Biology Contribution
Diversity and Complexity of Ceramide Generation After Exposure of Jurkat Leukemia Cells to Irradiation

https://doi.org/10.1016/j.ijrobp.2008.11.033Get rights and content

Purpose

To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to γ-ray exposure.

Methods and Materials

We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy.

Results

Irradiation of Jurkat leukemia cells resulted in the sequential triggering of sphingomyelin hydrolysis, followed by de novo synthesis that led to a late ceramide response (from 24 h) correlated with the triggering of apoptosis. At the subcellular level, pulse-label experiments, using [3H]-palmitate as a precursor, strengthened the involvement of the radiation-induced sphingomyelin breakdown and revealed a very early peak (15 min) of ceramide in plasma membrane rafts. A second peak in mitochondria was measured 4 h after irradiation, resulting from an increase of the sphingomyelin content relating to the targeting of acid sphingomyelinase toward this organelle.

Conclusion

These data confirm that ceramide is a major determinant in the triggering of radiation-induced apoptosis and highlight the complexity of the sequential compartment-specific ceramide-mediated response of Jurkat leukemia cells to γ-rays.

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.

References (45)

  • J.A. Rotolo et al.

    Caspase-dependent and -independent activation of acid sphingomyelinase signaling

    J Biol Chem

    (2005)
  • A. Charruyer et al.

    UV-C light induces raft-associated acid sphingomyelinase and JNK activation and translocation independently on a nuclear signal

    J Biol Chem

    (2005)
  • A.B. Abdel Shakor et al.

    Cell surface ceramide generation precedes and controls FcgammaRII clustering and phosphorylation in rafts

    J Biol Chem

    (2004)
  • C. Bezombes et al.

    Rituximab antiproliferative effect in B-lymphoma cells is associated with acid-sphingomyelinase activation in raft microdomains

    Blood

    (2004)
  • B.J. Pettus et al.

    Ceramide in apoptosis: An overview and current perspectives

    Biochim Biophys Acta

    (2002)
  • S. Grether-Beck et al.

    Ultraviolet A-induced signaling involves a ceramide-mediated autocrine loop leading to ceramide de novo synthesis

    J Invest Dermatol

    (2005)
  • G. Kroemer

    Mitochondrial control of apoptosis: An introduction

    Biochem Biophys Res Commun

    (2003)
  • V. Gogvadze et al.

    Mitochondrial regulation of apoptotic cell death

    Chem Biol Interact

    (2006)
  • C.R. Bollinger et al.

    Ceramide-enriched membrane domains

    Biochim Biophys Acta

    (2005)
  • X. Lin et al.

    Ceramide mediates radiation-induced death of endothelium

    Crit Care Med

    (2000)
  • A. Haimovitz-Friedman et al.

    Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis

    J Exp Med

    (1994)
  • G. Alphonse et al.

    Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to gamma-radiation but activation of this sequence is defective in radioresistant SQ20B cells

    Int J Radiat Biol

    (2002)
  • Cited by (0)

    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.

    View full text