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Oxidative stress-induced activation of Lyn recruits sphingomyelinase and is requisite for its stimulation by Ara-C¹

CHRISTINE BEZOMBES^*, ISABELLE PLO^*, VÉRONIQUE MANSAT-DE MAS, ANNE QUILLET-MARY^*, ANNE NÈGRE-SALVAYRE, GUY LAURENT^*, and JEAN-PIERRE JAFFRÉZOU^*2

^* INSERM E9910, Institut Claudius Réaud, Toulouse 31052;
Service d’Hématologie, Centre Hospitalier Universitaire Purpan, Toulouse 31059; and
Laboratoire de Biochimie Médicale, INSERM 466, Centre Hospitalier Universitaire Rangueil, Toulouse 31403, France

²Correspondence: INSERM E9910, Institut Claudius Régaud, 20 rue du Pont St Pierre, Toulouse 31052, France. E-mail: jaffrezou{at}icr.fnclcc.fr

SPECIFIC AIMS

Many studies have demonstrated that 1-ß-D-arabinofuranosylcytosine (Ara-C) triggers a wide spectrum of intracellular signals, which may contribute downstream of drug-DNA incorporation to modulate Ara-C cytotoxicity. Therefore, in this study we sought to determine the respective roles of reactive oxygen species (ROS) and p53/p56 Lyn kinase in the regulation of sphingomyelinase (SMase) activation in Ara-C-triggered JNK activation and apoptosis in leukemic myeloid cells.

PRINCIPAL FINDINGS

U937 and HL-60 cells were treated in kinetic experiments with 40 µM Ara-C and analyzed for cell viability and DNA fragmentation using the [³H]thymidine release assay, poly (ADP-ribose) polymerase (PARP) cleavage, and DAPI staining. Ara-C induced a significant loss of cell viability with DNA fragmentation of 9.7% as soon as 6 h and 45.9% at 24 h. We also observed typical morphological features of apoptosis by DAPI staining and PARP cleavage. The enumeration of morphologically apoptotic cells was estimated at 31% ± 5% and 62% ± 7% at 6 and 24 h, respectively. Finally, the nucleosides cytidine or deoxycytidine were noncytotoxic at 40 µM. Similar results were observed in HL-60 cells.

Since it has been described that anticancer drugs can induce apoptosis through the sphingomyelin (SM)-ceramide pathway, we measured SMase activity and ceramide generation in Ara-C-treated cells. Whereas cytidine and deoxycytidine had no effect, 40 µM Ara-C induced an increase in the neutral SMase activity with a peak at 20 min. This enzyme stimulation was accompanied by a concomitant generation of ceramide. Acid SMase remained unaffected throughout.

To investigate the effect of Ara-C on src-like kinases, we measured the activity of those expressed in myeloid cells, p55 Hck, and p53/p56 Lyn. After Ara-C treatment, we observed an increase in p53/p56 Lyn activity that peaked at about the 9th min (Fig. 1A ). p53/p56 Lyn activation was totally blocked by preincubation of cells with a potent tyrosine kinase inhibitor, herbimycin A. No change in p55 Hck activity was observed in Ara-C-treated cells.

View larger version (24K): [in this window] [in a new window]   Figure 1. ROS dependent activation of p53/p56 Lyn by Ara-C. a) Time course analysis of kinase activity in U937 cells treated with 40 µM Ara-C with or without preincubation with 0.5 µg/ml herbimycin A for 4 h. b) Flow cytometry analysis of H2O2 production in Ara-C-treated cells. Insert: H2O2 production measured at 5 min in H2O2-treated cells. C) The effect of antioxidants on p53/p56 Lyn activation was measured in cells preincubated with 25 mM N-Ac for 2 h or 100 µM PDTC for 30 min and then treated for 10 min by 40 µM Ara-C. 20 min incubation with 1 mM H2O2 was used as a positive control. Data (mean±SD) were derived from four (A, B) or five (c) independent experiments. *P < 0.05. **P < 0.01 compared with controls (Student’s t test).

In light of increasing evidence for a role of ROS in tyrosine kinase activation, we first measured the fluorescence distribution of the C2938 dye, revealing the presence of hydrogen peroxide, by flow cytometry in the viable cell population. We observed an increase in the mean C2938 fluorescence with a peak at 5 min in 40 µM Ara-C-treated cells relative to untreated cells (Fig. 1B ). The level of H₂O₂ generation induced by Ara-C was intermediate to that observed in cells treated by 8 and 16 µM H₂O₂. Ara-C-induced H₂O₂ generation was totally inhibited by preincubation of cells with the antioxidant pyrrolidine dithiocarbamate (PDTC), and 40 µM cytidine presented no significant H₂O₂ production (data not shown).

To further investigate the role of ROS in p53/p56 Lyn activation induced by Ara-C, we evaluated the effect of two antioxidants N-acetylcysteine (N-Ac) and PDTC. As shown in Fig. 1C , both N-Ac and PDTC totally inhibited Ara-C-induced p53/p56 Lyn activation, whereas treatment of cells with H₂O₂ alone induced an increase in p53/p56 Lyn activity.

To define the cascade of events induced by Ara-C, we evaluated the effect of the antioxidants N-Ac and PDTC as well as herbimycin A on neutral SMase activation induced by Ara-C or H₂O₂. In the absence of antioxidant, SMase activity increased in the presence of Ara-C from a basal activity of 10.8 ± 0.5 pmol/mg/h to 16.9 ± 0.22 pmol/mg/h. Preincubation of cells with either N-Ac and PDTC blocked the Ara-C induced elevation in SMase activity. Herbimycin A similarly inhibited SMase stimulation induced by Ara-C. Finally, SMase activation induced by H₂O₂ was completely blocked by herbimycin A, suggesting that p53/p56 Lyn is acting upstream of SMase and downstream of ROS production. Basal SMase activity was not effected by herbimycin A.

To precisely identify p53/p56 Lyn as the tyrosine kinase responsible for neutral SMase activation, we incubated U937 cells with a p53/p56 Lyn antisense oligonucleotide. Whereas Ara-C stimulated SMase activation in cells incubated with a random oligonucleotide, Ara-C had no effect on cells treated with p53/p56 Lyn antisense. The inhibition of p53/p56 Lyn translation by antisense treatment was confirmed by Western analysis.

To investigate the interaction between p53/p56 Lyn and neutral SMase, we immunoprecipitated p53/p56 Lyn and then measured SMase activation under Ara-C and H₂O₂ treatment. As shown in Fig. 2 , after Ara-C or H₂O₂ treatment, a significant increase in SMase activity was observed in the immunoprecipitates. To provide evidence for specificity, we preincubated U937 cells with herbimycin A, which blocked the Ara-C-triggered increase in p53/p56 associated SMase activity. Finally, we failed to observe SMase activity in p55 Hck immunoprecipitates. Western analysis of basal Lyn and Hck expression revealed that both src kinases are equivalently present in U937 cells (Fig. 2 , insert). These results suggest that Ara-C and H₂O₂ induced SMase stimulation by specifically activating p53/p56 Lyn, leading to its interaction with the SMase and consequently to its activation.

View larger version (32K): [in this window] [in a new window]   Figure 2. Interaction between p53/p56 Lyn and SMase. SMase activity was measured in control (assay performed in the absence of cell lysate) and in immunoprecipitates of p53/p56 Lyn or p55 Hck of U937 cells pretreated or not with 0.5 µg/ml herbimycin A for 4 h and then incubated with or without 40 µM Ara-C or 1 mM H2O2 for 10 min. Data (mean±SD) were derived from three independent experiments. *P < 0.01 compared with controls (Student’s t test). Inset: Western analysis of basal Lyn and Hck expression in U937 cells.

We next evaluated the effects of Ara-C (40 µM) on a ceramide target: JNK. We observed that Ara-C essentially stimulated JNK2 phosphorylation (and JNK1, but not significantly) as early as 10 min and reached a plateau at 20 min. To determine whether the inhibition of Ara-C-induced, p53/p56 Lyn-mediated sphingomyelinase stimulation and ceramide generation might influence JNK activation, we measured JNK phosphorylation in Ara-C-treated cells in the presence of herbimycin A and p53/p56 Lyn antisense. We observed that both herbimycin A and p53/p56 Lyn antisense completely abolished JNK2 phosphorylation. Furthermore, treatment with p53/p56 Lyn antisense significantly protected U937 cells from Ara-C-induced apoptosis.

CONCLUSION AND SIGNIFICANCE

Induction of apoptosis is considered to be the underlying mechanism that accounts for the efficiency of chemotherapeutic drugs. The identification of ceramide as a key mediator of apoptosis has led to the prospect of identifying cellular entities that regulate drug-induced sphingomyelinase activation. Using human leukemia cell lines, we demonstrate that Ara-C stimulated ceramide generation, which was dependent on ROS. Coimmunoprecipitation studies revealed that Ara-C-generated ROS activated the src kinase p53/p56 Lyn, which then recruited neutral sphingomyelinase activity. These effects were blocked by herbimycin A and Lyn antisense oligonucleotides. Hence, this study establishes a cascade of events in which early ROS generated by Ara-C leads to Lyn-dependent neutral sphingomyelinase activation, JNK phosphorylation, and apoptosis. These findings may have important clinical implications, since it is now conceivable that enhanced oxidative defenses may decrease Ara-C cytotoxicity. One could perhaps increase chemosensitivity of neoplastic cells by pharmacological manipulation.

View larger version (18K): [in this window] [in a new window]   Figure 3. Hypothetical schema of Ara-C-triggered SMase activation. This study establishes a cascade of events in which early ROS generated by Ara-C leads to p53/p56 Lyn activation, which then interacts and activates neutral sphingomyelinase, thereby generating ceramide, which is responsible for JNK phosphorylation and apoptosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0787fje ; to cite this article, use FASEB J. (May 9, 2001) 10.1096/fj.00-0787fje

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