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Resistance to TNF-Induced Cytotoxicity Correlates with an Abnormal Cleavage of Cytosolic Phospholipase A₂¹

Nour-Eddine El Mahdani^*, Maya Ameyar^*, Zhenzi Cai^*, Odile Colard, Joëlle Masliah and Salem Chouaib^2,*

^* Institut National de la Santé et de la Recherche Médicale Unité 487, Cytokines et Immunologie des Tumeurs Humaines, Institut Gustave Roussy, Villejuif, France; and Institut de la Santé et de la Recherche Médicale Unité 538, Paris, France

	Abstract

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To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [³H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A₂ (PLA₂) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA₂ inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA₂ (cPLA₂) and the Ca²⁺-independent PLA₂, respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA₂ activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA₂ activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA₂ in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA₂ was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA₂ cleavage.

	Introduction

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Tumor necrosis factor is a pleiotropic cytokine that elicits a wide variety of functions and plays a prominent role in the immune and antitumor response (1, 2). However, despite the major advance in understanding the early events in TNF signaling and the identification of molecules that are recruited to TNFR1, the mechanism of resistance to TNF observed in some tumor cells remains largely unknown. Therefore, understanding the molecular and biochemical mechanisms of tumor cell resistance to the cytotoxic action of TNF may ultimately provide new approaches to enhance its therapeutic efficiency against human malignancies. We have earlier shown that cell surface expression of TNF receptors was necessary but not sufficient to mediate an apoptotic response, and that postreceptor mechanisms are important in controlling cell susceptibility to the cytotoxic action of TNF (3). 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 (4, 5, 6, 7), the disruption of p53 wild-type function (8), and the activation of the transcription factor NF-B (9, 10, 11). However, these proteins confer only partial protection against TNF cytotoxicity, suggesting that additional resistance mechanisms exist.

Several second messengers have been proposed to mediate the biological effects of TNFR1 ligation, including various phospholipid breakdown products, arachidonic acid (AA)³ metabolites, free radicals, and increased intracellular Ca²⁺ (12). It is clearly established that TNF stimulates the activation of a number of phospholipases including phospholipase C (13) and phospholipase A₂ (PLA₂) (14) in addition to sphingomyelinase (15). Several lines of evidence suggest that cytosolic PLA₂ (cPLA₂) may play a role in TNF cytotoxicity in different cell types (16, 17). However, the way cPLA₂ is involved in the acquisition of human cell resistance to the TNF action remains unknown.

PLA₂ are important components in the regulation of various biological processes including inflammation, cancer, and apoptosis (18). These enzymes cleave membrane phospholipids to give rise to lipid second messengers and are known to be responsible for AA release, which is the main limiting step for the synthesis of eicosanoids (19). Purification and cloning of several PLA₂s have demonstrated differences between secreted and intracellular PLA₂s (20, 21, 22). The secreted PLA₂s are closely related proteins of low molecular mass (14 kDa), with calcium requirement in the millimolar range. In contrast, intracellular PLA₂s have a higher molecular mass: Ca²⁺-independent PLA₂ (iPLA₂, 80–85 kDa) or cPLA₂ (100–110 kDa). Little is known about iPLA₂ implication in TNF-induced cytotoxicity (23). cPLA₂, which plays a role in receptor-mediated production of eicosanoids, is regulated posttransductionally by phosphorylation and by its calcium-dependent translocation to membranes (24, 25). Recent evidence indicates that cPLA₂ regulation might also occur through its cleavage by some proteases such as caspases (26, 27, 28, 29, 30).

In the present study, we attempted to further investigate the role of cPLA₂ in the acquisition of human breast adenocarcinoma resistance to the cytotoxic action of TNF. We demonstrated that the acquired cell resistance to TNF was associated with an abnormal cPLA₂ cleavage and the subsequent alteration of its activity.

	Materials and Methods

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Reagents

Highly purified (>99%) recombinant TNF (TNF-, sp. act., 6.63 x 10⁶ U/mg protein) was kindly provided by I. Apfler (Bender Wien, Austria). [5,6,8,9,11,12,14,15-³H]AA (210 Ci/mmol), and L-3-phosphatidylcholine,1-stearoyl-2-[1-¹⁴C]arachidonyl (25 µCi/ml) were purchased from Amersham (Les Ulis, France). Methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), PLA₂ inhibitors, were obtained from TEBU (Le Perray-en-Yvelines, France). Polyclonal Abs against cPLA₂ and iPLA₂ were respectively from Santa Cruz and Cayman (Rueil Malmaison, France). HRP linked to anti-rabbit and anti-monkey Abs were purchased from TEBU. The ECL kit was obtained from Amersham (Les Ulis, France). All other reagents were purchased from Sigma (St. Louis, MO).

Cell culture

TNF-resistant cells were derived from a TNF-sensitive human breast carcinoma MCF7 cell line after continuous exposure to increasing doses of recombinant TNF (31). On selected resistant clones, human p55 TNFR1 cDNA cloned in mammalian expression vector pMPSVEH was used to transfect these resistant clones. After 10–14 days of selection in growth medium containing 200 µg/ml G418 (Sigma), C1001 clone was isolated and examined for human p55 TNFR1 expression by fluorescence-activated cell sorting. This clone was subsequently maintained in the same medium with G418 for more than 2 mo. The sensitivity of MCF7 and the C1001 clone to TNF was tested every 2 wk during culture. All cell lines were routinely cultured in RPMI 1640 medium containing 5% FCS, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified atmosphere with 5% CO₂.

Measurement of TNF-mediated cytotoxicity

Cell lines were trypsinized and incubated at a concentration of 7 x 10⁴ cells/ml in flat-bottom 96-well microtiter plates in 100 µl of complete medium. Recombinant TNF- (50 ng/ml) and PLA₂ inhibitors (10 µM MAFP and 10 µM BEL) in a total volume of 100 µl were then added. After 72 h of incubation at 37°C, medium was removed and replaced with 100 µl of 0.5% crystal violet solution. After a 10-min incubation at room temperature, the plates were washed and viable crystal violet-stained cells were lysed with a solution of 1% SDS. OD was then measured for each well using a test wavelength of 450 nm. Using this colorimetric procedure, TNF-mediated cell lysis could be assessed as compared with the viability of cells incubated in the presence of TNF using the following calculation: percent lysis = 100 x (1 - OD/OD₀), where OD and OD₀ were the OD obtained for treated and TNF-untreated cells, respectively.

[³H]AA release

Cell lines were incubated for 20 h in RPMI 1640 medium supplemented with 0.5 µCi/ml [³H]AA (sp. act., 218 Ci/mmol, Amersham, Arlington Heights, IL). At the end of the incubation, the labeling medium was removed and the monolayers were washed twice with PBS containing 0.1% of free fatty acid BSA and once with PBS. The cells were then incubated with TNF (50 ng/ml) or various doses of PLA₂ inhibitors (MAFP, BEL). Aliquots of media were collected at different times (0, 6, and 18 h). At the end, cells were washed and scraped. Media and cells were counted by liquid scintillation. Results are expressed as percentage of AA released from total AA incorporated into cells.

Quantification of AA

Cells were washed twice with PBS, harvested, and lipids were extracted by adding chloroform and methanol according to Bligh (cited in 42). The esterified fatty acids were hydrolyzed by alkaline hydrolysis. Briefly, lipid extract was resuspended in 0.5 ml of KOH (0.5 N)/methanol and incubated for 15 min at 56°C. After cooling, fatty acids were methylated by adding 0.5 ml of BF3 (20%)/methanol and incubated for 15 min at 56°C. The methyl esters were extracted with hexane and separated by gas chromatography on a capillary column containing supercowax 10 bonded phase (0.32 mm in diameter, 30 m long). The methyl esters were detected by mass spectrometry (model R10-10C; Nermag, Houston, TX) in the chemical ionization mode with ammonia (0.1 bar) reagent gas. The positive quasi-molecular ions were monitored and time integrated. Quantification was referred to heptadecanoic methyl ester as an internal standard.

[³H]AA distribution into phospholipids

Following incubation (20 h) with [³H]AA (0.5 µCi/ml), the cell lines were washed twice with PBS and counted. Total lipids were extracted as described elsewhere (32), and total radioactivity was determined by scintillation counting. To study [³H]AA distribution into phospholipids, labeled lipids were separated in duplicates on HPTLC plates. Known phospholipids were run in parallel. The mobile phase consisted of chloroform/methanol/acetic acid/water (75:45:12:6, v/v/v/v) for 25 min. Phospholipids visualized by autoradiography were phosphatidylcholine and a mixture of phosphatidylinositol/phosphatidylserine and phosphatidylethanolamine. The corresponding spots were scraped and counted for radioactivity. Results are expressed as percent radioactivity related to total cell incorporation.

Homogenate preparation

Cell lines were washed twice with PBS. The cells were scraped off in 40 mM Tris buffer (pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 1 mM PMSF, and 1 µg/ml leupeptin, 10 mM ß-glycerophosphate, 50 mM NaF, and 100 µM sodium orthovanadate and sonicated for 15 s. The homogenate was stored at -80°C until use.

Measurement of PLA₂ activity

The assay mixture (total volume, 250 µl) contained 1 mg/ml fatty acid free BSA, 5.5 mM CaCl₂, 30% glycerol, 50 µM Triton X-100, 10 mM DTT, 100 mM Tris buffer (pH 8.5), 50 µg of cell homogenate, 4 µM L-3 phosphatidylcholine,1-stearoyl-2-[¹⁴C-1]arachidonyl (sp. act., 200 GBq/mmol, 54 mCi/mmol; Amersham). The substrate was prepared by drying labeled (0.32 nmol) and unlabeled (0.68 nmol) phosphatidylcholine under a stream of nitrogen and resuspending in ethanol/ether (1:1), then in 100 mM Tris (pH 8.5) and briefly sonicated. The reaction mixture was incubated at 37°C for 15 min, resuspended in chloroform/methanol, and resolved by TLC on silica gel plates using chloroform/methanol/water (65:25:4). The spots were visualized with iodine vapor. Those corresponding to phosphatidylcholine and free fatty acid were scraped off and their radioactivity was determined by scintillation counting. The PLA₂ activity was expressed as picomoles per minute per milligrams of proteins as described previously (33). Secretory PLA₂ (sPLA2) activity was measured as described elsewhere (34).

Western blot analysis

Cell homogenates from MCF7 and C1001 cells (100 µg) after 18-h TNF treatment were subjected to SDS-PAGE (10% acrylamide) under reducing conditions. The proteins were transferred onto nitrocellulose membrane (Bio Blot-NC; Schleicher & Schüll, Dasser, Germany), for 3 h at 4°C, at a constant voltage (70 V). The blots were washed at room temperature in blocking buffer containing TBS, 3% nonfat dried milk, and 0.1% Tween 20 and then incubated for 2 h in the same buffer containing rabbit polyclonal anti-iPLA₂. Recombinant cPLA₂ from sf9 cells was used as control (32). Membranes were washed and incubated with a goat anti-IgG coupled to peroxidase for 1 h at room temperature. After an additional washing, immunoreactive bands were visualized by chemiluminescence (ECL; Amersham).

	Results

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Failure of TNF to induce AA release in TNF-resistant cells

We first compared TNF-induced cytotoxicity and stimulation of AA release in MCF7 cells and in their resistant counterpart C1001 clone. As shown in Fig. 1A, although TNF induced MCF7 cell death, no effect was observed in C1001 cells. Time course of AA release from prelabeled cells was compared in both MCF7 and C1001 cells (Fig. 1B). No increase in AA release was observed in both cell lines under stimulation with TNF (50 ng/ml) during short-term incubation (0–1 h). However, AA release increased in MCF7 cells after 6 h of incubation with TNF and this increase was sustained at 18 h. In contrast, AA release in C1001 cells was not increased following TNF treatment of C1001 cells, suggesting that the acquisition of resistance to the cytotoxic action of TNF by these cells correlated with a failure of AA release.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. AA release in TNF-sensitive cells and their resistant derivatives. A, Cytotoxic effect of TNF- was quantified by the viability of parental MCF7 and resistant C1001 cells. Seven x 103 cells/well were incubated for 72 h with the indicated doses of recombinant TNF (50 ng/ml). Cell lysis was measured using crystal violet assay as described in Materials and Methods. Data are the means ± SD of quadruplicate experiments. B, AA release course between 0 and 18 h. MCF7 and C1001 cells were incubated for 20 h with 0.5 µCi/ml [3H]AA. Cells were washed and treated with medium (control) or 50 ng/ml TNF. After 20 h of incubation, cells were harvested and aliquots of supernatants and cells were counted to determine the levels of released radioactivity. Results presented as percentage of control.

To determine whether the difference in AA release observed in TNF-sensitive and TNF-resistant cells was due to a difference in availability of AA-containing phospholipids, we evaluated its content in both cell lines. Fig. 2A unexpectedly showed that TNF-resistant cells (C1001) contained a higher amount of AA as compared with MCF7 cells (15 vs 9%). Moreover, incorporation of radiolabeled AA at tracing doses for 20 h was also lower in MCF7 cells as compared with C1001 cells (Fig. 2B). Analysis of the distribution of labeled AA into the various phospholipid classes showed no difference between the two cell lines (Table I). Altogether, these results showed that the failure of TNF to induce AA release in C1001 cells was not due to a decreased availability of AA but may involve a decreased activation of phospholipid hydrolysis.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. AA content and distribution into phospholipid pools in TNF-resistant (C1001) and TNF-sensitive (MCF7) cells. A, AA content of MCF7 and C1001 was quantified by gas liquid chromatography/mass spectrometry as described in Materials and Methods. Data are from a representative experiment made in duplicate. B, AA incorporation. MCF7 and C1001 cells were incubated for 20 h with 0.5 µCi/ml [3H]AA before lipid extraction. Total cell radioactivity was determined by scintillation counting as described in Materials and Methods. Values are means ± SD of two independent determinations.

Different PLA₂s, including sPLA₂ and cPLA₂ are induced by TNF in several cell types. We first measured the PLA₂ activity secreted in the absence or in the presence of TNF. Media from MCF7 and C1001 cells exhibited a similar sPLA₂ activity under basal conditions. Such activity did not increase when cells were pretreated with TNF (50 ng/ml) for 18 h and incubated in the presence or absence of either heparin or 1 M NaCl (data not shown). To discriminate between intracellular PLA₂s, we used two inhibitors, MAFP and BEL, known to inhibit respectively cPLA₂ and iPLA₂. Fig. 3 shows that cell incubation with 10 µM MAFP or BEL had no effect on the basal release of [³H]AA in both TNF- sensitive and -resistant cells. In contrast, MAFP totally inhibited the stimulation of AA release induced by TNF in MCF7 cells, whereas BEL had no significant effect (p < 0.05). To confirm the involvement of cPLA₂, we measured the Ca²⁺-dependent PLA₂ activity on radiolabeled arachidonyl phosphatidylcholine in both C1001 and MCF7 cells. Data depicted in Fig. 4 indicated that under basal conditions, PLA₂ activity of cell homogenates was unaffected by MAFP or BEL. In contrast, following TNF stimulation, it was increased in TNF-sensitive, but not in TNF-resistant cells. This PLA₂ activity increase was abolished following EGTA or MAFP treatment whereas BEL had no effect. Altogether, these results indicate in this cell system that the PLA₂ involved in TNF-induced AA release is Ca²⁺ sensitive and rule out both Ca²⁺-independent PLA₂ and sPLA₂ activities. On the other hand, to confirm an assignment of cPLA₂ activation to TNF-induced cell death, we measured the effect of MAFP on TNF-induced cytotoxicity. Fig. 5 illustrates that pretreatment of MCF7 cells with MAFP reduced by 5-fold the TNF-induced MCF7 lysis, whereas BEL had no effect. These findings exclusively incriminate cPLA₂ activation in TNF-induced cytotoxicity in our cell system.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Effect of PLA2 inhibitors on basal and TNF-induced AA release. Cells were prelabeled with 0.5 µCi/ml [3H]AA as described in Materials and Methods. They were stimulated with TNF (50 ng/ml) or vehicle for 18 h. MCF7 and C1001 were exposed to 10 µM of each PLA2 inhibitor or vehicle. Data are means ± SD of three independent experiments. *, p < 0.05; NS, corresponding to p > 0.07.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Effect of PLA2 inhibitors on PLA2 activity in TNF-resistant and TNF-sensitive cells. Cell homogenates were prepared as described in Materials and Methods. PLA2 activity was determined in vitro using 1-stearoyl-2-[14C-1]arachidonyl (sp. act., 200 GBq/mmol, 54 mCi/mmol) as substrate incubated in the presence of 10 mM DTT. Cell homogenates were prepared from cells treated with TNF (50 ng/ml) for 18 h, and when necessary 4 mM EGTA, 10 µM MAFP, and 10 µM BEL were added to the incubation medium. Data are presented as means ± SD of three independent experiments. *, p < 0.05.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Effect of PLA2 inhibitors on TNF-induced cell death in MCF7 cells. Cells (7 x 103 cells/well) were incubated for 72 h with the indicated concentrations of TNF and PLA2 inhibitors (MAFP, BEL). Cell lysis was measured using the crystal violet assay as described in Materials and Methods. Data presented are the means ± SD of three separates experiments.

The mechanism by which TNF triggers cPLA₂ activation is not clearly established. Our results show a parallel between AA release induced by TNF and an increase in cPLA₂ activity. To evaluate whether this increase was due to an elevated cPLA₂ expression, we performed Western blot analysis. Fig. 6 shows the constitutive expression of p70cPLA₂ and p50cPLA₂ bands in MCF7 and C1001, respectively. The 100-kDa native cPLA₂ band was lower in MCF7 than in C1001 cells. TNF treatment of MCF7 cells resulted in a significant increase of the p70cPLA₂ band. In contrast, the p50cPLA₂ band remained unchanged after TNF treatment in C1001 cells. Fig. 6 also showed that TNF treatment of sensitive and resistant cells had no effect on the iPLA₂ expression. These different patterns observed in the two cell lines might account at least in part for different levels of cPLA₂ activation.

View larger version (60K): [in this window] [in a new window]   FIGURE 6. Western blot analysis of cPLA2 and iPLA2 in TNF-resistant cells (C1001) and TNF-sensitive cells (MCF7). Cell homogenates were prepared after an 18-h TNF treatment as described in Materials and Methods. One hundred micrograms of proteins of each cell line was loaded into 10% polyacrylamide gel. The blots were then incubated for 2 h in the same buffer containing rabbit polyclonal anti-human cPLA2 or polyclonal anti-iPLA2. Recombinant cPLA2 from SF9 cells was used as control.

	Discussion

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It has been reported that different forms of PLA₂ were activated by TNF according to the cell type: iPLA₂ (14), cPLA₂ (16, 17), or sPLA₂ (40). To determine the nature of PLA₂ involved in the control of MCF7 cell susceptibility to TNF, we have used specific inhibitors and tested the PLA₂ activity in conditions allowing distinction between the three forms of PLA₂ (intracellular or extracellular localization, sensitivity to calcium) and their determined activity in both TNF-sensitive and -resistant cells. Our data indicated that the failure of AA release in response to TNF was selectively correlated with an alteration of cPLA₂. The lack of stimulation of cPlA₂ in TNF-resistant cells was in accordance with previous reports showing the involvement of cPLA₂ in TNF cytotoxicity in different cell types (16, 17). However, the mechanism associated with the alteration of cPLA₂ stimulation in TNF-resistant cells remains unclear. It has been shown that exogenous ceramide could induce cPLA₂ transcription (41). In a previous study, we provided evidence indicating that the acquisition of MCF7 resistance to the cytotoxic action of TNF correlated with an alteration in sphingomyelin and the subsequent ceramide generation (42). In this study, the long-term effect on AA release in response to TNF puts the stimulation of PLA₂ downstream activation of ceramide production. In contrast, Jayadev et al. (43) have reported that AA release resulted in ceramide generation in murine L929 cells after TNF treatment. Although these observations strongly suggest the existence of a link between PLA₂ activation and ceramide generation, a difference in the time course of AA release and cPLA₂ stimulation in MCF7 cells was observed as compared with murine L929 (43). Whether ceramide and AA release function independently or together to transduce TNF cytotoxic effect requires further investigations. It has also been reported that cPLA₂ may interact with other components involved in TNF signaling. In this regard, Thommesen et al. (44) have reported data suggesting the involvement of cPLA₂ in the TNF signal transduction pathway leading to NF-B translocation. Nevertheless, using the TNF-resistant C1001 cells, we demonstrated here that the activation of cPLA₂ did not occur, despite a normal activation of NF-B in these cells by TNF (42). These observations suggest that in our cell model, PLA₂ inhibitors alter TNF-induced cell lysis by a mechanism at least in part independent from the NF-B pathway. Among the reported mechanisms implicated in cPLA₂ regulation, its cleavage was found to be an essential requirement in TNF- and Fas-induced apoptosis. We found that cPLA₂ was differentially cleaved in TNF-resistant vs TNF-sensitive cells. Comparative analysis of the pattern of cPLA₂ in TNF-resistant and -sensitive cells showed distinct immunoreactive bands. Indeed, cPLA₂ is one of very few so far described proteins that can be cleaved by several caspases at multiple cleavage sites (45). In this regard, it has been shown that cPLA₂ was sensitive to caspases which are able to hydrolyze protein at specific sites, producing fragments from 50 to 70 kDa (28). This in vitro cPLA₂ cleavage study established that caspase-7 and -8 exclusively generated the p70 (70-kDa) cleaved form, and this specific cleavage was enhanced by TNF treatment, mainly for caspase-7. Furthermore, the p50 (50-kDa) cleaved form was exclusively generated by caspase-1 and -3. These data are consistent with our unpublished results indicating an alteration of caspase-8 in TNF-resistant cells. Given the fact that these cells are deficient in caspase-3 (46) and present only the p50 cleaved form, it is likely that caspase-1-like activity may play a role in p50 band generation. In TNF-sensitive cells, we exclusively observed an increase with the 70-kDa band after TNF stimulation. In contrast, in resistant cells, only the p50 cleaved form was observed. Our findings fit well with the report of Wissing et al. (30) demonstrating that the 70-kDa band corresponds to the activated form of PLA₂. It has been reported that cPLA₂ can be cleaved by a caspase-3-like activity, leading to the inactivation of its enzymatic function (23, 26). However, the role of caspase-3 in this cleavage, as reported by Wissing et al. (30), is ruled out in MCF7 cells. Using the universal caspase inhibitor ZVAD, we could demonstrate the existence of a relationship between cPLA₂ cleavage by caspases, AA release, and TNF-induced cell death (data not shown).

Future investigations will focus on the nature of caspase involved in the TNF cell death. It remains unclear whether the proteolytic fragments of cPLA₂ are produced as a result of its activation or whether it may prevent its activation. Nevertheless, the differential pattern of proteolytic products observed in TNF-sensitive and TNF-resistant cells suggests the possible existence of different caspase activity in the two cell lines which might result in a differential activation of cPLA₂.

The data of the present study may have potential relevance for a better understanding of the intracellular apoptotic effectors and the involvement of cPLA₂ in apoptosis promoted by TNF. Defining relevant transducers of the apoptotic signal triggered by TNF may be of major interest to manipulate the sensitivity of tumors for therapeutic interventions.

	Footnotes

 
¹ This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Association pour la Recherche sur le Cancer (Contract 6227).

² Address correspondence and reprint requests to Dr. Salem Chouaib, Institut National de la Santé et de la Recherche Médicale Unité 487, Cytokines et Immunologie des Tumeurs Humaines, Institut Gustave Rousssy, 94805 Villejuif, France.

³ Abbreviations used in this paper: AA, arachidonic acid; PLA₂, phospholipase A₂; cPLA₂, cytosolic PLA₂; sPLA₂, secretory PLA₂; iPLA₂, Ca²⁺-independent PLA₂; MAFP, methylarachidonyl fluorophosphate; BEL, bromoenol lactone.

Received for publication April 6, 2000. Accepted for publication September 15, 2000.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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