Original Contribution
Radioresistance of human carcinoma cells is correlated to a defect in raft membrane clustering

https://doi.org/10.1016/j.freeradbiomed.2007.04.031Get rights and content

Abstract

In addition to DNA damage, exposure to irradiation involves the plasma membrane in the early phases of γ-ray-induced cell death. The involvement of raft microdomains following γ-radiation derives essentially from the role of ceramide as a critical component leading to apoptosis. It is demonstrated here that γ-irradiation of a radiosensitive human head and neck squamous carcinoma cell line (SCC61) results in the triggering of raft coalescence to larger membrane platforms associated with the externalization of an acid sphingomyelinase (A-SMase), leading to ceramide release in raft, 30 min postirradiation. For the first time, we show that this structural rearrangement is defective in the radioresistant SQ20B cells and associated with the lack of A-SMase activation and translocation, a result which could explain in part their resistance to apoptosis following ionizing radiation. Moreover, we show that SQ20B are protected against radiation injury through a fivefold upper level of endogenous glutathione compared to SCC61. Overcoming the endogenous antioxidant defenses of SQ20B through either H2O2 treatment or GSH depletion triggers A-SMase activation and translocation, raft coalescence, and apoptosis. On the contrary, ROS scavengers abolished these events in radiosensitive SCC61 cells. Translation of this concept to tumor biology suggests that manipulation of rafts through redox equilibrium may provide opportunities for radiosensitization of tumor cells.

Introduction

Exposure of tumor cells to ionizing radiation leads to the formation of reactive oxygen species that are known to induce double-strand breaks either directly or by forming hydroxyl radicals after water radiolysis [1]. In addition to DNA damage, exposure to irradiation triggers intracellular signaling cascades, probably involving, at least in part, the plasma membrane through alterations at the cell surface. It is now becoming evident that lipid microenvironments on the cell surface, known as detergent-insoluble glycolipid-enriched complexes (DIGs) or lipid rafts, also play an important part in signal transduction processes [2], [3], [4], [5]. Many studies support the model in which rafts merge into larger membrane domains on hydrolysis of sphingomyelin (SM) to ceramide (CER) following various stimuli [7], [8], [9], [10], [11], [12]. Indeed, CER molecules dramatically change the biophysical properties of plasma membrane, which results in spontaneous self-association of rafts to larger domains called platforms [13], [14], [15]. Many receptors or stimuli are able to induce the formation of CER-enriched membrane domains or activation of sphingomyelinase(s) (acidic A-SMase and/or neutral N-SMase), or require the expression of SMase for transmission of the specific biological effect [16].

Previous work in our laboratory showed that γ-irradiation of SCC61, a radiosensitive human squamous carcinoma cell line, resulted in the triggering of CER-induced apoptosis 24 h postirradiation [17]. On the contrary, this apoptotic pathway is not functional in the radioresistant counterpart SQ20B cell line, as after Fas induction [18]. Moreover, although the upstream signaling of apoptosis remains uncertain, CER has been implied as a second messenger for a variety of stresses. Indeed, our group demonstrated the existence of a direct relationship between resistance to radiation-induced apoptosis and defective CER signaling in squamous carcinoma cells. This defect of CER release after irradiation did not result from either deficient SMase activity or a deviation to CER metabolites [19]. Moreover, the addition of three different inhibitors of sphingolipid metabolism (imipramine, D,L-PDMP, MAPP) artificially increased the endogenous CER signal and was able to overcome the radioresistant phenotype of SQ20B cells [19].

Nevertheless, definitive proof that CER generation is the primary mediator of the apoptotic response to irradiation is lacking. The exact mechanism by which γ-radiation activates SMase is still unknown, despite studies that have reported radiation-induced activation of SMase in isolated cell membranes, therefore suggesting that DNA is not absolutely required [20]. Moreover, the formation of CER-enriched membrane domains has not been formally shown for all events, but essentially for CD95 [21], CD40 [9], DR5/TRAIL [22], CD20 [11], FcγRII [12], P. aeruginosa [23], rhinovirus [24], UV light [25], and cisplatin [10]. Concerning γ-radiation, studies of Kolesnick's group [26], [27] showed that γ-radiation triggered apoptosis of wild-type endothelial cells, while A-SMase-deficient endothelial cells of tumor vessels were resistant to irradiation-induced apoptosis, resulting in resistance of the tumor to radiation [27]. However, the exact topology and the kinetics of CER generation in the plasma membrane, the identity of SMase catalyzing its release, and the reorganization of microdomains into signaling platforms following γ-irradiation are still hypothetical. Although numerous studies have indicated a central role of the A-SMase for CD95-induced apoptosis, others studies have reached different conclusions about the function of the A-SMase in CD95 [28] or γ-irradiation-triggered apoptosis [26]. Indeed, both N-SMase and A-SMase have been implied in CER production and apoptosis, depending on the cellular origin and the experimental conditions [29], [30], [31].

Based on these considerations, we investigated in this study whether CER-enriched membrane platforms mediate the effects of γ-radiation, and then function as a general motif in signaling transduction, in a model of HNSCC. In order to understand the mechanisms regulating resistance to apoptosis following irradiation, we investigated whether this resistance in SQ20B cells might be related to a failure in the activation of membrane signaling through raft microdomains and to try to overcome this resistance.

Section snippets

Drugs and reagents

Drugs and reagents, unless specified, were purchased from Sigma Aldrich (St. Quentin Fallavier, France). Silica gel 60 thin-layer chromatography plates were purchased from Merck (France). [2-14C]Acetic acid, sodium salt (53.0 mCi/mmol) was purchased from Amersham Bioscience and [9,10 (n)-3H]palmitic acid (60.0 Ci/mmol) was obtained from American Radiolabeled Chemicals. Emulsifier Scintillator Plus scintillation mixture was purchased from Packard. Rabbit monoclonal anti-caveolin-1, rabbit

Isolation and characterization of caveolae-enriched microdomains

The present study was performed on two human head and neck squamous carcinoma cell lines SCC61 and SQ20B. In order to establish the presence of caveolin-1 in these two cell lines, Western blotting experiments were conducted on the cell homogenates with anti-caveolin-1 and anti-GAPDH antibodies, control of protein loading (Fig. 1A). Detergent-resistant microdomains were isolated from these two cell lines using extraction with cold 1% Triton X-100, followed by density-gradient centrifugation. The

Discussion

In this work, we show for the first time that the radioresistance of squamous carcinoma cells can be linked to a defect in the structural rearrangement of the plasma membrane leading to the formation of signaling platforms. Our comparative study was conducted with a radiosensitive squamous carcinoma cell line (SCC61) that had been previously reported to undergo CER-induced apoptosis 24 h postirradiation [18]. Here, our data clearly demonstrate that γ-radiation results in the coalescence of

Acknowledgments

This work was supported by grants from the French ETOILE project of Hadrontherapy. We thank Dr. Christine Bezombes for help and advice (INSERM U563, CHU Purpan, Toulouse, France) during the course of this study, Dr Kobayashi who provided the RFP-lysenin (Lipid Biology Laboratory, RIKEN, Saitama, Japan), and the Department of Immunology (Prof Bienvenu) of the Hospices Civils of Lyon, France.

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