Distinct stress and cell destruction pathways are engaged by TNF and ceramide during apoptosis of MCF-7 cells

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Abstract

Ceramide has been proposed to be an important signaling intermediate in tumor necrosis factor (TNF)-induced apoptosis in human MCF-7 breast adenocarcinoma cells. We compared cell death and signal transduction pathways induced by TNF and ceramide in TNF-sensitive, parental MCF-7 cells to those in TNF-resistant, MCF-7 cells (3E9). TNF caused proteolysis of the caspase substrate, polyADP-ribose polymerase (PARP) in parental cells, but not in 3E9 cells. Both apoptosis and PARP cleavage were strongly prevented by co-incubation with caspase inhibitors. In contrast, ceramide-induced cell death was neither affected by TNF resistance nor was it associated with PARP cleavage, and death could not be prevented by co-incubation with caspase inhibitors in either cell line. TNF was able to activate JNK/SAPK approximately 30-fold and approximately 5-fold in parental MCF-7 and 3E9 cells, respectively; in contrast, cell-permeable ceramide only weakly stimulated JNK/SAPK activity in either cell type. Although JNK was activated by TNF, pharmacological blockade of the JNK pathway did not inhibit TNF- or ceramide-mediated cell death. Using mass spectroscopic analysis for ceramide, no increase, rather, a decrease in total ceramide content in TNF-treated parental cells was observed. These results suggest that the cell death signaling and execution pathways utilized by ceramide are distinct from those activated by TNF in MCF-7 cells.

Introduction

Tumor necrosis factor (TNF) stimulates diverse biological activities through engagement with two types of specific cell surface receptors which couple to several signal transduction cascades. In tumor cells, TNF activates at least three distinct pathways that transmit signals to the nucleus or activate cytoplasmic and membrane-directed signals that initiate the cell death response [1]. Activation of this cell death or apoptotic cascade is mediated by receptor recruitment of accessory proteins, including caspases, that directly form signaling complexes capable of inducing apoptosis. However, other TNF-activated nuclear and cytoplasmic events can attenuate, promote or otherwise regulate the apoptotic response. These events include activation of the NF-κB transcription factor [1], [2], stimulation of stress kinases, including JNK/SAPK [3], [4], activation of oxidative pathways [5], [6] and liberation of the membrane lipid, ceramide, by activation of sphingomyelinase (SMase; [7], [8]). Recently, a linkage between the latter two pathways in MCF-7 cells has been proposed, wherein TNF treatment induces oxidation of cellular glutathione, thereby relieving a glutathione-driven constitutive inhibition of SMase and allowing ceramide liberation [9], [10]. These downstream components may mediate several of the biologic activities of TNF, including apoptotic regulation or promotion, but may have distinct levels of participation in tumor cells of different origin. Importantly, elevation of intracellular ceramide levels may initiate its own signaling process that induces several of the activities associated with TNF action. However, distinctions in ceramide and TNF signaling have not been thoroughly examined.

The direct mode of action of ceramide and other sphingolipids is unknown. The ability of ceramide to bind membrane components and proteins through a specific side-chain-mediating recruitment of signaling molecules to the membrane fraction may underlie the multiple and diverse signals that have been associated with ceramide action [11]. Specifically, disruption of ras–raf association by some forms of ceramide has been described [12], as has evidence for Raf-1/ERK as the specific target for de novo-synthesized ceramide [13]. Evidence also suggests that ceramide may directly suppress activation of Akt kinase activity [14] and can therefore disrupt signaling pathways controlling cell survival. Further, PKCξ has been shown to directly interact with ceramide during modulation of the TNF response [15]. More recently, ceramide has been proposed to be a key effector molecule in assembly of caveolin-rich micro-domains or rafts into larger domains, wherein receptor clustering may be facilitated during induction of signal transduction cascades [16], [17].

Cells undergoing a variety of environmental stresses respond by activation of JNK/SAPK [3], [4], [18], [19]. A connection between ceramide elevation and JNK/SAPK activation was proposed because in several systems, cell-permeable ceramide analogues stimulated 2- to 3-fold activation of JNK/SAPK [20], [21], [22], [23]. However, other studies, on the contrary, support the dissociation of JNK/SAPK activation by TNF- and ceramide-induced cell death [24].

In this report, the apoptotic and signaling properties of TNF and cell-permeable ceramide are compared in TNF-sensitive parental MCF-7 cells [25] and in a TNF-resistant MCF-7 cell variant, 3E9 [26]. The results demonstrate that TNF- and ceramide-mediated cell death pathways are not subject to the same regulatory mechanisms, occur with different degrees of involvement of caspase and JNK/SAPK stress cascades, and mediate distinct patterns of DNA damage. Further, evidence is provided for the dissociation of ceramide from TNF signaling. Collectively, these results define marked distinctions in both upstream and downstream processes mediating TNF and ceramide activity in human MCF-7 breast adenocarcinoma cells.

Section snippets

Cell lines, cytokine, growth factor, ceramides and antibodies

MCF-7 cell lines [25] available from various laboratories and other sources are known to vary widely in their TNF sensitivities. The parental MCF-7 breast carcinoma cell line used in the current studies was from two sources: the first was originally provided to us by Dr. Kapil Dhingra, Department of Medical Breast Oncology, The University of Texas M.D. Anderson Cancer Center. However, we have in addition confirmed the similar TNF sensitivity of MCF-7 cells available from the ATCC.

These cells

Cytotoxic susceptibility to TNF vs. C6-ceramide of parental MCF-7 and 3E9 cells

MCF-7 and 3E9 cells demonstrated marked differences in susceptibility to TNF (Fig. 1). Following 48 h of incubation, TNF caused significant dose-dependent loss of survival in parental MCF-7 cells (15.5 ± 3.5% and 59.5 ± 5.7% of control survival with 75 and 2.4 ng/ml TNF, respectively); in contrast, TNF reduced survival of 3E9 cells only to 64.4 ± 5.4% of control at 75 ng/ml. This indicated a 32-fold difference in susceptibility to TNF between these cell lines. Even at the high dose level of

Discussion

Much interest has recently focused on the possible intracellular signaling role of the sphingolipid, ceramide, derived either via de novo synthesis or via SMase-mediated hydrolysis of SM [7], [8], [9], [10], [32], [35], [36]. The mechanism of SMase activation is unknown, but may involve its recruitment to signaling complexes, including those activated by TNF receptor cross-linking [37]. Activation of the acidic, endosomal SMase by the p55 TNF receptor has been attributed to a domain within the

Acknowledgements

This research was supported by NIH CA73018 (NJD) and grants from The University of Texas M.D. Anderson Cancer Center Breast Cancer Research Program (JK) and the DOD Breast Cancer Research Program (DAMD17-01-0310; NJD and DAMD17-99-1-9265; JK). We thank Elena Leroux and Edmond Auzenne for expert technical assistance, Doug Weidner for flow cytometric analyses, and Ed Felix for LC/MS analyses.

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