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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 21, 2002 as doi:10.1096/fj.01-0794fje. |
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* INSERM E9910, Institut Claudius Régaud, 31052 Toulouse, France;
Laboratoire de Biochimie Médicale, INSERM U466, CHU Rangueil, 31403 Toulouse, France; and
Service d’Hématologie, CHU Purpan, 31059 Toulouse, France
3Correspondence: INSERM E9910, Institut Claudius Régaud, 20 rue du Pont St. Pierre, 31052 Toulouse France. E-mail: jaffrezou{at}icr.fnclcc.fr
SPECIFIC AIMS
In the present study, we show that 1-ß-D-arabinofuranosylcytosine (Ara-C) and daunorubicin (DNR) rapidly induce activation of Lyn and its translocation into membrane rafts, leading to neutral sphingomyelinase activation and raft-associated sphingomyelin hydrolysis with concomitant generation of the proapoptotic lipid second messenger ceramide.
PRINCIPAL FINDINGS
1. Ara-C- and DNR-induced activation of SMase in rafts
To examine whether neutral SMase was activated by Ara-C and DNR in U937 rafts, cells were treated with 40 µM Ara-C for 15 min or with 1 µM DNR for 10 min (corresponding to peak Ara-C- and DNR-triggered SMase stimulation, respectively), then lysed in cold Triton X-100 and fractionated on a sucrose density gradient. Neutral and acid SMase were then analyzed in the low- and high-density part of the gradients (fractions 1, 2, and 10–12) and in the SM- and CD36-enriched rafts (fractions 4–6). We observed basal neutral and acid SMase activity throughout the gradient, with significant neutral SMase enrichment in the raft microdomains (79±2 pmol/h·mg fraction protein) compared to low- (16.4±1.8 pmol/h·mg fraction protein) and high-density (2.8±0.9 pmol/h·mg fraction protein) parts of the gradient. Only in rafts did we measure significant neutral SMase activation (>35%) under Ara-C treatment. No significant increase in acid SMase activity was observed under Ara-C treatment. Similar results were observed with Ara-C and DNR using a nondetergent extraction protocol. By prelabeling U937 cells with [9, 10-3H]palmitic acid to equilibrium for 48 h before Ara-C or DNR treatment and cell fractionation, we were able to observe that only in the SM-rich raft domains was there significant CER generation (Fig. 1
A, B) and SM hydrolysis (Fig. 1A
, insert). These results also show that in untreated U937 cells there was a significant basal concentration of CER in rafts, which was further increased by Ara-C and DNR treatment.
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2. Ara-C- and DNR-induced Lyn translocation into rafts
We have previously shown that the SMase/CER pathway triggered by Ara-C in U937 cells is dependent on activation and binding of the Src kinase Lyn to SMase. Since Ara-C induced SMase activation in rafts, it was essential to show that this correlated with Lyn activation within these same microdomains. As shown in Fig. 2
A, inuntreated U937 cells Lyn was found essentially in the high-density fractions, with a small constitutive amount observed in the Triton insoluble fraction. Treatment of U937 cells with 40 µM Ara-C or 1 µM DNR induced a rapid (within 10–15 min) translocation of Lyn from the high-density fractions to the raft domains. Similar results were observed using the murine pro-B lymphoid cell line Ba/F3 (data not shown). Pretreatment of U937 cells with the tyrosine kinase inhibitor herbimycin (which has been shown to completely block drug-induced Lyn activation, SMase stimulation, and apoptosis) not only inhibited Lyn translocation into rafts, but displaced the basal Lyn levels. This suggested that tyrosine phosphorylation of Lyn was necessary for its compartmentalization (and therefore its interaction with SMase) into rafts. Indeed, Ara-C treatment led to a significant increase in tyrosine phosphorylation events in rafts that was significantly blocked by herbimycin (Fig. 2B
); as expected, Ara-C SMase stimulation was inhibited by herbimycin (Fig. 2C
). Since we had demonstrated that Lyn directly interacts with neutral SMase, we evaluated whether activated Lyn could stimulate raft-associated neutral SMase activity in an in vitro assay. We observed that raft-associated neutral SMase activity was significantly increased when measured in the presence of activated Lyn (increasing from 89±2 to 130±12 pmol/h·mg protein).
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CONCLUSION AND SIGNIFICANCE
Two of the most effective agents used to treat acute myelogenous leukemia are Ara-C and DNR. However, it is unclear how drug-induced DNA damage relates to programmed cell death signaling. Many studies have demonstrated that Ara-C and DNR both trigger a wide spectrum of intracellular signals that may contribute, downstream of drug–DNA interaction, to modulate cytotoxicity. We previously demonstrated that Ara-C at 40 µM and DNR at 1 µM (clinically relevant doses) induced in the U937 myeloid cell line, after 6 h of treatment, a loss of cell viability with characteristic apoptotic features (DNA fragmentation and PARP cleavage). Under these conditions, Ara-C and DNR induced rapid activation of a neutral SMase within 10 to 15 min with concomitant CER generation. Finally, we demonstrated that p53/p56 Lyn activation required for apoptosis signaling by Ara-C and DNR was requisite for SMase stimulation.
Since Src kinases are generally associated with the inner surface of the plasma membrane, we decided to investigate the possible implication of rafts in Ara-C and DNR signaling. In this study, we show that U937 plasma membrane rafts are highly enriched, as expected in SM, but also in neutral SMase activity. Treatment of U937 cells with Ara-C or DNR induced within 10–15 min significant stimulation of SMase activity with concomitant CER generation only in the raft fractions.
It is clearly established that in most cells SM is found essentialy within the outer leaflet of the plasma membrane. Present knowledge strongly suggests that under stress-induced apoptosis signaling, a pool of SM becomes accessible to a cytosolic SMase (thereby generating CER), possibly by flipping to the inner leaflet due to phospholipid scrambling. Moreover, the absence of such a hydrolyzable SM pool within the plasma membrane inner leaflet has been associated with resistance to apoptosis. It therefore appears that SMase activation is substrate dependent and that the spacial SM organization within the plasma membrane may be essential for the initiation of a SMase-dependent apoptotic pathway. These findings may have important implications, since it is now conceivable that the lack of apoptosis signaling in drug-resistant cells is perhaps linked to the absence and/or constitution of rafts. Further investigations are necessary to determine to what extent the compartmentalization of these signaling processes is modified/absent in drug-resistant cells.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0794fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.01-0794fje 
2 S.G. and N.M. contributed equally to this work.
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) or treated (
) with 40 µM Ara-C for 15 min. Cells were then lysed in cold Triton X-100 and fractionated on a sucrose density gradient. Fractions were analyzed for ceramide content (expressed as % of total ceramide). Insert: SM levels in control (•) and Ara-C treated (
) cells. B) U937 cells labeled to isotopic equilibrium with [9, 10-3H]palmitic acid were either untreated (open bar) or treated with 40 µM Ara-C for 15 min (gray bar) or with 1 µM DNR for 10 min (filled bar). Cells were lysed in cold sodium carbonate buffer and fractionated on a sucrose density gradient. In each case, raft fractions were collected and analyzed for CER content. Results are mean of triplicate determinations of a representative experiment (1 of 3 independent experiments) ± SE. *P < 0.01.
