Analysis of Human Breast Adenocarcinoma MCF7 Resistance to Tumor Necrosis Factor-induced Cell Death

Considerable progress has been made in the understanding of tumor necrosis factor (TNF) signaling; however, the molecular and biochemical basis of tumor resistance to the cytotoxic action of TNF are still not definitively identified yet. Although a role of c-Jun N-terminal kinase (JNK) pathway has been suggested as an effector in TNF signaling, its exact relative contribution and its interaction with ceramide pathway and tumor resistance to TNF remain unknown. The relationship between JNK activation and human breast adenocarcinoma MCF7 resistance acquisition to the cytotoxic action of TNF was therefore investigated. We demonstrate that TNF triggers JNK activation in both TNF-sensitive MCF7 cells and its resistant derivative, RA1/1001. In addition, when MCF7 cells were stably transfected with mitogen-activated protein kinase kinase 4 (MKK4) dominant-negative cDNA or transiently transfected with a dominant-negative c-Jun mutant (TAM 67), their susceptibility to the cytotoxic action of TNF remains comparable with control cells. We also demonstrated that JNK activation does not require ceramide generation since in MCF7 cells transfected with a dominant-negative derivative of FADD (FADD-DN), which are resistant to the cytotoxic action of TNF, TNF induced JNK activation in the absence of ceramide generation. Furthermore, our data indicate that exogenous permeable synthetic ceramide C-6 induced the killing of MCF7 cells transfected with MKK4 dominant-negative cDNA. These results provide strong evidence indicating that tumor acquisition of resistance to the cytotoxic action of TNF may occur either independently or at a level downstream of JNK activation and suggest that JNK activation is not linked to ceramide pathway in TNF-mediated apoptosis.

Tumor necrosis factor ␣ (TNF) 1 is a cytokine with powerful direct tumor-killing capability (1,2). This cytokine has also been shown to play a role in tumor regression mediated by cytotoxic T cells. In fact, TNF may be released by cytotoxic T cells clones and significantly contributes to the local immune response to the tumor (3). Thus, when its secretion is confined to the area of tumor growth, TNF may fulfill its promise as an anticancer agent. It is well established that release of cytotoxic cytokines such as TNF triggered by T cell receptor engagement may be even more important to tumor destruction than direct lysis by cell-cell contact. This factor is now recognized as the most pleiotropic cytokine acting as a host defense factor in immunological responses and may contribute to tumor cell destruction (4).
Whereas expression of the TNF receptor 1 (TNFR1) alone is necessary for providing a biological response, it is not sufficient to induce the cytolytic process (5,6). Despite the major advance in understanding the early events in TNF signaling and the identification of molecules that are recruited to TNFR1, the mechanisms of resistance to TNF observed in some tumor cells remain largely unknown. It has become clear that the initiation of intracellular signaling events through TNFR1 depends on protein intermediates that interact with specific cytoplasmic domains of this receptor. In fact, the death domain motif of TNFR1 plays a central role in the interactions between TRADD (TNFR1-associated death domain) (7,8) and its association with FADD (Fas-associated death domain) (8 -10), RIP (receptor-interacting protein) (11,12), and TRAF2 (TNFR-associated factor 2) (8,13). It is well established that among the early downstream effects of TNF are the activation of several kinases (4) and the transcription factors NF-B (14) and AP-1 (15).
Evidence has been provided indicating that signaling pathways initiated by TNF include the activation of neutral or acidic sphingomyelinases (16 -18) leading to the elevation of cellular ceramide that induces apoptosis in several cell types (19). The targets of ceramide are multiple (20 -21), and above all, recent studies have suggested the implication of c-Jun N-terminal kinases (JNKs)/SAPK as a critical mediator in apoptosis triggered by ceramide (22)(23)(24)(25). The prototypical JNK/ SAPK pathway involves the sequential activation of mitogenactivated protein kinase kinase kinase 1, mitogen-activated protein kinase kinase 4 (MKK4), JNK, and c-Jun. The activated JNKs translocate to the nucleus where they phosphorylate transcription factors such as c-Jun and ATF2 (26 -29). JNK activation requires phosphorylation at 2 residues, Thr-183 and Tyr-185, by MKK4, a dual-specific protein kinase (30 -32) that is structurally related to mitogen-activated protein kinase kinase. MKK4 itself is phosphorylated and activated by the up-stream mitogen-activated protein kinase kinase kinase 1 (30,34).
The original observation that apoptosis may be linked to the activation of JNK cascade was made in PC-12 pheochromocytoma cells during nerve growth factor withdrawal (22). Moreover, introduction of constitutively active mitogen-activated protein kinase kinase kinase 1 resulted in increased apoptosis in PC-12 cells, whereas dominant interfering mutants of c-Jun, a downstream target of the JNK cascade, blocked apoptosis induced by nerve growth factor withdrawal (22). The requirement of JNK signaling for TNF-induced cell death remains controversial (22)(23)(24)(25)(35)(36)(37)(38)(39), and the involvement of JNK activation and its interaction with ceramide pathway in the control of cell susceptibility to the cytotoxic action of TNF remain unknown.
The present data demonstrate that the resistance of MCF7 cells to the cytotoxic action of TNF is not associated with a defect in SAPK/JNK activation and emphasizes the absence of interaction between JNK and ceramide pathways in TNF-mediated apoptosis.
Cell Lines and Culture-TNF-resistant cells, 1001, were derived from RA-1 cells transfected by p55 TNF receptor cDNA as described (6). MCF7 FADD dominant-negative cells were kindly provided by V. Dixit (University of Michigan Medical School, Michigan). MKK4-DN-expressing cells were obtained by stable transfection of the MCF7 cells with an expression vector encoding MKK4 dominant-negative cDNA. All cell lines were routinely cultured in RPMI 1640 medium containing 5% fetal calf serum, 1% penicillin-streptomycin, 1% L-glutamine at 37°C in a humidified atmosphere with 5% CO 2 .
Determination of Cell Viability-Cells were seeded in 96-well plates (7 ϫ 10 4 cells/ml) and treated either with human recombinant TNF, synthetic cell-permeable C 6 -ceramide, or with C 6 -dihydroceramide. After incubation for 72 h with TNF or 48 h with ceramide at 37°C, the medium was replaced with 0.5% crystal-violet solution. Plates were then incubated for 10 min at room temperature and washed, and viable crystal-violet-stained cells were lysed with 1% SDS. Absorbance (A), proportional to cell viability, was then measured at 540 nm. Cell lysis was assessed by comparing the viability of untreated cells versus treated cells using the following calculation: cell viability (%) ϭ 100 ϫ (A1/Ao); cell lysis (%) ϭ 1 Ϫ cell viability (%), where A1 and Ao were the absorbance obtained from treated and untreated cells, respectively. The main value of quadruplicate was used for analysis.
Metabolic Labeling, Extraction, and Analysis of Cellular Phospholipids-Cells were incubated in RPMI medium containing 5% fetal calf serum and labeled with 0.5 Ci/ml [9,10-3 H]palmitic acid (35.9 Ci/ mmol) for ceramide analysis or 0.5 Ci/ml [methyl-3 H]choline for sphingomyelin analysis. After 48 h of incubation, the medium was removed, and cells were washed several times with phosphate-buffered saline. Cells (5 ϫ 10 6 ) were then resuspended and treated with TNF (50 ng/ml) for various times. Lipids were extracted by the method of Bligh and Dyer (41) and separated by thin layer chromatography (TLC) using as developing solvent systems for ceramide analysis chloroform/methanol/ water (100:42:6) followed by a second step using hexane/diethyl ether/ formic acid (55:45:1). For sphingomyelin analysis, lipids were separated using chloroform/methanol/water (70: 35:5). Radioactive lipid spots were detected upon exposure to iodine vapor, scraped into scintillation fluid, and counted. The positions of ceramide on TLC plates were determined by comparison with concomitantly run nonradioactive ceramide (type III) (Sigma). Statistical analysis was performed using Student's t test.
Western Blotting-Proteins were separated on 10% SDS-polyacrylamide gels and electroblotted onto hybond TM membranes (Amersham Pharmacia Biotech). After blocking, the membranes were probed with anti-JNK polyclonal antibody (Santa Cruz Biotech) as described elsewhere (43). The complexes were detected using enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) and autoradiography.
Transfection and Apoptosis Assays-For transient transfection, cells were plated overnight at a density of 10 5 /60-mm plate and then cotransfected with vector encoding the dominant-negative mutant of Jun pCMVTAM67 (27,44) or the dominant-interfering pc-DNA3-Flag-MKK4 (Ala) mutant (27) together with the pE-GFP vector (CLON-TECH) expression vector encoding for the green fluorescent protein, according to the manufacturer instructions (Qiagen). After 24 h, cells were subsequently treated or not with TNF (50 ng/ml) for 16 h. For apoptosis analysis, cells were stained with DAPI at a concentration of 1 mM (Sigma) and examined with a Zeiss Axiophot fluorescent microscope. For stable transfection, MCF7 cells were transfected with the MKK4 dominant-negative cDNA inserted in the mammalian expression vector pc-DNA3 or with an empty vector (control) and selected for 3 weeks with G418 sulfate (Sigma) at a concentration of 0.5 mg/ml. Individual clones were pooled, and cells were then subjected to TNF treatment and harvested for the cytotoxic or JNK assays.

Effect of TNF on JNK Activation in TNF-sensitive (MCF7)
and -resistant (RA1/1001) Cells-To further analyze the biochemical basis of cell resistance acquisition to the cytotoxic action of TNF, we first performed experiments to investigate the possible implication of the JNK/SAPK pathway in the acquisition of MCF7 resistance to the cytotoxic action of TNF. For this purpose, we have used the TNF-sensitive human breast adenocarcinoma MCF7 cell line and its well characterized TNF-resistant variant (RA1/1001) established by prolonged culture of MCF7 in the presence of increasing concentrations of TNF (6). As expected, over 70% MCF7 cells undergo apoptotic cell death in response to TNF, whereas less than 10% RA1/1001 clone exhibited such a phenotype (Table I). The resistance exhibited by these cells was not due to a lack of TNF receptor expression or TNF signaling (6,45).
To determine whether the RA1/1001 cell resistance to the cytotoxic action of TNF interferes with JNK activation, JNK activity was examined in these cells by an immunocomplex assay with GST-c-Jun as substrate (46 -47). As shown in Fig.  1A, JNK was similarly activated in both MCF7 and RA1/1001 clone. Kinetic studies showed that optimal induction of JNK was observed after 15-30 min of exposure to TNF. Immunoblotting of cell lysates with anti-JNK antibody revealed that JNK1 protein level remained constant under TNF treatment (Fig. 1C). The data in Fig. 1, panel C, also indicate that RA1/ 1001 cells contain more JNK protein than in MCF7 cells. These data suggest the absence of a causal correlation between activation of JNK and the acquisition of resistance to the cytotoxic action of TNF by MCF7 cells.

Inactivation of JNK Cascade Had No Effect on TNF-induced Cell
Death-Experiments were performed to examine whether inactivation of the JNK pathway interferes with MCF7 resistance to the cytotoxic action of TNF. We therefore examined the ability of dominant interfering mutants of MKK4 to inhibit JNK activation and cell death in response to TNF. Therefore, we stably transfected MCF7 cells with an expressing vector encoding a dominant-negative mutant of MKK4 (MKK4 ala), which has a single mutation at the ATP-binding site, abrogating the kinase activity (31). Fig. 2A shows that stable expression of MKK4 ala inhibited (70%) the activation of JNK by TNF, indicating that TNF signaling leading to JNK activation was specifically blocked in MCF7-transfected cells. Furthermore, when expressed with an hemagglutinin epitope-tagged JNK in a transient transfection assay, MKK4 ala suppressed TNF-induced JNK activation (Fig. 2B). Interestingly, the data depicted in Fig. 3B demonstrate that such transfection did not result in the alteration of MCF7 sensitivity to the cytotoxic action of TNF, as compared with control cells (MCF7/pc-DNA3). A similar conclusion could be drawn when these experiments were performed using MCF7 cells transiently co-transfected with MKK4 ala and an expression vector encoding green fluorescent protein, which allows the identification of transfected cells. DAPI staining showed that inhibition of the JNK pathway in MKK4-DN-transfected cells did not interfere with TNF-induced apoptosis in these cells (data not shown). It is therefore unlikely that JNK activation plays a direct role in TNF-induced programmed cell death in MCF7 cells.
To provide further evidence that inactivation of JNK cascade does not significantly contribute to apoptosis triggered by TNF, we examined the effect of the dominant interfering mutant of c-Jun (TAM 67) on TNF-induced MCF7 cell death. TAM 67 acts as a dominant-inhibitor due to a deletion in the N-terminal transactivation domain of c-Jun that includes the binding site for JNK (27,44). MCF7 cells were transiently co-transfected with TAM 67 and green fluorescent protein. As shown by DAPI staining, treatment of transfected cells with TNF induced condensation and fragmentation of the nucleus (Fig. 2C), consistent with apoptosis, irrespective of whether these cells are transfected with control expression vector or TAM 67. To provide evidence that MCF7 cells expressed sufficient levels of TAM 67 to block c-Jun activity, we tested the ability of TAM 67 to inhibit the transcriptional activity of c-Jun. When fused to the DNA binding domain of the yeast transactivator Gal4 (1-147), Jun proteins can activate transcription from a promoter containing a luciferase reporter gene under the control of Gal4 binding sites linked to an E1b TATA. As expected, expression of TAM 67 inhibits the activation of Gal4-Jun reporter in response to TNF. These results, together with the inability of MKK4 ala to block TNF-mediated cell death, are consistent with a model in which TNF-mediated JNK activation and cell death occur through independent mechanisms in MCF7 cell model. (22,23), the interaction between ceramide pathway and JNK activation in the control of TNF-induced apoptosis or resistance to the cytotoxic action of TNF remains, respectively, controversial and unknown. To elucidate the relationship between ceramide generation and JNK activation, we stably transfected MCF7 cells with the dominant interfering mutant of FADD (FADD-DN) (10). The data shown in Fig.  3A clearly demonstrate that in FADD-DN-expressing cells, which are resistant to the cytotoxic action of TNF (data not shown), TNF was inefficient in inducing sphingomyelin hydrolysis and ceramide generation (Fig. 3B). In contrast, in MCF7 cells transfected with the control vector, a sphingomyelin hydrolysis and a significant boost in intracellular ceramide were concomitantly observed (126%) (Figs. 3B). Interestingly, when MCF7 cells transfected with FADD-DN or the control vector were treated with TNF, a similar JNK activation was observed in both cells (Fig. 3, C and D). These data suggest that the abrogation of ceramide generation does not result in the alteration of JNK activation, indicating that ceramide release and JNK activation can occur independently.

JNK Activation Occurs In the Absence of Ceramide Generation in MCF7 FADD-DN Cells-Although a coordinated regulation of apoptosis via the sphingomyelin pathway and JNK has been suggested
Exogenous Ceramide-induced Cell Death in the Absence of JNK Activation-It has been reported that ceramide-induced apoptosis in U937 leukemia cells and bovine aorta endothelial cells was associated with induction of JNK activity (22). It is becoming increasingly clear that exogenous ceramide induced JNK activation through phosphorylation of MKK4 (48). We reasoned if ceramide induced JNK activation, which in turn leads to apoptosis, then expression of MKK4 dominant-interfering mutant would block ceramide-induced cell death. Using MCF7 transfected with MKK4 dominant-interfering mutant, we demonstrated that exogenous synthetic cell-permeable C 6ceramide-induced cell death was not altered in these cells. Data depicted in Fig. 4 show a significant cell death of MCF7 MKK4-DN cells (60% of lysis), comparable with cells trans- fected with control vector (Figs. 4, A and B), despite a strong inhibition of JNK activation (Fig. 4C). These data suggest that JNK activation is not required for ceramide-induced MCF7 cell killing. DISCUSSION 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. Overexpression of several TNFinduced early response genes such as MnSOD, A20, HSP 70, and IAPs has been reported to protect cells against TNF cytotoxicity (49 -54). Recently, we provided evidence indicating that the alteration of sphingomyelinase activation and the subsequent ceramide generation may represent a potential additional mechanism by which human tumor cells may escape TNF-mediated apoptosis (6).
It is well established that several protein kinases are rapidly activated in response to TNF, including JNKs (55)(56). Recently, much emphasis has been placed on the potential role of JNK as mediator of TNF signaling. Although JNK and its target c-Jun were suggested as critical mediators of apoptosis induced by TNF (22)(23)(24)(25), their involvement in the control of TNF-induced cell death remains controversial. The relationship of JNK activation with respect to the acquisition of tumor resistance to TNF was particularly investigated. We used a cell model that is a valid tool to further dissect the biochemical mechanisms associated with the acquisition of tumor resistance to TNF. The present studies provide direct evidence indicating that TNF-induced JNK activation similarly occurs in TNF-sensitive MCF7 cells and its resistant counterpart (RA-1/ 1001), suggesting that JNK activation was not abrogated in TNF-resistant cells. However, it should be noted that in TNFresistant cells, more endogenous JNK protein was observed than in TNF-sensitive cells. Whether JNK plays a role in TNF resistance has to be determined. Our data are consistent with the concept that the JNK cascade does not play a role in TNF-induced cell death and are in agreement with previous reports indicating that induction of apoptosis by TNFR1 or by Fas was not hindered by disruption of the JNK cascade (e.g. by introduction of dominant-interfering TRAF2 or JNK mutants) (35). Since similar JNK activation by TNF occurs in MCF7 and its resistant derivative, it is tempting to speculate that the activation of the JNK cascade by TNFR1 is a bystander event that follows rather than leads to apoptosis, as suggested previously by others (35). In addition, the findings that CD40 ligation, which protects B cells against apoptosis, causes potent JNK activation are also in support of the view that apoptosis can be triggered in the absence of JNK activation (57)(58). We also obtained data demonstrating that transient TAM 67 expression had no effect on TNF-induced apoptosis in MCF7. In contrast, Verheij et al. (23) have shown that C 2 -ceramide-induced apoptosis was inhibited in transiently transfected U937 and bovine aorta endothelial cells with TAM 67. It should be noted that the role of JNK cascade in stress-induced apoptosis should consider the type of cellular stress that is involved. Recently, Chauhan et al. (60) suggested that there are at least two different apoptotic pathways in multiple myeloma cell lines, one that involves activation of JNK as induced by irradiation and another that is independent of JNK as triggered by dexamethasone. In addition, evidence has been provided indicating the existence of tissue specificity differences in the role of JNK in apoptosis (59). Taken together, our data clearly indicate that the JNK cascade does not appear to interfere with the acquisition of MCF7 resistance to the cytotoxic action of TNF and confirm that TNF can induce MCF7 cell death in a JNK-independent fashion. It is conceivable that the JNK cascade may play a role in TNF-induced apoptosis presumably in association with other signaling pathways that may be altered in TNF-resistant cells. In this regard, it has been reported that activation of JNK and concurrent inhibition of extracellular signal-regulated kinase are critical for induction of apoptosis in rat PC-12 pheochromocytoma cells upon nerve growth factor withdrawal. A dynamic balance between extracellular signalregulated kinase and JNK pathways in determining cell survival or cell death has been reported (22).
The coordinated regulation of apoptosis via the sphingomyelin and SAPK pathway has been suggested (20). In this context, direct evidence has been provided for an important role of sphingomyelinase in UV-induced activation of JNK (61) and for the implication of TGF ␤-activated kinase (TAK1) in mediating the ceramide signaling to SAPK/JNK (47). It has also previously been shown that a dominant-negative derivative of FADD (FADD-DN) blocked CD95-induced ceramide generation (10). This prompted us to investigate whether the sphingomyelin metabolism initiated by TNF in MCF7 cells has been affected by FADD-DN. We demonstrated in the course of these studies that JNK activation can occur in the absence of ceramide generation. Indeed, in our system, whereas dominant-negative FADD inhibited ceramide generation in MCF7, it does not interfere with JNK activation. These data confirm that FADD, but not JNK, is required for TNF-induced ceramide generation and apoptosis and that TNF-induced apoptotic pathway is separate from the pathway that leads to JNK activation. Furthermore, it has been reported that activation of SAPK/JNK can occur by TNFR1 through a noncytotoxic TRAF2-dependent pathway (35)(36)(37). This is consistent with the view that in TNFresistant cells, JNK activation may occur through a pathway that is not required or linked to TNFR1-induced apoptosis. In addition, we obtained data showing that the C 2 -ceramide-induced apoptosis in MKK4-DN-transfected MCF7 cells was not prevented, suggesting that ceramide may induce apoptosis in the absence of JNK activation. Whether another kinase such as the recently reported MKK7 (33,(62)(63)(64) is involved in JNK activation in MCF7 cells has to be determined.
Overall, our observations are consistent with the notion that JNK activation does not interfere with the acquisition of cell resistance to TNF and that MKK4-induced JNK activation is not a crucial mediator of TNF-and ceramide-induced cell death.