from healthy human volunteers. We investigated the role of CERS6 in chemo-resistance in T-ALL cell lines. Stable knockdown of CERS6 in CCRF-CEM and MOLT-4 cells resulted in increased sensitivity to ABT-737, a pan-BCL-2 inhibitor, while CCRF-CEM cells with exogenous CERS6 expression showed resistance to ABT-737 relative to the vector control. The cytotoxic activity of ABT-737 in CERS6 knockdown cells was significantly reduced by the addition of a caspase-8 inhibitor Z-IETD, suggesting that CERS6 alters the cytotoxicity via extrinsic pathway of apoptosis. By co-immunoprecipitation of CERS6 in CCRF-CEM cells, we identified CD95/Fas, a mediator of extrinsic apoptotic pathway, as a novel CERS6 binding partner. In Fas pull-down samples, FADD (Fas-associated protein with death domain) was detected at higher levels in cells with CERS6 knockdown compared with control cells when treated with ABT-737, and this was reversed by the overexpression of CERS6, demonstrating that CERS6 interferes with Fas–FADD DISC assembly. CERS6 may serve as a biomarker in determining the effectiveness of anticancer agents acting via the extrinsic pathway in T-ALL.
Highlights
Acute lymphoblastic leukemia (ALL) is the most common childhood and adolescent cancer, and approximately 2900 new cases of pediatric ALL are diagnosed annually in the United States[1]
The results showed that ceramide synthase-6 (CERS6) binds to Fas death receptor, known as CD95, when the final pull-down sample was probed with Fas antibody (Fig. 5c)
These results suggest that CERS6 binds to Fas and inhibits its assembly with FADD leading to decreased apoptosis via the extrinsic pathway
Summary
Acute lymphoblastic leukemia (ALL) is the most common childhood and adolescent cancer, and approximately 2900 new cases of pediatric ALL are diagnosed annually in the United States[1]. Much progress has been made in understanding cellular responses to standard chemotherapeutic agents, approximately 10% of pediatric patients with ALL do not respond to treatment, and Sphingolipids are bioactive lipids and primarily include sphingosines, ceramides and sphingomyelins that control a variety of cellular functions[7]. Key intermediates in the biosynthesis of all the complex sphingolipids, have a significant role in the regulation of cell growth, differentiation, apoptosis and Official journal of the Cell Death Differentiation Association. CERSs produce ceramides using either the de novo synthesized sphinganine[10] or by acylation of sphingosines from the salvage pathway (Fig. 1a). Ceramides are generated by breakdown of sphingomyelins using sphingomyelinases
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