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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0716fje. |
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INSERM U526 Activation des Cellules Hematopoietiques, Physiopathologie de la Survie et de la Mort Cellulaires et Infections Virales, Equipe Labellisée Ligue Nationale contre le Cancer, IFR50, Faculté de Médecine, 06107 Nice-Cédex 2, France; and
* Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California, USA
2Correspondence: INSERM U526 Activation des Cellules Hematopoietiques, Physiopathologie de la Survie et de la Mort Cellulaires et Infections Virales. Equipe Labellisée Ligue Nationale contre le Cancer, IFR50, Faculté de Médecine, Ave. de Valombrose, 06107 Nice-Cédex 2, France. E-mail: auberger{at}unice.fr
SPECIFIC AIMS
Engagement of the B cell receptor antigen (BCR) triggers apoptosis on immature B cell lines. We previously found that cross-linking of death receptors on T and B lymphocytes induces caspase-dependent cleavage and relocation of the Src kinase family members Fyn and Lyn. The present study was thus designated to elucidate the potential role of Lyn cleavage in the course of BCR-induced cell death.
PRINCIPAL FINDINGS
1. BCR ligation on Ramos cells leads to procaspase 9 and 7 activation and to Lyn cleavage
Fifteen hours after stimulation with anti-IgM, p56/53 Lyn was cleaved, generating a characteristic 51 kDa fragment. Lyn cleavage was maximal after 36 h of anti-IgM treatment and paralleled that of PARP. The pan-caspase inhibitor Z-VAD-fmk was found to inhibit BCR-mediated Lyn proteolysis, implicating caspases in Lyn cleavage. Accordingly, 24 h after stimulation we also observed a significant cleavage of both procaspase 9 and 7. Together, these results strongly suggest that antigen receptor-mediated caspase activation, Lyn cleavage, and apoptosis are mitochondria dependent in Ramos B cells.
2. The soluble form of Lyn generated by caspases during BCR-induced apoptosis is a negative regulator of B lymphocyte death
To investigate the potential role of Lyn cleavage and relocation during the B cell death process, we generated by deletion of the 18 amino-terminal amino acids of the p56Lyn cDNA, stable transfectants overexpressing the soluble form of Lyn (p54 Lyn-
-N). Compared with cells transfected with p56 Lyn (Lyn-WT), where endogenous p53 and p56 Lyn were present, the Lyn--N clone exhibited high amount of p54Lyn (Fig. 1
A). Stimulation of Ramos Lyn-WT and Lyn--N clones with anti-IgM for 36 h resulted in loss of 
m in 53.5% and 10.6% of each B cell clones, respectively (Fig. 1B
). Apoptosis was detected after 24 h of anti-IgM stimulation in 36% of Lyn-WT cells and was maximal after 36 or 48 h (42 to 52% of dead cells), whereas cell death occurred in < 5% of Lyn--N transfected cells after 24 h and in 9 and 24% after 36 or 48 h of stimulation (Fig. 1C
). Impaired B cell receptor-mediated apoptosis in Lyn--N transfectant was confirmed by measurement of overall caspase activity (Fig. 1D
).
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3. Cell signaling during BCR-induced cell death
To address whether the apoptotic defect of Lyn--N clone was reflected by modulation of the main survival pathways, we performed Western blot analysis of phospho p42/44 MAPK, phospho-Akt, and phospho-p90Rsk. Phosphorylation of the three kinases was detected rapidly after anti-IgM addition in both cell clones and was maintained for 6 h or more, depending on the kinases. Moreover, no significant difference in term of kinase phosphorylation was observed in each cell clone. Thus, the poor sensitivity of Lyn--N cell clones to BCR-mediated apoptosis cannot be accounted for by an increased activation of the main survival pathways.
4. c-DNA array analysis of BCR-mediated apoptosis in Lyn-WT and Lyn--N cells
As BCR-induced apoptosis requires gene transcription, we used a cDNA array approach to compare gene modulation in Lyn-WT and Lyn--N expressing cells. We focused on a restricted panel of 200 genes rather than on a larger collection. Globally, cDNA arrays identified 
20 genes that were regulated differently in control and anti-IgM stimulated Ramos cells. Expression of six relevant genes was evaluated semiquantitatively by RT-PCR. Among them, four (Bcl-2, Flip-L, Bik, and Bim) were up-regulated and two (c-Myc and survivin) were down-regulated after anti-IgM treatment. Increased expression of Bcl-2, Bim, and Flip-L and underexpression of c-Myc and survivin were confirmed at the protein level. Although BCR-mediated gene expression was found to be identical for the quasi totality of the regulated gene, c-Myc expression behaved differently in Lyn-WT and Lyn--N clones.
5. c-Myc is involved in BCR-mediated apoptosis
In murine and human B cell lymphoma lines, apoptosis induced by surface IgM cross-linking correlates with down-regulation of c-Myc. c-Myc expression was thus monitored by Western blot in Lyn-WT and Lyn--N cells incubated for various times with anti-IgM. In Lyn-WT cells, c-Myc expression decreased progressively until 7 h to reach a steady-state level after 15 h of stimulation with anti-IgM (Fig. 2
). By contrast, the kinetic of c-Myc expression in Lyn--N-overexpressing cells was biphasic, with a very rapid decrease during the first 7 h and a progressive increase from 15 to 36 h (Fig. 2)
. Indeed, the c-Myc protein level decreased much more rapidly in Lyn--N cells compared with Lyn-WT cells, an effect slowed down by the PI3K inhibitor Ly294002. Ly294002 also maintained high level of c-Myc expression at 36 h, more particularly in Lyn--N cells. Finally, c-Myc levels remained very high after 15 h in Lyn--N cells treated with the combination of Ly294002 and anti-IgM.
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DISCUSSION AND SIGNIFICANCE
The mechanisms by which BCR signals to differentially transduce B cell responses such as proliferation, anergy, and apoptosis are a matter of debate. Here, we show, in a B cell lymphoma, that upon BCR triggering, p56 and p53 Lyn isoforms are cleaved by caspases to generate two products of 54 and 51 kDa. We also observed a preferential activation of procaspases 9 and 7, 24 h after BCR cross-linking. To address the role of Lyn in BCR-induced apoptosis, we generated stable transfectants in the B lymphoma cell line Ramos overexpressing either Lyn-WT or Lyn--N, the cleaved form of Lyn released into the cytoplasm during BCR-induced apoptosis. Careful examination of both transfectants revealed that Lyn--N expressing cells were refractory to BCR-induced apoptosis. Altogether, our data strongly suggest that the cleaved form of Lyn generated after B cell receptor engagement behaves as a negative regulator of cell death.
As differential activation of the main survival pathways compared with Lyn-WT cells is unlikely to explain the resistance of the Lyn--N clone to BCR-mediated apoptosis, the possibility of a transcriptional control was hypothesized. To investigate gene expression events, we used a selected cDNA array approach. Among the 200 genes tested, 10% were modulated, 5 were down-regulated, and 15 were induced on BCR triggering. These genes belong to three main families: proteins involved in the regulation of apoptosis, cell cycle, or gene transcription. We then focused our attention on c-Myc protein regulation because c-Myc gene expression is regulated differently in Lyn-WT and Lyn--N clones. Thus, we performed detailed kinetics of c-Myc expression on anti-IgM treatment. In a first phase (0–7 h), upon BCR engagement c-Myc expression decreased much more rapidly in Lyn--N-overexpressing cells. On the other hand, the level of c-Myc protein then remained low and constant in Lyn-WT cells, but the amount of the protein increased in Lyn--N cells from 15 h. These results suggest that differential modulation of c-Myc expression could be responsible for the sensitivity or refractoriness of both cell clones to BCR-induced apoptosis.
We investigated the effect of blocking the PI3K/Akt pathway on c-Myc expression in both cell clones. We show that maintenance of a high level of c-Myc correlates with BCR-mediated apoptosis whereas a low level of c-Myc is compatible with resistance to apoptosis. Thus, c-Myc protein amount in IgM-stimulated B cell represents a good index of the sensitivity or the refractoriness to apoptosis. Up-regulated c-Myc expression is generally associated with proliferation, but numerous studies have linked either up-regulated or down-regulated c-Myc expression to apoptosis. The importance of c-Myc in regulating the sensitivity of B cells to apoptosis is strengthened by the observation that in 20 genes modulated identically upon anti-IgM treatment of Lyn-WT and Lyn--N cells, only c-Myc exhibited a differential expression in both clones. The mechanisms by which Lyn--N may interfere with c-Myc expression remain to be established. However, our data indicate that BCR triggering through P42/44 MAPK and PI3K activation induces modulation of gene expression, more particularly Bcl-2 homologs and c-Myc, thus favoring mitochondria-dependent caspase activation and cell death (Fig. 3
). Once activated, caspases cleave Lyn, a mechanism that orchestrates its relocation from the membrane into the cytoplasm, where Lyn--N may act on c-Myc or c-Myc-interacting proteins to block the apoptotic signal (Fig. 3)
. Taken together, our data show that the p54 cleaved form of the tyrosine kinase Lyn generated by caspases during BCR-induced cell death in B lymphoma acts as a negative regulator of apoptosis, most likely by modulating c-Myc expression.
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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.02-0716fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0716fje 
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-N in Ramos cells. Cells were lysed and proteins separated by electrophoresis on 11% polyacrylamide gels. Proteins were then blotted onto PVDF membranes, which were incubated with an anti-Lyn antibody. Cells incubated for 36 h (B) or for different times (C) with 10 µg/mL anti-IgM were stained with DiOC6(3) (50 nM, 15 min, 37°C) and propidium iodide. Percentage in the lower left panel indicates apoptotic cells. D) Caspase activity was assessed on cell lysates prepared from cells incubated for different times with 10 µg/mL goat anti-IgM using 0.2 mM Ac-DEVD-pNA as substrate. Ac-DEVD-pNA hydrolysis was determined in quadruplicate in the presence or absence of 10 µM Ac-DEVD-CHO.
