Alteration of the Sphingomyelin/Ceramide Pathway Is Associated with Resistance of Human Breast Carcinoma MCF7 Cells to Tumor Necrosis Factor-alpha -mediated Cytotoxicity*

(Received for publication, July 12, 1996, and in revised form, December 30, 1996)

Zhenzi Cai Dagger §, Ali Bettaieb par , Nour El Mahdani **, Luc G. Legrès Dagger **, Rodica Stancou Dagger , Joëlle Masliah Dagger Dagger and Salem Chouaib Dagger §§

From Dagger  INSERM Contrat Jeune Formation 94-11 "Cytokines et Immunité Antitumorale," Institut Gustave Roussy, 94805 Villejuif,  INSERM CJF 95-03, Centre Claudius Régaud, 31052 Toulouse, and Dagger Dagger  CNRS Unité de Recherche associée 1283, Centre Hospitalier et Universitaire Saint-Antoine, 75012 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappa B (NF-kappa B) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.


INTRODUCTION

Tumor necrosis factor-alpha (TNF),1 originally described for its antitumor activity, is now recognized as one of the most pleiotropic cytokine to act as a host defense factor in immunological and inflammatory responses (1-3). Like other biological activities of TNF, its antitumor activity is exerted through binding to two distinct but structurally related cell surface receptors, p55 (TNF-R1) and p75 (TNF-R2). Gene knockout experiments and the use of receptor-specific agonistic antibodies confirmed that the two TNF receptors generate nonoverlapping signals (4-7). Although there is now considerable evidence that p55 is the TNF receptor that directly mediates cytotoxicity in a wide variety of cell types (8), recent studies have indicated that p75-mediated signals may cooperate with p55 to facilite cell death in some cell types (9). Structure function analysis of p55 TNF-R signaling demonstrated that an 80-amino acid region within the cytoplasmic domain is required for initiation of apoptosis and NF-kappa B activation (10).

The cytotoxic effect of TNF toward tumor cells can be affected by both intrinsic and acquired cell resistance. However, the current understanding of the molecular mechanisms critical for tumor resistance to TNF and for subsequent tumor progression remains limited. Cell surface expression of TNF receptors is necessary but not sufficient to induce a biological response, and post-receptor mechanisms are important in controlling the susceptibility to the cytotoxic action of TNF. The elucidation of the TNF signaling transduction pathway is particularly challenging because of the extremely wide variety of TNF responses. It is established that TNF regulates the transcription of several genes, many of which are regulated by NF-kappa B (11). It is also clear that the activation of this transcription factor is a pivotal and integral event for the transfer of the TNF signal to the nucleus. Several mechanisms have been reported to contribute to cellular resistance to TNF-induced cell killing, including the constitutive expression of several protective proteins in resistant tumor cells, such as MnSOD, endogenous TNF, major heat shock protein hsp70, A20 zinc finger protein (12-15). However, these proteins confer only partial protection against TNF cytotoxicity, suggesting that additional resistance mechanisms exist.

Recently, ceramide was reported to be an important lipid messenger in various pathways of TNF action (16, 17). Ceramide can be generated from sphingomyelin (SM) hydrolysis by two types of early TNF-responsive sphingomyelinases (SMases), a membrane-associated neutral (N-)SMase and an endosomal acidic (A-)SMase (18, 19). Ceramide targets may include a membrane-associated ceramide-activated protein kinase (20), a cytosolic ceramide-activated protein phosphatase (21), the mitogen-activated protein kinase cascade (22), and the stress-activated protein kinases (23). Recent studies established that ceramide generated by N-SMase directed the activation of proline-directed serine/threonine protein kinase(s) and phospholipase A2 (PLA2), while ceramide generated by A-SMase triggers the activation of NF-kappa B, suggesting that the two SMases control important yet dissociable and nonoverlapping pathways of TNF receptor signaling (24). Because of the potential role of ceramide in mediating the cytotoxic effect of TNF, we examined its possible involvement in cell resistance to TNF. Our findings indicate that stable transfection of p55 TNF receptor restores TNF signaling including NF-kappa B activation in TNF-resistant MCF7 variant R-A1, but does not restore the susceptibility of these cells to TNF cytotoxicity. The data presented in this study further support the notion that the apoptotic effect of TNF is probably dissociated from NF-kappa B activation, and suggest that an alteration of sphingomyelinase activation and subsequent ceramide generation may represent an important additional mechanism by which human tumor cells may escape TNF-mediated apoptosis.


EXPERIMENTAL PROCEDURES

Cytokines, Monoclonal Antibodies, and Reagents

Highly purified (>99%) recombinant TNF-alpha (TNF, specific activity 6.63 × 106 units/mg protein) was kindly provided by A. G. Knoll (BASF, Ludwigshafen, Germany). Monoclonal antibodies htr-9 and utr-1 directed against TNF receptors p55 and p75, respectively, were generously provided by Dr. M. Brokhaus (Hoffman-La Roche, Ltd., Basel, Switzerland). Sphingomyelinase (Staphylococcus aureus) and phospholipase D (cabbage) were obtained from Sigma. N-Hexanoyl-D-sphingosine (C6-ceramide) and C6-dihydroceramide were purchased from Matreya (Pleasant Gap, PA).

Cell Cultures and Stable Transfection of p55 TNF-R cDNA

TNF-resistant R-A1 cells were derived from a TNF-sensitive human breast carcinoma MCF7 cell line after continuous exposure to increasing doses of recombinant TNF-alpha (25). The wild-type human p55 TNF-R cDNA cloned in a mammalian expression vector pMPSVEH (26) was used to transfect R-A1 cells by the calcium phosphate precipitation method (27). Briefly, 1000 cells/10-cm tissue culture plate were plated. After 10-14 days of selection in growth medium containing 200 µg/ml G418 (Sigma), 4-5 resistant colonies were isolated from each plate and examined for human p55 TNF-R expression by fluorescence-activated cell sorting. The positive clones were subsequently maintained in medium with 100 µg/ml G418 for more than 2 months. The sensitivity of clones to TNF was tested every 2 weeks during culture. All cell lines were routinely cultured in RPMI 1640 medium containing 5% FCS, 1% penicillin-streptomycin, 1% L-glutamine at 37 °C in a humidified atmosphere with 5% CO2.

Determination of Cell Viability and DNA Fragmentation

Cell viability was determined using the crystal violet staining method as described previously (25). Absorbance (A), which was proportional to cell viability, was measured at 540 nm. Cell viability (%) = 100 × (A1/A0), cell lysis (%) = 1 - cell viability (%), where A1 and A0 were the absorbance obtained from treated and untreated cells, respectively. The mean value of quadruplicate was used for analysis. Quantitative DNA fragmentation was determined as described previously (28). TNF- or ceramide-treated and untreated cells (1 × 106) were pelleted and washed in PBS. Cells were then resuspended in lysis buffer (0.5% v/v Triton X-100, 20 mM EDTA and 5 mM Tris-HCl, pH 8.0) and centrifuged at 27,000 × g for 20 min to separate the chromatin pellet from fragmented DNA. Both the pellet (resuspended in 1 mM EDTA and 10 mM Tris-HCl, pH 8.0) and supernatant were assayed to determine DNA by the spectrofluorometric DAPI procedure (29).

Flow Cytometric Analysis

Indirect immunofluorescence was performed by incubating of 1 × 106 cells with TNF receptor antibodies (htr-9 and utr-1) for 1 h on ice in PBS, 1% FCS. Cells were then washed and stained with 1:50 dilution of affinity-purified biotinylated goat anti-mouse IgG for 30 min. After three washes with PBS, 1% FCS, cells were incubated with 50 µl of streptavidin-phycoerythrin solution for 30 min. After additional washing with PBS, stained cells were analyzed using an EPICS profile II Coulter (Coultronic, Margency, France). Fluorescence data were collected on 5 × 103 viable cells, as determined by forward light scatter intensity. Background fluorescence was determined using PBS, 1% FCS instead of the TNF-R monoclonal antibody.

Measurement of Internalization and Degradation of Cell-bound TNF

TNF ligand internalization and degradation were estimated, as described previously by Tsujimoto et al. (30). Cells (1 × 106) were exposed to 125I-TNF (Amersham) at 4 °C for 2.5 h and then washed with ice-cold medium and shifted to 37 °C by adding prewarmed medium and further incubated at 37 °C. At the times indicated, culture fluids were harvested, and trichloroacetic acid was added to a final concentration of 10% to quantitate degradation of internalized TNF by the cells. The soluble counts were determined after removal of the precipitate by centrifugation at 1500 × g for 20 min. The cells were washed once with ice-cold PBS and incubated for 5 min at 4 °C with 2 ml of 50 mM glycine-HCl buffer (pH 3.0) containing 150 mM NaCl. After removal of the glycine buffer, cells were washed twice and solubilized in 0.1% SDS. 125I radioactivity found in the glycine buffer and that found in solubilized cells, respectively, represented surface-bound TNF and internalized intracellular TNF.

Nuclear Extracts and Electrophoretic Mobility Shift Assays

Human breast carcinoma MCF7 cells (15 × 106) were incubated for 90 min in the presence or absence of TNF. Adherent cells were then trypsinized, and nuclear extracts were prepared according to the procedure of Dignam et al. (31). Gel mobility shift assays were performed with a synthetic double-stranded 31-mer oligonucleotide containing the kappa B sequences of the human immunodeficiency virus long terminal repeat, 5'-end-labeled with [gamma -32P]ATP using the T4 kinase (32).

RNA Extraction and Northern Blot Analysis

Total RNA was extracted from the tumor cell lines according to the method of Chomczynski and Sacchi (33). RNA (15 µg/lane) were electrophoresed in a 1.2% agarose gel and transferred to nitrocellulose membrane Hybond-C (Amersham). The membrane was then hybridized overnight at 42 °C with the probe labeled with [alpha -32P]dCTP using a Megaprime DNA labeling system (Amersham). The hybridized membrane was washed and exposed to Hyperfilm-MP (Amersham). The blot was stripped by boiling in 0.1% SDS and probed again with a beta -actin probe as a control for equal RNA loading.

Metabolic Labeling, Extraction, and Analysis of Cellular Phospholipids

For phospholipid labeling, cells were incubated with RPMI medium containing 5% FCS and 1 µCi/ml [9,10-3H]palmitic acid (35.9 Ci/mmol, NEN du Pont, France). After 48 h of incubation, the radioactive medium was removed and cells were incubated for another 2-4 h in culture medium. Cells (3 × 106) were resuspended in 1 ml of culture medium supplemented with 10 mM HEPES and treated with TNF at various times. Lipids were extracted by the method of Bligh and Dyer (34) and were separated by thin layer chromatography (TLC) using chloroform/methanol/water (100:42:6, by volume) followed by a second step using petroleum ether/diethylether/acetic acid (80:20:1, by volume) or hexane/diethylether/formic acid (55:45:1, by volume) as developing solvent systems. Radioactive lipid spots, detected with a Berthold radiochromatoscan and upon exposure to iodine vapor, were scraped into scintillation fluid and counted. The positions of ceramide on TLC plates were determined by comparison with concomitantly run 3H-lipid extracts from MCF7 cells treated with exogenous bacterial SMase (100 milliunits/1.5 × 106 cells). Statistical analysis was performed using Student's t test.

Analysis of Cellular SM Content

Cells (5 × 106) were washed twice in phosphate-buffered saline, and lipids were extracted by the method of Bligh and Dye (34) and separated on TLC using chloroform/methanol/water (70:35:5, by volume). The various spots detected after exposure to iodine vapor were determined for phosphorus content according to Böttcher et al. (35).

Assay for SMase Activity

The SMase assay was performed, as described previously by Wiegmann et al. (24). Cells were incubated with or without TNF for various times, and the stimulation was stopped by placing 2-3 × 106 cell aliquots in a methanol-dry ice bath. To measure acidic SMase activity, cell pellets were resuspended in 0.1% Triton X-100 and incubated for 15 min at 4 °C before homogenization. About 100 µg of cellular lysate protein were incubated for 1 h at 37 °C in a buffer containing 250 mM sodium acetate (pH 5.0), 1 mM EDTA, and [choline-methyl-14C]SM (54.5 mCi/mmol, NEN DuPont; 100,000 dpm/assay). To measure neutral SMase activity, cell pellets were resuspended in 0.1% Triton X-100, 20 mM HEPES (pH 7.4), 10 mM MgCl2, 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM Na3VO4, 0.1 mM Na2MoO4, 10 mM beta -glycerophosphate, 750 µM ATP, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin. After incubation for 5 min at 4 °C, cells were homogenized and 100 µg of cellular lysate protein were incubated for 2 h at 37 °C in 20 mM HEPES (pH 7.4), 1 mM MgCl2, and [choline-methyl-14C]SM (100,000 dpm/assay). Radioactive phosphocholine produced from [choline-methyl-14C]SM was then extracted with chloroform/methanol (2:1) and quantitated by scintillation counting (36).

Measurement of [3H]Arachidonic Acid Release

According to the method of Mutch et al. (37), cells (2 × 105) were plated in 35-mm dishes containing 1 ml of culture medium to attach overnight and were then incubated with 0.5 µCi/ml [3H]arachidonic acid ([5,6,8,9,11,12,14,15-3H]) (209 Ci/mmol, NEN du Pont, France) at 37 °C for 18 h. Unincorporated [3H]arachidonic acid was removed and the cells were washed twice with PBS supplemented with 0.1% BSA. Radiolabeled cells were incubated with medium or TNF. The medium was then removed and centrifuged at 2000 × g for 5 min to separate cell debris. 0.1 ml of the supernatant fluids was mixed with 3 ml of scintillation fluid and counted. The amount of 3H release was determined in triplicate. The relative release of 3H was calculated as the percentage of arachidonic acid released in the supernatant with respect of total [3H]arachidonic acid content of cells.


RESULTS

Lack of Signaling through TNF Receptors in the TNF-resistant Variant of MCF7

In an attempt to examine the mechanism of TNF-resistance acquisition by tumor cells, we established a TNF-resistant variant R-A1, derived from MCF7 cells (25). As shown in Fig. 1A, R-A1 cells were resistant to TNF compared to the parental MCF7 cells. Flow cytometry analysis (Fig. 1B) indicates that while p55 TNF-R was highly expressed in parental MCF7 cells (80%), a lower level of p55 expression (30%) was observed in R-A1. Both cell lines displayed marginal expression of the p75 receptor (10%). Data of binding experiments (Fig. 1C) using 125I-radiolabeled TNF show that receptor-bound TNF was rapidly internalized by TNF-sensitive MCF7 cells. In contrast, very little TNF binding and no TNF internalization were detected in R-A1 cells. Electrophoretic mobility shift assays for NF-kappa B activation were performed to further evaluate the response of these cells to TNF. Treatment with TNF induced NF-kappa B translocation in MCF7 but had no effect in R-A1 cells (Fig. 1D). The data of binding and internalization experiments are consistent with the failure of TNF to induce NF-kappa B activation in R-A1. The hypothesis that resistance to TNF exhibited by R-A1 cells may be due to altered signaling through TNF receptors was examined next.


Fig. 1. Lack of TNF signaling in TNF-resistant MCF7-derived R-A1 cells. A, effect of TNF on the viability of parental MCF7 (square ) and R-A1 (black-square) cells. Cells (7.5 × 103 cells/well) were incubated for 72 h with the indicated doses of recombinant TNF-alpha . Cell viability was measured using the crystal violet assay as described under "Experimental Procedures." Data presented are the means ± S.D. of quadruplicate. B, flow cytometric analysis of TNF receptor expression on MCF7 and R-A1. Cells (1 × 106) were detached from the culture and stained with the htr-9 and utr-1 monoclonal antibodies directed against the p55 and p75 TNF receptors, respectively, as described under "Experimental Procedures." C, internalization and degradation of 125I-TNF bound to MCF7 and R-A1 cells. 125I-TNF (50 pM) was allowed to bind to cells for 2 h at 4 °C. Thereafter, cells were washed free of TNF and shifted to 37 °C for indicated incubation times. Surface-bound (open circle ), internalized (bullet ), and degraded (triangle ) 125I-TNF were determined, as described under "Experimental Procedures." Data presented are representative of one of three independent experiments. D, effect of TNF on NF-kappa B activation in MCF7 and R-A1 cells. Cells (10 × 106) were incubated for 90 min in the presence or absence of TNF (50 ng/ml). Nuclear proteins (15 µg) extracted from untreated cells (lane 1) or TNF-treated cells (lane 2) were submitted to electrophoretic mobility shift assay as described under "Experimental Procedures." TNF-treated extracts were competed with 100-fold excess of unlabeled oligonucleotide (lane 3).
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p55 TNF-R Expression in R-A1 Cells by Gene Transfection Restored NF-kappa B Activation but Not TNF-induced Cell Lysis

Based on the above observations and as the p55 TNF receptor was reported to be responsible for TNF cytotoxicity signaling in most cellular models, we attempted to correct TNF signaling by transfecting R-A1 cells with a wild-type p55 expression vector, pMPSVEH-hup55 TNF-R. Following screening by fluorescence-activated cell sorting analysis using anti-TNF-R-p55 monoclonal antibody (htr-9), the stable transfected cells expressing a high level of cell surface p55 receptor (more than 70%) were selected for further study. Gel shift experiments were first performed to examine early response to TNF, i.e. NF-kappa B activation, in the transfected cells. As shown in Fig. 2A, exposure of three representative R-A1 p55-transfected clones (clones 1001, 2101, and 3024) to TNF (50 ng/ml) resulted in the activation of NF-kappa B, indicating that the TNF signaling pathway leading to NF-kappa B activation was functional. Interestingly, despite NF-kappa B activation, these p55-transfected clones remained resistant to TNF, even when a high concentration of TNF (200 ng/ml) was used (Fig. 2B). These data clearly indicate that wild-type p55 receptor expression and the resulting NF-kappa B activation in R-A1 cells are not sufficient to trigger TNF cytotoxic activity.


Fig. 2. A, effect of TNF on NF-kappa B activation in vector-transfected control R-A1 cells and pMPSVEH-hup55-transfected R-A1 clones (clones 1001, 2101, and 3024). Cells (10 × 106) were incubated for 90 min in the presence or absence of TNF (50 ng/ml). Nuclear proteins (15 µg) extracted from untreated cells (-) or TNF-treated cells (+) were submitted to electrophoretic mobility shift assay as described under "Experimental Procedures." NF-kappa B activation by TNF was undetectable in control vector-transfected R-A1 cells. B, effect of TNF on the viability of parental MCF7 and transfected R-A1 cells. Cells (7.5 × 103/well) were incubated for 72 h with the indicated doses of recombinant TNF-alpha . Cell viability was measured using the crystal violet assay as described under "Experimental Procedures." Data presented are the means of quadruplicate.
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The TNF Resistance of p55-transfected R-A1 Cells Is Not Associated with MnSOD and A20 Gene Expression

Strong evidence has been provided about the role of MnSOD and A20 in cell protection against the cytotoxic action of TNF (12, 38). We therefore investigated the mRNA expression of these genes in the p55-transfected R-A1 cells to determine whether they were involved in the resistance to TNF cytotoxicity observed in these cells. Northern blot analysis showed that TNF treatment induced a similar increase in MnSOD and A20 mRNA expression in sensitive MCF7 and in p55-transfected R-A1 cells (Fig. 3). As expected, no increase in the mRNA expression level of either gene was detectable in control R-A1 cells due to the absence of TNF signaling. These data suggest that the resistance of these transfected cells was not directly related to MnSOD and A20 gene expression and further emphasize TNF signaling efficiency in these p55-transfected cells.


Fig. 3. Northern blot analysis of MnSOD and A20 gene expression following TNF treatment. Cells (10 × 106) were incubated with medium (-) or 50 ng/ml TNF (+) for 6 h at 37 °C. Total RNA (15 µg/lane) were electrophoresed in a 1.2% agarose gel and hybridized with 32P-labeled MnSOD and A20 specific cDNA probes. beta -Actin probe was used to confirm comparable loading of RNA sample.
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Failure of TNF to Induce SMase Activation and Ceramide Generation in TNF-resistant p55-transfected Cells

Ceramide, the product resulting from the breakdown of sphingomyelin, serves as the second messenger in the apoptotic signaling pathway (17). Recently, ceramide was reported to be an essential mediator in TNF-induced cell killing (39, 40). We therefore investigated the possible interference of the sphingomyelin/ceramide pathway with the mechanism of TNF resistance in MCF7-derivative cells. As shown in Fig. 4A, a concomitant increase in intracellular ceramide (~2.4-fold increase) was detected in MCF7 at 10-20 min of TNF incubation, preceded by a rapid SM hydrolysis (>30% decrease in SM content) that reached the maximum level within 5-10 min after TNF treatment. The measurement of neutral and acidic SMases indicates that the SM breakdown and ceramide generation in MCF7 cells was correlated with a significant induction of these two SMase activities (20% and 40% of control for neutral and acidic SMases, respectively) after 5-15 min of TNF incubation (Fig. 4B). In contrast, TNF did not induce neither ceramide generation nor SM hydrolysis in R-A1 and p55-transfected cells (clones 1001 and 3024) (Fig. 4A). This is consistent with the failure of TNF to stimulate both SMase activities in these cells (Fig. 4B). However, the defect of the SMase activation was not related to a decrease in basal SMase activities in the resistant cells, since the basal SMase activities were not significantly different in the three cell lines tested (e.g. N-SMase activities were 231, 385, and 258 pmol/h/mg proteins for MCF7, R-A1, and clone 1001 cells, respectively).


Fig. 4. Induction of sphingomyelin metabolism in the parental TNF-sensitive MCF7 and in its resistant counterparts. A, ceramide and SM levels were evaluated in cells (3 × 106) prelabeled with [9,10-3H]palmitic acid for 48 h. Cells were then treated with 50 ng/ml TNF for the indicated time intervals. Aliquots were then collected to prepare lipid extracts. Labeled ceramide and SM were resolved by TLC as described under "Experimental Procedures." Results are expressed as a percentage of untreated controls. Control ceramide counts were 11,076 ± 230 and 10,190 ± 540 cpm for MCF7 and R-A1, respectively. For MCF7, ceramide data are the mean ± S.E. of three independent experiments. *, p < 0.045; **, p < 0.002, relative to time zero. SM data are from one representative of three independent experiments. For R-A1- and p55-transfected cells (clones 1001, and 3024), data are from one representative of three independent experiments. B, for neutral (N-) and acidic (A-) SMase activities, aliquots were collected after TNF treatment (50 ng/ml) and enzyme assays were performed as described under "Experimental Procedures." Data are from one representative of two independent experiments, and they are expressed as a percentage of controls. The results for A-SMase are the mean ± S.D. of duplicate measurements. The data (not shown) for R-A1 and clone 3024 are similar to that of clone 1001.
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Cellular SM Content in TNF-sensitive and -resistant MCF7 Cells

In order to compare the basal level of SMase substrate in TNF-sensitive and resistant cells, the analysis of cellular SM content was performed. As shown in Table I, the percentage of SM (as compared to total phospholipids) was significantly higher in parental MCF7 cells (28-30% of increase) than in R-A1 and clone 1001 cells. Moreover, the basal SM content was 2-fold higher in MCF7 than in the two resistant counterparts (20.9 nmol for MCF7 versus 10.7 nmol and 12.4 nmol for R-A1 and clone 1001 cells, respectively). This result suggests that the different responses induced by TNF in these cells may be dependent on the cellular SM content.

Table I.

Cellular SM content in TNF-sensitive and -resistant MCF7 cells

SM contents were measured as described under "Experimental Procedures." The data presented are the mean ± S.D. of three independent determinations. IP, inorganic phosphorus; PL, total cellular phospholipids.
Cells SM SM (compared to PL)

nmol IP/mg protein %
MCF7 20.9  ± 0.3 14.96  ± 0.20
R-A1 10.7  ± 1.3 11.46  ± 1.60a
Clone 1001 12.4  ± 0.3 11.66  ± 0.50b

a Decreased vs. % of MCF7 SM (p < 0.02).
b Decreased vs. % of MCF7 SM (p < 0.001).

Exogenous SMase and Ceramide Trigger Cell Death in TNF-resistant Cells

To determine whether the activation of SMase and the production of ceramide could overcome the resistance of transfected R-A1 cells, we examined the susceptibility of these cells to exogenous bacterial-derived SMase and synthetic cell-permeable ceramide. The addition of SMase (Fig. 5A) or C6-ceramide (Fig. 5B) was able to induce the killing of p55-transfected cells as well as that of control R-A1 and parental MCF7 cells in a dose-dependent manner. The cytolytic effect of SMase and C6-ceramide on these cells was specific, since the addition of phospholipase D (data not shown) or C6-dihydroceramide (Fig. 5B) at equivalent concentrations failed to induce cell killing. Furthermore, DNA fragmentation analysis indicates that C6-ceramide killed both TNF-sensitive and TNF-resistant MCF7 cells through an apoptotic pathway (Fig. 6). After 24 or 48 h of treatment of TNF-sensitive MCF7 cells, more apoptotic cells were observed upon C6-ceramide treatment than that upon TNF treatment, indicating that ceramide triggers apoptosis of these cells in a more direct manner than TNF.


Fig. 5. Effects of exogenous bacterial SMase and ceramide on parental TNF-sensitive MCF7 and its resistant counterparts. Cells were treated (7500 cells/well) with bacterial SMase (A) or synthetic cell-permeable C6-ceramide (closed circles) or C6-dihydroceramide (open circles) (B) at indicated concentrations. Cell viability was measured after 48 h of treatments using the crystal violet assay as described under "Experimental Procedures." Data presented are the means of quadruplicate determinations. Experiments were repeated twice with similar results.
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Fig. 6. Analysis of DNA fragmentation in MCF7 and p55-transfected R-A1 cells. Cells were incubated with TNF (50 ng/ml) or C6-ceramide (10 µM) for 24 and 48 h. Quantitative DNA fragmentation was determined by the spectrofluorometric DAPI procedure as described under "Experimental Procedures." Data (means ± S.D.) represent triplicate determinations from one of two similar experiments.
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TNF Induces the Release of Arachidonic Acid in Sensitive MCF7 but Not in p55-transfected R-A1 Cells

TNF has been described to be capable of activating PLA2 and inducing the release of arachidonic acid (AA) from membrane phospholipids in several sensitive target cells (41, 42). In addition, ceramide was recently reported to be capable of activating the PLA2/AA pathway (24). Initial experiments using dexamethasone, an inhibitor of PLA2, indicated that the addition of this component at a subtoxic concentration (100 ng/ml) efficiently inhibited (4-fold) the killing of parental MCF7 cells by TNF (Fig. 7A). Therefore, the involvement of PLA2 in TNF signaling in MCF7 and transfected R-A1 cells was assessed by measuring the release of AA. As shown in Fig. 7B, TNF induced a significant release of 3H-labeled AA metabolites (165% of control) in sensitive MCF7 cells after 18 h of incubation. No stimulation of AA release was observed during short term (0-6 h) incubations (data not shown). In contrast, the increase in the release of 3H-labeled AA in R-A1- and p55-transfected clones was not detected at any of times tested (Fig. 7B), suggesting that the resistance of these cells to TNF may also be related to altered AA release.


Fig. 7. A, inhibition of TNF cytotoxicity by dexamethasone in parental MCF7 cells. Cells (7500 cells/well) were preincubated with 100 ng/ml dexamethasone for 15 min. TNF was then added at indicated concentrations. After a 72-h incubation at 37 °C, cell viability was measured using the crystal violet assay. Data presented are the means ± S.D. of quadruplicate determinations. Experiments were repeated three times with similar results. B, relative release of AA induced by TNF. Cells were labeled with [3H]AA as described under "Experimental Procedures." Cells were then washed and treated with medium (control) or 50 ng/ml TNF. After 18 h of incubation, cells were harvested and aliquots of supernatant and of cells were counted to determine levels of released radioactivity. Experiments were performed with triplicate measurements, and the results are the means ± S.D. of three independent experiments.
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DISCUSSION

In contrast to the rapid progress that has been made in defining gene products capable of regulating TNF-induced cell death, the knowledge of the molecular components involved in cell resistance to TNF remains limited. We attempted to delineate the functional role of some second messengers in the acquisition of tumor resistance to TNF by comparing a TNF-sensitive human breast cancer cell line MCF7 with its R-A1 variant selected for resistance to TNF. This TNF-resistant variant derived from MCF7 was found to be susceptible to anti-Fas-induced cell lysis as well as to the chemotherapeutic drug adriamycin (data not shown), compelling evidence that the intracellular cell death pathway is functional in these cells. As compared to the parental MCF7 cells, the resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF-Rs. Although functional wild-type p55 receptor expression was reestablished in R-A1 cells by gene transfer as well as subsequent NF-kappa B activation in response to TNF, these cells remained totally resistant to the cytotoxic action of TNF. It should be noted that transfection of rodent cells using the same vector efficiently triggered the cytotoxic effect of TNF (26). Our observations suggest that NF-kappa B is not sufficient to induce cell killing, and confirm that the activation of this nuclear factor and apoptosis are coincidental but that these two activities are separable. This is also in agreement with the report of Dbaibo et al. (43), indicating that the growth inhibitory effect of TNF is dissociated from the activation of NF-kappa B in Jurkat lymphoblastic leukemia cells.

Cell resistance to the cytotoxic action of TNF is thought to be an active process requiring the synthesis of TNF-inducible proteins, since this resistance can be overcome by protein synthesis inhibitors in some experimental systems (44). Overexpression of several TNF-inducible early response genes, such as MnSOD and A20, has been reported to protect cells against TNF cytotoxicity (12, 15). MnSOD acts as a superoxide radical scavenger, and its presence correlates with cellular protection against TNF cytotoxicity. A20 zinc finger protein is the product of a cytokine-induced primary response gene, and its overexpression inhibits TNF-induced activation of PLA2 and TNF-mediated apoptosis (38). Although the involvement of MnSOD and A20 in cellular protection has been established, our data indicate that both genes can be induced by TNF in p55-transfected resistant cells at similar level as compared to that in TNF-sensitive MCF7 cells, suggesting that the resistance of these cells is not related to overexpression of these two genes.

Ceramide has emerged as a potent second messenger in TNF signaling, and a substantial amount of evidence has been accumulated in favor of ceramide functioning as a selective mediator of the cytotoxic/cytostatic effect of TNF (16, 17). Ceramide generated by the activation of sphingomyelinase has been reported to mediate TNF-induced apoptosis in the human monocyte-like U937, human leukemic HL-60, and murine fibrosarcoma cell lines (39, 40). This study shows that TNF can activate both neutral and acidic SMases in human breast tumor MCF7 cells. A defect in the activation of these enzymes is apparently sufficient to confer resistance to TNF in R-A1 cells. Indeed, when this defect was overcome by adding exogenous SMase or ceramide, the susceptibility of R-A1 cells to apoptosis was restored. This suggests that the stimulation of ceramide-activated enzymes may constitute an important step in the regulation of programmed cell death. It is worthy to note that in our study N-SMase was more rapidly activated than A-SMase, confirming that the activation requirements of A-SMase differ from that of N-SMase. This is also in agreement with the report by Wiegmann et al. (24), which suggests that N- and A-SMase activations may be triggered by distinct pathways. When specific inhibitors of two SMases become available, the determination of the nature of SMase involved in TNF-induced apoptotic cell death would be of major interest. It is tempting to speculate that the failure of TNF to induce DNA fragmentation and apoptosis in resistant cells may be related to structural membrane organization of SM, which may constitute a limiting step for the generation of this second messenger. Our data also demonstrate that the basal level of total SM content was higher in sensitive parental MCF7 cells, as compared to the resistant counterparts (R-A1 and clone 1001 cells). On the other hand, both sensitive and resistant cells showed a similar basal level of SMase activity. Thus, the lower level of basal SM content and the absence of SMases activation in resistant cells may represent a double blockage for ceramide generation. These results confirm the hypothesis from our previous report indicating that the failure of TNF to induce either SM hydrolysis or apoptosis in resistant myeloid leukemia KG1a cells was correlated with the SM pool used for TNF signaling, which is predominantly located in the inner leaflet of the plasma membrane (45). One could speculate that the lower TNF-hydrolyzable SM pool in p55-transfected R-A1 cells and the absence of SMase activation would explain the failure of TNF signaling to induce SM hydrolysis and ceramide generation in these cells. Although the sphingomyelin/ceramide pathway was reported to be capable of signaling NF-kappa B translocation in HL-60 cells (46, 24), we showed here that TNF induced NF-kappa B translocation in p55-transfected resistant cells in the absence of ceramide generation. This is in agreement with other reports demonstrating that exogenous addition of a short chain ceramide to Jurkat cells or the inhibition of ceramide pathway had no effect on NF-kappa B activation (47, 48), and further confirming that NF-kappa B activation by TNF can be independent of endogenous cellular ceramide generation.

Alternatively, non-induction of apoptosis in the p55-transfected cells could be due to an abnormal expression of other genes such as the proto-oncogene bcl2, involved in the regulation of apoptosis (49, 50). However, the involvement of bcl2 in the resistance of R-A1 cells can be excluded, since the parental TNF-sensitive MCF7 cells displayed a higher bcl2 expression level than the resistant R-A1 cells (data not shown), suggesting that there is no correlation between bcl2 expression and the magnitude of TNF-induced apoptosis in these cells. In addition, we and others have reported that bcl2 acts downstream of ceramide preventing ceramide-induced cell death but not ceramide accumulation in at least two models of chemotherapy-induced cell death (51).2 Our demonstration that TNF failed to generate ceramide in p55-transfected R-A1 clones overrides the implication of bcl2 in this phenomenon.

A complex pattern of integrated signals may be generated in response to an elevation of cellular ceramide content. Indeed, recent studies support the view that ceramide produced by N-SMase triggers the mitogen-activated protein kinase cascade via ceramide-activated protein kinase, which presumably results in the activation of PLA2 (24, 52, 53). This is consistent with several lines of evidence suggesting the involvement of PLA2 in the cytotoxic pathway of TNF (37, 41, 46, 54). In addition, the AA generated by the activation of PLA2 was reported to activate sphingomyelin hydrolysis in HL-60 cells (55). Data obtained in our studies indicate that the release of arachidonic acid is altered in p55-transfected TNF-resistant cells. This might be a consequence of the defect in ceramide generation. Although ceramide generation occurred rapidly (10-20 min) following TNF treatment in the sensitive cells, the activation of PLA2 and the AA release induced by TNF could not be detected in the first hours (0-6 h) after TNF treatment. The AA release probably does not precede TNF-stimulated SM hydrolysis in our system, but is involved as a later biochemical response to the action of TNF. This is consistent with the report of Wiegmann et al. (24), suggesting that PLA2 activation occurs as a later event in the TNF signaling pathway. Whether ceramide and AA function independently or in coordination to transduce TNF cytotoxic effect requires further investigation. Taken together, our data suggest that a selective defect in TNF signaling associated with an alteration in sphingomyelinase activation can, at least in part, confer resistance to TNF-induced cell death.

An insight into signal transduction by p55 TNF-R1 has resulted from the identification of the TNF-R1-associated protein TRADD, which interacts with the death domain of p55 and signals both cell death and NF-kappa B activation (56). Following TNF treatment, the association of TRADD and TNF-R1 occurs rapidly in U937 cells (57). In addition, TRADD interacts with TRAF2 (58) and FADD/MORT1 (59, 60) leading, respectively, to NF-kappa B activation and apoptosis induction in the overexpression systems. The importance of the signaling complex assembly is also demonstrated by the fact that dominant-negative derivative of FADD/MORT1 abrogated CD95(Fas)-induced apoptosis and ceramide generation in a B lymphoma cell line (61). One can suggest that in the case of TNF, ceramide generation may also be a downstream event, e.g. post-TRADD and/or post-FADD/MORT1 activation. We have obtained data indicating a comparable TRADD protein expression level in parental MCF7 as well as in resistant R-A1 and clone 1001 cells, and that transient TRADD-transfection induced apoptosis and NF-kappa B activation in all these cells. However, no effect of TRADD overexpression on ceramide accumulation could be detected (data not shown). It seems unlikely that TRADD alone triggers cell death signaling through ceramide pathway. Whether TRADD/FADD complex formation occurs and interacts with the SM/ceramide pathway under physiological conditions remains to be determined. It would be of major interest to decipher the possible cross-talk between diverse TNF-R associated signaling molecules, such as FAN protein (62), and the sphingolipid messengers implicated in the TNF cytotoxic signaling pathway. Understanding of the molecular and biochemical mechanisms of tumor cell resistance to the cytocidal effect of TNF may ultimately provide new approaches to enhance the therapeutic efficacy of TNF against human malignancies.


FOOTNOTES

*   This work was supported in part by grants from INSERM and Grant 6627 from the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a grant from the Institut de Formation Supérieure BioMédicale and the Association pour la Recherche sur le Cancer.
par    Recipient of a grant from the Societé Française d'Hématologie.
**   Recipient of a grant from La Ligue Nationale Contre le Cancer.
§§   To whom correspondence should be addressed. Tel.: 33-1-42-11-45-47; Fax: 33-1-42-11-52-88; E-mail: chouaib{at}igr.fr.
1   The abbreviations used are: TNF, tumor necrosis factor-alpha ; TNF-R, TNF receptor; NF-kappa B, nuclear factor-kappa B; MnSOD, mitochondrial manganous superoxide dismutase; SM, sphingomyelin; SMase, sphingomyelinase; PLA2, phospholipase A2; AA, arachidonic acid; FCS, fetal calf serum; PBS, phosphate-buffered saline; A- and N-SMase, acidic and neutral SMases, respectively; DAPI, 4,6-diamidino-2-phenylindole.
2   Allouche, M., Bettaieb, A., Vindis, C., Rousse, A., Grigon, C., and Laurent, G. (1997) Oncogene, in press.

Acknowledgments

We thank Drs. X. J. Ma (Wistar Institute), A. Harel-Bellan (CNRS URA 1156, Villejuif), G. Laurent and J. P. Jaffrézou (INSERM CJF 9503, Toulouse) for critical comments, and L. Saint-Ange (Institut Gustave-Roussy, Villejuif) for editing the manuscript. We are also grateful to Dr. D. Wallach (Rehovot, Israel) for providing pMPSVEH-hup55 TNF-R constructs.


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