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(The FASEB Journal. 1998;12:101-109.)
© 1998 FASEB

RESEARCH COMMUNICATION

Restoration of TNF-{alpha}-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833

Christine Bezombesa, Nicolas Maestrea, Guy Laurenta,b, Thierry Levadec, Ali Bettaïeba, and Jean-Pierre Jaffrézoua,1

a CJF INSERM 9503, Centre Claudius Régaud, Toulouse Cédex 31052, France
b Service d'Hématologie, Centre Hospitalier Universitaire Purpan, Toulouse 31059, France
c Laboratoire de Biochimie Médicale, INSERM 466, Centre Hospitalier Universitaire Rangueil, Toulouse 31403, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF-{alpha}) is a cytokine with antitumor activity against several cellular models. TNF-{alpha}-induced apoptosis seems to be mediated by a signaling pathway termed `sphingomyelin-ceramide' pathway, which consists of the hydrolysis of sphingomyelin and the production of its breakdown product ceramide. Our study shows that KG1a cells, which are inherently resistant to TNF-{alpha} and do not produce ceramide upon cytokine stimulation, can be sensitized by the use of the P-glycoprotein inhibitor PSC833. Coincubation with 1 µM of this cyclosporin derivative restored the apoptotic potential of 10 ng/ml TNF-{alpha}. This effect was associated with the restoration of ceramide generation (315%) and activation of neutral, but not acid sphingomyelinase activity (143%). Furthermore, we demonstrate that treatment of KG1a cells with 1 µM PSC833 led to a threefold increase in inner plasma membrane sphingomyelin content and basal neutral sphingomyelinase activity. These results support the hypothesis whereby resistance to TNF-{alpha}-mediated apoptosis of certain leukemic cells is linked to the disposability of the sphingomyelin pool. These data also suggest a role for P-glycoprotein in sphingomyelin transverse plasma membrane asymmetry.— Bezombes, C., Maestre, N., Laurent, G., Levade, T., Bettaïeb, A., Jaffrézou, J.-P. Restoration of TNF-{alpha}-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833. FASEB J. 12, 101–109 (1998)


Key Words: TNF-{alpha} • sphingomyelin asymmetry • sphingomyelinase • ceramide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TUMOR NECROSIS FACTOR {alpha} or cachectin (TNF-{alpha}),2 which is produced essentially by monocytes and macrophages, is a cytokine whose antitumor activity in vitro and in vivo has been well documented in many cellular models (1). In hematopoietic cells, TNF-{alpha} generally exerts a cytotoxic or cytostatic effect (2). However, in myeloid leukemias, the extent of this cytotoxic effect is largely dependent on the cellular model. For example, Munker and co-workers (3) observed in clonogenic tests that HL-60 and U937 cell lines are very sensitive to TNF-{alpha}, whereas KG1, KG1a, and HEL are up to 100-fold resistant even though these cells share similar receptor levels. From these studies, it is apparent that certain leukemic cells present a `natural' resistance to TNF-{alpha}, a trait that may provide a leukemic cell a proliferative advantage.

Identification of the molecular basis for resistance to TNF-{alpha} has given rise to many studies. For example, it has been proposed that the overexpression of certain proteins such as manganous-dependent superoxide dismutase (4), heat shock protein hsp70 (5), and Bcl-2 (6) can confer resistance to TNF-{alpha}. However, these proteins give rise to only low levels of resistance, which argues for the existence of other resistance mechanisms in myeloid cells (4–6).

The nature of the cell death process induced by TNF-{alpha} in sensitive cell lines such as U937 has been well characterized. In these cells, TNF-{alpha} triggers apoptosis (7, 8). Several studies suggested that ceramide (CER) may act as a mediator of TNF-{alpha}-triggered apoptosis. Indeed, TNF-{alpha} elicits a sphingomyelin (SM) -CER pathway, which activates a sphingomyelinase (SMase) that hydrolyses SM to produce CER (9). These events appear minutes after TNF-{alpha} ligation. The role of ceramide as an apoptotic mediator in this process is strongly supported by the observation that cell-permeant CER analogs or exposure to bacterial SMase can induce apoptosis (9–11).

If one concedes that CER originates from SM hydrolysis, then it becomes evident that any perturbation in SM metabolism or disposability may affect CER generation and hence TNF-{alpha}-induced apoptosis. SM or N-acylsphingosine-1-phosphocholine is a major constituent of mammalian cell membranes. In most cells, SM is found essentially within the plasma membrane and concentrated mostly in its outer leaflet (SM transverse asymmetry) (12, 13). Recent studies by our group and others (14, 15) have demonstrated that in response to TNF-{alpha}, SM is hydrolyzed within the plasma membrane inner leaflet. Therefore, modifications of this particular metabolic pool may have consequences for apoptosis.

We recently proposed (16) that the natural resistance to TNF-{alpha}-triggered apoptosis in KG1a cells was at least in part due to a significant decrease in inner leaflet SM content. Indeed, although KG1a cells are resistant to TNF-{alpha}-mediated cell death, they remain sensitive to CER-triggered apoptosis. However, compared to the TNF-{alpha}-sensitive cell line U937, KG1a cells exhibit a sevenfold decrease in the cytosolic leaflet SM content (16). Therefore, we proposed that the absence of CER generation in TNF-{alpha}-challenged KG1a cells could be related to a deficiency in an SM-hydrolyzable pool. If this hypothesis were valid, one could concede that by modifying SM transverse distribution it would be possible to restore an apoptotic pathway in KG1a cells.

A very recent report demonstrated that human MDR1 and mouse mdr1a P-glycoprotein (P-gp) can translocate short-chain lipids (including short-chain SM analogs) at the cell surface across the plasma membrane, and that P-gp inhibitors blocked lipid translocation across the membrane (17). From these important observations, it has been proposed that MDR1 P-gp may function as a lipid translocase of broad specificity (17, 18).

Since KG1a cells express functional P-gp (19), which may potentially be an SM translocator, we hypothesized that P-gp inhibition may limit SM movement from the inner to the outer leaflet of the plasma membrane, leading to an increase of hydrolyzable SM pool, and thus sensitize KG1a cells to TNF-{alpha}. We now describe the effects of the cyclosporin derivative PSC833, a potent and specific P-gp blocker (20), on TNF-{alpha}-mediated apoptosis in KG1a cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and reagents
Human recombinant TNF-{alpha} was supplied from PeproTech-Tebu (Le Perray-en-Yvelines, France). PSC833 was generously provided by Sandoz (Rueil Malmaison, France). Ultroser HY was purchased from Gibco-BRL (Cergy-Pontoise, France). Silica gel 60 thin-layer chromatography plates were from Merck (Darmstadt, Germany). All other drugs and reagents were purchased from Sigma Chemical Co. (St. Louis, Mo.), Carlo Erba (Rueil-Malmaison, France), or Prolabo (Paris, France).

Cell culture
The human myeloblastic cell lines U937 and KG1a, purchased from the ATCC (Rockville, Md.), were cultured in IMDM medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (all from Eurobio, Les Ulis, France). Cell stocks were screened routinely for Mycoplasma (Stratagene Mycoplasma PCR kit, La Jolla, Calif.).

Cytochemical staining
Changes in cellular nuclear chromatin were evaluated by fluorescence microscopy by DAPI (4', 6'-diamino 2-phenylindol) staining (21).

DNA analyses
DNA was resolved on a 1.8% agarose gel and visualized with ethidium bromide as previously described (22). Quantitative DNA fragmentation was determined by the spectrofluorometric DAPI procedure as previously described (23, 24).

Pulsed-field gel electrophoresis
To detect high molecular weight DNA fragmentation (25), 5 x 106 control and treated cells were embedded in 0.7% low-melting agarose plugs and incubated for 48 h at 40°C in 0.2 M ethylene diaminetetraacetic acid (EDTA) containing 1 mg/ml proteinase K. The agarose plugs were washed three times for 1 h in 0.2 M EDTA and stored at 4°C prior to analysis. The samples, including a 50–1000 kbp lambda DNA standard (Promega Corporation, Madison, Wis.), were analyzed on a 1% agarose gel (SeaKem FastLane) by using a SwitchBack Pulse Controller pulsed-field electrophoresis system (Hoefer Scientific Instruments, San Francisco, Calif.) at 100 V, with pulse ramping times from 1 to 50 s (F/R ratio: 2.5:1), for 18 h in a 0.5x TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8). The buffer was recirculated continuously at room temperature. The gels were stained with ethidium bromide (0.5 mg/ml) and photographed under ultraviolet illumination.

Metabolic cell labeling and CER quantitation
Total cellular CER quantitation was performed by labeling cells to isotopic equilibrium with 1 µCi/ml of [9, 10-3H]palmitic acid (53.0 Ci/mmol, Amersham, Les Ulis, France) for 48 h in complete medium, as previously described (26, 27). Cells were then washed and resuspended in serum-free medium for kinetic experiments. Lipids were extracted and resolved by thin-layer chromatography (28); CER was scraped and quantitated by liquid scintillation spectrometry. Statistical analyses were performed by Student's t test.

Neutral and acid SMase assay
SMase activities were assayed as previously described using [choline-methyl-14C]SM (DuPont NEN, Les Ulis, France) (120,000 dpm/assay) as substrate (26, 29). Membrane-bound neutral SMase was assayed after lysing cells in 20 mM Hepes-NaOH, pH. 7.4, 10 mM MgCl2, 2 mM EDTA, 0.1 mM Na3VO4, 10 mM ß-glycerophosphate, 0.2% Triton X100, 30 mM paranitrophenylphosphate, 5 mM dithiothreitol, 0.1 mM Na2MoO4, 750 µM ATP, 1 mM phenylmethylsulfonylfluoride, 10 µM leupeptin, and 10 µM pepstatin, centrifugation at 100 000 g for 45 min at 4°C, and removal of supernatant (cytosolic extract).

Analysis of cellular SM content and transverse distribution
Analysis of transverse SM distribution in the plasma membrane was performed as previously described (16, 30–32). This method has been applied to various cell models such as Krebs-II ascitic cells, HL-60 cells, A431 cells, and neutrophils (31–35). Briefly, cells (50–100 x 106) resuspended in 5 ml phosphate-buffered saline were treated with 0.5 µCi 14C-concanavalin A (Sigma) for 10 min at room temperature in order to label plasma membranes. 14C-labeled concanavalin surface-labeled cells were resuspended in 100 mM KCl, 5 mM MgCl2, 1 mM ATP, 25 mM Tris-HCl (pH 9.6), and homogenized in a nitrogen cavitation bomb (Kontes, Vineland, NJ) after a 5 min period with 40 atm. of N2 at 4°C. Four milliliters of postnuclear supernatant was loaded onto a mixture of 11 ml Percoll, 2.2 ml distilled water, buffered with 4.8 ml of 400 mM KCl, 20 ml MgCl2, 100 mM Tris-HCl (pH 9.6). Membranes were then separated by centrifugation at 1.29 x 1010 rad2 x s-1, i.e., 160,000 g for 10 min at plateau in a Beckman Ti 60 rotor. Fractions of 2 ml were harvested from the top of each tube, diluted to adjust the pH to 7.4, and recentrifuged using a 50 Ti rotor at 200,000 g for 45 min to eliminate Percoll. Plasma membranes, corresponding to the peak of 14C-labeled concanavalin A across the gradient, were then collected. Efficiency of the plasma membrane isolation and purity of the preparation have already been reported (30, 36).

Analysis of transverse SM distribution in the plasma membrane was performed in KG1a cells according to the two-step procedure previously described (30, 32). In the first step, whole cells (1.5 x 106/ml) were incubated in the presence or absence of 100 mU/ml of S. aureus SMase at 37°C. The reaction was stopped by the addition of 10 mM EDTA on ice. After harvesting the cells, lipids were extracted by the method of Bligh and Dyer (37) and separated on TLC using chloroform/methanol/water (70:35:5, by vol). The phosphorus content of the various spots, detected after exposure to iodine vapor, were quantified according to Böttcher et al. (38).

Alternatively, SM quantitation was performed by labeling cells to isotopic equilibrium with 0.5 µCi/ml of [methyl-3H]choline (specific activity 81.0 Ci/mmol, DuPont-NEN, Les Ulis, France) for 48 h in complete medium, as previously described (26, 27). Cells were then washed and resuspended in serum-free medium for kinetic experiments. Aliquots were taken for protein determination (39). Cells were treated with or without SMase, and plasma membranes were purified as described above. Radioactive SM was extracted (27, 40, 41) and quantified by scintillation counting.

In both cases, plasma membrane inner leaflet SM content was deduced by subtracting the SM content of SMase-treated plasma membranes from the total plasma membrane SM content (16).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PSC833 and TNF-{alpha} on KG1a cell viability
Treated or untreated KG1a cells were seeded at a cellular concentration of 3 x 105/ml. Under these conditions KG1a cells grew exponentially, with a doubling time of approximately 24 h ( Fig. 1). In the continuous presence of 1 µM PSC833 or with daily treatment with 10 ng/ml TNF-{alpha} the growth kinetics were essentially unchanged. However, KG1a cells pretreated for 24 h with 1 µM PSC833 and then challenged every 24 h with 10 ng/ml TNF-{alpha} exhibited a dramatically different response. Indeed, cotreated KG1a cell growth was unaffected during the first 24 h; then cell growth was reduced for the next 24 h, after which a gradual loss of cell viability was observed; no viable cell was detectable after 72 h.



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  		Figure 1. Growth curves for KG1a cells incubated in the absence ({circ}) or presence of 1 µM PSC833({bigtriangleup}), 10 ng/ml TNF-{alpha} ({square}), or preincubated for 24 h with 1 µM PSC833, followed by cotreatment with 1 µM PSC833 and 10 ng/ml TNF-{alpha} ({bullet}). Cells cultured in 1% FCS supplemented with 2% Ultroser HY were enumerated each 24 h, using trypan blue. Points are mean of triplicate determination, SE < 10% (P<0.01).

Characterization of PSC833/TNF-{alpha}-induced cell death
Seventy-two hours after cotreatment with 1 µM PSC833 and 10 ng/ml TNF-{alpha}, DAPI staining revealed morphological changes characteristic of apoptosis in KG1a cells, with reduction of cell volume and chromatin fragmentation ( Fig. 2). Spectrofluorometric measurement showed a maximum of 31% DNA fragmentation at 72 h (data not shown).



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  		Figure 2. TNF-{alpha}-triggered apoptosis. U937 (A, B) and KG1a (C–F) cells were incubated in the absence (A–D) or presence of 1 µM PSC833 (E, F) with (B, D, F) or without 10 ng/ml TNF-{alpha} (A, C, E) for 72 h. Cells cultured in 1% FCS supplemented with 2% Ultroser HY were stained with 0.1 µg/ml DAPI for 1 h and viewed at x100.

Conventional DNA agarose gel electrophoresis confirmed that PSC/TNF-{alpha} induced oligonucleosomal DNA fragmentation in KG1a cells at 72 h ( Fig. 3A). In contrast, no DNA laddering was observed in KG1a cells treated with PSC833 or TNF-{alpha} alone. Since it has been reported that internucleosomal DNA fragmentation can be absent during apoptosis, leaving only large DNA fragments (42), we also investigated DNA macrofragmentation by using pulsed-field gel electrophoresis. These experiments showed large DNA fragments of between 600 and 50 kbp, which appeared as soon as 48 h in PSC833/TNF-{alpha}-treated cells ( Fig. 3B), but not in cells treated with PSC833 or TNF-{alpha} alone (data not shown).




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  		Figure 3. Apoptosis-associated DNA fragmentation in TNF-{alpha}-treated KG1a cells. KG1a cells incubated in 1% FCS supplemented with 2% Ultroser HY in the absence or presence of 1 µM PSC833, 10 ng/ml TNF-{alpha}, or preincubated for 24 h with 1 µM PSC833 followed by cotreatment with 1 µM PSC833 and 10 ng/ml TNF-{alpha}. The formation of oligonucleosomal fragments DNA macrofragmentation was determined as described in the Materials and methods section. A) DNA laddering in KG1a cells treated in the absence (lanes 1 and 3) or in the presence of PSC833 (lanes 2 and 4–6) and TNF-{alpha} (lanes 3–6) for 24 (lane 4), 48 (lane 5) and 72 (lanes 1–3 and 6) h. B) Kinetics of DNA macrofragmentation in KG1A cells treated with TNF-{alpha} in the absence or presence of PSC833 for 96 h. Results are representative of three independent experiments.

Analysis of ceramide generation
Since we and others (8, 16) have previously described that in TNF-{alpha} sensitive myeloid leukemic cells such as U937, apoptosis was triggered via CER generation, we investigated whether CER was generated in KG1a cells treated with both PSC833 and TNF-{alpha}. To ascertain whether the combination of PSC833 and TNF-{alpha} could activate the SM cycle in KG1a cells, cells were prelabeled with [3H]palmitic acid to equilibrium for 48 h. KG1a cells were then pretreated for 24 h with 1 µM PSC833 and then challenged every 24 h with 10 ng/ml TNF-{alpha}. CER production was monitored at 48 h, 10 min after the addition of TNF-{alpha} (corresponding to the peak CER generation in TNF-{alpha} sensitive U937 cells) (16). As shown in Fig. 4A, we found a significant burst in intracellular CER concentrations (>300%) at 48 h of cotreatment. No CER production was detected before this time (data not shown) or in cells treated with PSC833 or TNF-{alpha} alone.



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  		Figure 4. Effect of TNF-{alpha} and PSC 833 on ceramide levels and SMase activity in KG1a cells. KG1a cells (which were prelabeled with [9, 10-3H]palmitic acid for ceramide analysis) were incubated for 48 h in 1% FCS supplemented with 2% Ultroser HY in the absence or presence of 1 µM PSC833, 10 ng/ml TNF-{alpha}, or preincubated for 24 h with 1 µM PSC833, followed by cotreatment with 1 µM PSC833 and 10 ng/ml TNF-{alpha}. After the last stimulation with TNF-{alpha} for 10 min, aliquots were collected. Ceramide (A) and SMase assays (B) were performed as described in Materials and methods. Results are the mean of triplicate determinations and are representative of two to three independent experiments; they are expressed as a percentage of untreated cells. Basal acid and neutral SMase activities were 315 and 107 pmol/(h/mg), respectively.

Effect of PSC833 and TNF-{alpha} on SMase activity in KG1a cells
To identify the origin of CER generation, we measured neutral and acidic SMase activities. KG1a cells were then pretreated for 24 h with 1 µM PSC833 and challenged every 24 h with 10 ng/ml TNF-{alpha}. SMase activities were monitored 10 min after the addition of TNF-{alpha} (corresponding to the peak neutral SMase activation in TNF-{alpha}-sensitive U937 cells) (16). As shown in Fig. 4B, only neutral SMase was significantly activated at 48 h in KG1a cells cotreated with PSC833 and TNF-{alpha} (>140%). PSC833 and TNF-{alpha} alone had no detectable effect (data not shown).

Transverse distribution of SM in KG1a cells
The lack of apoptotic response of KG1a cells to TNF-{alpha} and the ability of exogenous ceramide to restore apoptosis in these cells (16) suggest that the SM-CER pathway is altered in KG1a cells upstream of the CER generation. Since we demonstrated that the inner leaflet SM pool in KG1a cells was relatively low compared to that of TNF-{alpha} sensitive cells such as U937 (16), we elected to measure the asymmetrical SM distribution in PSC833-treated KG1a cells. The total mass of cellular SM was 26.1 ± 3.6 nmol/mg protein of KG1a cells. Treatment by PSC833 alone did not significantly change this amount (data not shown). Moreover, no significant changes in SM asymmetry, with or without PSC833, were observed at 24 or 48 h (data not shown). However, whereas untreated KG1a cells presented only about 17% of the plasma membrane SM on the inner leaflet, PSC833 cells presented more than 54% at 72 h ( Fig. 5).



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  		Figure 5. Transverse distribution of SM in plasma membrane of KG1a cells. KG1a cells were incubated in 1% FCS supplemented with 2% Ultroser HY in the absence (left) or presence (right) of 1 µM PSC833 for 72 h. After treatment, aliquots were collected and SM measurements were performed as described in Materials and methods. Data are the mean of duplicate determinations and are representative of four independent experiments. Total outer membrane SM content in untreated and PSC833-treated KG1a cells was 9.6 ± 0.78 and 4.9 ± 1.66 nmol/mg, respectively. Insert) Basal membrane-bound neutral SMase activity in KG1a cells treated in the absence ({square}) or presence ({blacksquare}) of PSC833 for 72 h.

Basal membrane-bound neutral SMase activity in KG1a cells
Since PSC833 treatment of KG1a cells led to both an increase in inner plasma membrane leaflet SM content and activation by TNF-{alpha} of neutral SMase, we speculated that these two events were intimately related. Therefore, we measured basal neutral SMase activity in membrane preparations of KG1a cells with and without 1 µM PSC833. As shown in Fig. 5 (insert), PSC833 treatment of KG1a cells increases the basal membrane-bound neutral SMase activity by more than threefold (from 24.3±1.6 to 83.0±18.0 pmol·h-1·mg-1) at 72 h concurrently with the increased inner plasma membrane leaflet SM content. No significant changes in basal membrane-bound neutral SMase activity, with or without PSC833, were observed at 24 or 48 h (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study confirms that KG1a cells are highly resistant to TNF-{alpha}, as previously reported, although these cells express both type I (p55) and type II (p75) TNF-{alpha} receptors (43, 44). In a recent report we have shown that this resistance may be explained by the inability of TNF-{alpha} to induce apoptosis (16). Indeed, even at doses as high as 250 ng/ml and for incubation times as long as 96 h, TNF-{alpha} alone induced no apoptosis in KG1a cells, whereas much lower doses resulted in rapid apoptotic cell death in other myeloid leukemia cells, such as U937 or HL-60 cells. We and others (45, 46) have recently reported that KG1a cells are also resistant to various anti-cancer agents, including chemotherapeutic drugs and ionizing radiation, whereas these antitumor agents induced apoptosis in the TNF-{alpha}-sensitive U937 and HL-60 cells. These observations suggest that KG1a cells are naturally resistant to various anticancer agents that share a common apoptotic signaling pathway. Recently, it has been suggested that TNF-{alpha} (8), certain chemotherapeutic drugs (26, 47), and ionizing radiation (48, 49) may induce apoptosis through ceramide by stimulating SM hydrolysis. Therefore, notwithstanding the role of diverse signaling proteins such as FADD/MORT1, TRADD, and IAP, which may play important roles in the sensitivity to TNF-{alpha} (50–52), we hypothesized that, in KG1a cells, TNF-{alpha} was unable to trigger CER production. In fact, we have recently reported that TNF-{alpha} triggered intracellular SM hydrolysis and intracellular CER generation in TNF-{alpha}-sensitive U937 cells, but not in TNF-{alpha}-resistant KG1a cells, whereas these two cell lines were equally sensitive to synthetic C2- or C6- cell-permeant CER as well as to natural CER generated by bacterial SMase (16). These results strongly suggested that the lack of apoptotic response to TNF-{alpha} by KG1a cells was most likely linked to a lack of TNF-{alpha}-induced CER generation.

At least two hypotheses can be put forward to explain the lack of CER generation in TNF-{alpha}-treated KG1a cells. First, the lack of CER generation in KG1a cells could be due to the inability of TNF-{alpha} to stimulate SMase (53). Alternatively, a decreased substrate (SM) availability may account for the absence of CER generation in TNF-{alpha}-treated KG1a cells. It has been shown that the SM pool used for TNF-{alpha} signaling is located predominantly in the inner leaflet of the plasma membrane (14, 15), and reduced CER generation could be due to a decreased level of inner leaflet-associated SM pool. In fact, we found that the size of this pool was sevenfold lower in KG1a cells than in U937 cells (16).

To test the hypothesis whereby altered plasma membrane SM distribution plays a role in the natural resistance of KG1a cells to TNF-{alpha}, we evaluated the effects of pharmacological agents identified as modulators of lipid transport across the membrane. In two recent studies, van Meer's group (17, 18) demonstrated that outer translocation of a variety of short-chain glycerophospholipids and sphingolipids, including SM, was sensitive to P-gp inhibitors, suggesting a lipid flippase action of the MDR1 P-gp (54). In fact, phosphatidylcholine translocation mediated by other members of the MDR familly, human MDR3 and mouse mdr2, has previously been demonstrated (55, 56). Although whether MDR1 P-gp can translocate endogenous long-chain membrane lipids such as natural SM is still debated, we hypothesized that inhibition of P-gp in KG1a (which is highly active in these cells) (19) may interfere with SM transport and lead to SM enrichment of the plasma membrane inner leaflet. To test this hypothesis, we used PSC833, a potent and specific inhibitor of P-gp function (20).

This study shows that PSC833 dramatically sensitized KG1a cells to TNF-{alpha}. Indeed, whereas daily TNF-{alpha} exposure had no effect on KG1a cell growth over a 72 h period, cotreatment with TNF-{alpha} and PSC833 induced rapid loss of cell viability after 48 h. Characterization of the cell death process of KG1a cells cotreated with PSC833 and TNF-{alpha} showed that they died of apoptosis. Furthermore, monitoring of CER content and neutral SMase activity performed a few minutes after each TNF-{alpha} challenge showed that pretreatment with PSC833 restored the ability of TNF-{alpha} in generating CER and stimulating SMase activity.

The present work shows that prolonged exposure of PSC833 increased plasma membrane inner leaflet-associated SM content. The fact that total SM content was unmodified suggests that the equilibrium between SM synthesis and SM degradation was not influenced by PSC833. The effect of PSC833 on inner leaflet SM content may support the hypothesis for a function of P-gp as an SM translocase in KG1a cells. To test this hypothesis, we evaluated a series of P-glycoprotein inhibitors (30 µM verapamil, 100 nM perhexiline, 10 µM tamoxifen, 1 µM chlorpromazine, 5 µM cyclosporine A, and 1 µM PSC 833) (57). Of all these agents, only the most potent and specific inhibitor of P-gp function, PSC833 (20), had any effect on sensitizing KG1a cells to TNF-{alpha} cytotoxicity or on SM translocation (data not shown). Furthermore, whereas PSC833 concentrations could not be pushed above 1 µM (2 µM PSC833 induced a 10–20% toxicity level at at 72 h), the lower dose of 0.5 µM, which only modestly inhibits P-glycoprotein function (20), had no significant effect on TNF-triggered cell death or SM translocation (data not shown). Nevertheless, we cannot totally rule out that PSC833 may, independently of P-glycoprotein, have interfered with an intracellular SM transport mechanism. Indeed, there is some evidence for a vesicular anterograde transport of SM from the Golgi (where SM is synthesized) to the plasma membrane (15, 18).

Our study suggests that the modifications of membrane lipid composition induced by PSC833 have two important consequences for CER generation and, therefore, apoptosis: 1) increase of SM hydrolyzable pool; and 2) restoration of TNF-{alpha}-induced SMase stimulation. Indeed, whereas KG1a cells present significant basal SMase activities, TNF-{alpha} alone did not induce SMase stimulation (16). However, a combined treatment of KG1a cells by PSC833 and TNF-{alpha} resulted in SMase activation. The identification of the SMase (or SMases) involved in CER generation has proved to be equivocal. Three forms of SMases are candidates for SM hydrolysis. Acid SMase (pH optimum 4.5–5.0) has been associated in apoptosis signaling triggered by TNF-{alpha}, CD28, and Fas; activation of a membrane-bound neutral SMase (pH optimum 7.4) has also been described with TNF-{alpha}, anti-Fas antibody, IL-1ß, and daunorubicin. Finally, a cytosolic neutral SMase has been implicated in vitamin D3 induced differentiation (9). Our study designated neutral, but not acid SMase, as being responsible for CER generation in PSC833/TNF-{alpha}-treated KG1a cells.

PSC833 treatment of KG1a cells leads to two apparently distinct effects: increased inner plasma membrane leaflet SM content and activation by TNF-{alpha} of neutral SMase. We demonstrate that PSC833 treatment of KG1a cells increased basal membrane-bound neutral SMase activity by more than threefold concurrently with increased inner plasma membrane leaflet SM content. From these observations, we speculate that translocation of neutral SMase to the inner plasma membrane leaflet is SM dependent, and we propose the following cascade of events: PSC833 blocks P-glycoprotein-mediated SM translocation, leading to increased SM content within the inner plasma membrane leaflet. Neutral SMase is translocated to this SM enriched membrane, and TNF-{alpha} is now able to stimulate the membrane-bound neutral SMase activity. This hypothesis is being further investigated in our laboratory.

In conclusion, this study demonstrates that the natural resistance of KG1a cells to TNF-{alpha} can be reversed by a pharmacological agent, PSC833. This phenomenon appears to be the consequence of the restoration of an apoptotic response, which in turn may be linked to an increased hydrolyzable/signaling SM pool and SMase stimulation. KG1a cells are a model of natural resistance to various anti-cancer agents, including chemotherapeutic drugs and ionizing radiation, which makes this cell line an interesting model of multitoxicant-resistant leukemia cells (45). Indeed, CD-34 positive myeloid leukemia cells express functional P-glycoprotein (19) that protect cells not only against xenobiotic substances, but also against endogenous effectors such as complement-mediated autolysis (58). It would be of particular interest to investigate whether the PSC833-mediated redistribution of SM would allow other agonists of the SM-CER signaling pathway to induce an apoptotic response in KG1a cells. These studies are ongoing in our laboratory.


   ACKNOWLEDGMENTS
 
Our gratitude is extended to Dr. Berthand (Novartis, Pharma SA, France) for generously providing us with PSC833, and to Drs. M. Record and G. Ribbes (INSERM U 326, Toulouse) for their critical comments and A. Moisand (CNRS UPR 9062, Toulouse) and S. Vermeersch (INSERM U 466) for expert technical assistance. We also thank Drs. H. Chap (INSERM U 326, Toulouse), A. Quillet-Mary, and D. Lautier, as well as V. Mansat and C. Bordier (CJF INSERM 9503, Toulouse), for helpful discussions. This work was supported by a grant from the Actions Concertées Coordonnées-Sciences du Vivant ACCSV8:9508008 (G.L.) and by La Fédération Nationale des Centres de Lutte Contre le Cancer (J.P.J. and T.L.), and in part by the Conseil Régional Midi-Pyrénées (T.L. and J.P.J.), l'Association pour la Recherche sur le Cancer Grants 6749 (G.L.), 3002 (T.L.), and 2069 (J.P.J.), and La Ligue Nationale Contre le Cancer (G. L.). C.B. is the recipient of a Ligue Nationale Contre le Cancer fellowship.


   FOOTNOTES
 
1 Correspondence: CJF INSERM 9503, Centre Claudius Régaud, 20 rue du Pont St. Pierre, Toulouse Cédex 31052 France. E-mail: jaffrezou{at}regaud-tlse.fnclcc.fr

2 Abbreviations: TNF-{alpha}, tumor necrosis factor {alpha}; SM, sphingomyelin; SMase, sphingomyelinase; CER, ceramide; DAPI, 4', 6'-diamino 2-phenylindole; EDTA, ethylene diaminetetraacetic acid; P-gp, P-glycoprotein.

Received for publication July 1, 1997. Accepted for publication October 1, 1997.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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