International Journal of Radiation Oncology*Biology*Physics
Biology ContributionDiversity and Complexity of Ceramide Generation After Exposure of Jurkat Leukemia Cells to Irradiation
Introduction
In response to γ-ray exposure, numerous normal and cancer cells are killed after activation of the apoptotic program (1), a mode of cell death involving multiple subcellular compartments, including mitochondria as a central control point. Membranes and DNA represent the main cellular targets initially damaged after γ-ray exposure. Among others, mechanisms behind both have been demonstrated to involve ceramide, a stress response sphingolipid mediator, as a critical component of irradiation-induced apoptosis 2, 3, 4. Ceramide is generated through two main pathways: (1) the de novo synthesis that starts in the endoplasmic reticulum, and (2) the hydrolysis of membrane sphingomyelin by acid and/or neutral sphingomyelinases stimulated by physiologic or environmental stimuli 5, 6. Irradiation-dependent ceramide neosynthesis requires several hours 7, 8 and/or depends on the DNA damage signaling pathway 4, 9. Since the pioneering work of Santana et al.(10), which reported the link between the response of normal B lymphoblasts to ionizing radiation and the hydrolysis of sphingomyelin to ceramide leading to apoptosis, several studies have shown that different normal and cancer cell types seem to use various mechanisms for ceramide generation in response to irradiation, including either neutral 11, 12, 13 or acidic sphingomyelinase(s) 10, 14. A transitory increase of ceramide is observed within minutes after irradiation as a consequence of DNA-independent sphingomyelinase activation 2, 4, but the increase seems insufficient to induce the apoptotic program in the absence of additional signals (15). Several hours after irradiation, a second wave of ceramide accumulation was observed in different cell types 2, 4, corresponding to the DNA damage–dependent activation of ceramide synthase. Apart from their role in the execution of the apoptotic program, mitochondria have also been reported to contain enzymes involved in ceramide synthesis and hydrolysis 16, 17, 18. In addition, ceramide has been demonstrated as having numerous effects on mitochondria, including the enhanced generation of reactive oxygen species, the alteration of mitochondrial calcium homeostasis, the inhibition and/or activation of various components of the electron transport chain, and the collapse in the inner membrane potential (see Siskind [19] for a review). Moreover, an increase in mitochondrial ceramide preceding cytochrome c release and apoptosis has been reported under various cellular stresses 20, 21, 22. The hydrolysis of a specific mitochondrial pool of sphingomyelin induced by mitochondrial targeting of bacterial sphingomyelinase (23) led to the proposal that ceramide may induce cell death specifically when generated in mitochondria. Although this hypothesis is attractive, no similar data have been reported in response to cellular stresses, including irradiation. The aim of this study was to propose a scheme to integrate the different pools of ceramide generated in response to γ-rays in Jurkat leukemia cells. Accordingly, we show that irradiation-induced ceramide production is, on the one hand, a complex event involving both sphingomyelin hydrolysis and de novo synthesis, and on the other hand a multiwave event involving different subcellular compartments, including plasma membrane and mitochondria, for the initiation of apoptosis in these cells.
Section snippets
Cell culture and irradiation
Jurkat E6.1 cells derived from human acute T leukemia were obtained from the European Collection of Cell Cultures (Salisbury, United Kingdom). Cells were grown in Dubelcco's modified Eagle's medium (DMEM) GlutaMAX I (Invitrogen, Carlsbad, CA) as previously described 3, 24. Irradiation of culture flasks was performed at room temperature using a Saturne 42 irradiator (Varian Medical Systems, Palo Alto, CA) at a single dose of 10 Gy (dose rate, 2.4 Gy/min).
Radiolabeling of cells
Labeling was performed by culturing cells
Time course of γ-ray–induced ceramide generation in whole cells
Exposure of Jurkat leukemia cells to a single dose (10 Gy) of γ-rays resulted in a time-dependent increase in ceramide (Fig. 1A), beginning 8 h after irradiation and followed by a constant and sustained increase up to 72 h (+430% vs. control). The kinetics of ceramide release after irradiation correlated with the apoptotic response of the cells (Fig. 1B): 50% of cells were recovered in the sub-G1 phase 48 h after irradiation. To determine the contribution of sphingomyelin hydrolysis and/or de
Discussion
Over the past decade, the involvement of ceramide in several physiopathologic responses has been well documented, with several studies focusing on its role in inducing cell death under various cellular stresses, including both ultraviolet (UV) and γ-irradiation (see Pettus et al.[35] for a review). The recent emergence of the sphingomyelin pathway as a mediator of radiation-induced apoptosis 2, 3, 4, 11, 13, 36 has provided new tools for modulating the radiosensitivity of tumor cells 36, 37.
Acknowledgments
The authors thank Dr. Gérard Morel (Centre National de la Recherche Scientifique 5123, Université Lyon 1) for electron microscopy.
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This work was supported by the French ETOILE project of Hadrontherapy and The Ligue Contre le Cancer (Section de l'Ain).
Conflict of interest: none.