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Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma.

Boukhiar MA, Roger C, Tran J, Gressin R, Martin A, Ajchenbaum-Cymbalista F, Varin-Blank N, Ledoux D, Baran-Marszak F - Exp Hematol Oncol (2013)

Bottom Line: Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1.Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival.Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM, UMR U978, Adaptateur de Signalisation en Hématologie, F-93000, Bobigny, France. fanny.baran-marszak@avc.aphp.fr.

ABSTRACT

Background: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL.

Methods: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry.

Results: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

Conclusions: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

No MeSH data available.


Related in: MedlinePlus

Kinetics of BCR-induced expressions of EGR-1 and c-MYC in MCL cell lines and primary MCL cells. (A) Kinetics of BCR-induced mRNA expression of EGR-1 and c-MYC in Granta-519 and HBL-2 cell lines. 3×106 cells/ml were stimulated with 10 μg/ml of immobilized anti-IgM antibody for 15 min, 30 min, 1 h, 3 h and 6 h. EGR-1 and c-MYC expressions were analyzed by qRT-PCR. (B) Induction of EGR-1 protein upon BCR engagement was confirmed by western-blot. (C) The same experiments were performed on primary patients’ cells. EGR-1 and c-MYC expressions were analyzed by qRT-PCR (left panel) and western-blot (right panels). (D) EGR-1 and c-MYC mRNA expressions upon anti-IgM stimulation (1 h) were analyzed by qRT-PCR from 7 patients’ samples (UPN2, 3, 4, 5, 6, 7 and 8). Fold increase of mRNA level were calculated compared with unstimulated cells in all experiments. All measurements were done in duplicate and the mean is provided.
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Figure 1: Kinetics of BCR-induced expressions of EGR-1 and c-MYC in MCL cell lines and primary MCL cells. (A) Kinetics of BCR-induced mRNA expression of EGR-1 and c-MYC in Granta-519 and HBL-2 cell lines. 3×106 cells/ml were stimulated with 10 μg/ml of immobilized anti-IgM antibody for 15 min, 30 min, 1 h, 3 h and 6 h. EGR-1 and c-MYC expressions were analyzed by qRT-PCR. (B) Induction of EGR-1 protein upon BCR engagement was confirmed by western-blot. (C) The same experiments were performed on primary patients’ cells. EGR-1 and c-MYC expressions were analyzed by qRT-PCR (left panel) and western-blot (right panels). (D) EGR-1 and c-MYC mRNA expressions upon anti-IgM stimulation (1 h) were analyzed by qRT-PCR from 7 patients’ samples (UPN2, 3, 4, 5, 6, 7 and 8). Fold increase of mRNA level were calculated compared with unstimulated cells in all experiments. All measurements were done in duplicate and the mean is provided.

Mentions: BCR-induced expressions of c-MYC and EGR-1 were then confirmed by kinetic experiments in MCL cell lines (Granta-519 and HBL-2) (Figure 1A and B) and in MCL patients’ samples (Figure 1C). For MCL cell lines, basal levels of EGR-1 mRNA was rapidly increased within 30 min upon BCR ligation, peaked at 1 h and gradually returned to basal level within 3 to 6 hours. Similarly, EGR-1 protein levels increased upon anti-IgM stimulation and returned to basal level within 6 h (Figure 1B). A similar increase was observed for primary cells (UPN7 and UPN10) with EGR-1 proteins still detectable at 6 hours (Figure 1C). C-MYC expression was significantly induced upon BCR engagement in patients’ cells only (Figure 1A for Granta-519 and HBL-2 cells, Figure 1C for UPN7). The pattern of c-MYC mRNA induction differed from that of EGR-1 and displayed a constant increase at least up to 3 h associated with an increase of c-MYC protein (Figure 1C). We next evaluated the impact of BCR stimulation on a series of 7 patients’ samples (Figure 1D). Upon 1 h of anti-IgM stimulation, EGR-1 mRNA expression was highly upregulated in 4 out of 7 cases and clearly with a major extent as compared to induction of c-MYC (median fold increase: 30.1 and 2.98 for EGR-1 and c-MYC respectively). No correlation was observed between IGHV mutational status and intensity of BCR-induced responses (not shown).


Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma.

Boukhiar MA, Roger C, Tran J, Gressin R, Martin A, Ajchenbaum-Cymbalista F, Varin-Blank N, Ledoux D, Baran-Marszak F - Exp Hematol Oncol (2013)

Kinetics of BCR-induced expressions of EGR-1 and c-MYC in MCL cell lines and primary MCL cells. (A) Kinetics of BCR-induced mRNA expression of EGR-1 and c-MYC in Granta-519 and HBL-2 cell lines. 3×106 cells/ml were stimulated with 10 μg/ml of immobilized anti-IgM antibody for 15 min, 30 min, 1 h, 3 h and 6 h. EGR-1 and c-MYC expressions were analyzed by qRT-PCR. (B) Induction of EGR-1 protein upon BCR engagement was confirmed by western-blot. (C) The same experiments were performed on primary patients’ cells. EGR-1 and c-MYC expressions were analyzed by qRT-PCR (left panel) and western-blot (right panels). (D) EGR-1 and c-MYC mRNA expressions upon anti-IgM stimulation (1 h) were analyzed by qRT-PCR from 7 patients’ samples (UPN2, 3, 4, 5, 6, 7 and 8). Fold increase of mRNA level were calculated compared with unstimulated cells in all experiments. All measurements were done in duplicate and the mean is provided.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585857&req=5

Figure 1: Kinetics of BCR-induced expressions of EGR-1 and c-MYC in MCL cell lines and primary MCL cells. (A) Kinetics of BCR-induced mRNA expression of EGR-1 and c-MYC in Granta-519 and HBL-2 cell lines. 3×106 cells/ml were stimulated with 10 μg/ml of immobilized anti-IgM antibody for 15 min, 30 min, 1 h, 3 h and 6 h. EGR-1 and c-MYC expressions were analyzed by qRT-PCR. (B) Induction of EGR-1 protein upon BCR engagement was confirmed by western-blot. (C) The same experiments were performed on primary patients’ cells. EGR-1 and c-MYC expressions were analyzed by qRT-PCR (left panel) and western-blot (right panels). (D) EGR-1 and c-MYC mRNA expressions upon anti-IgM stimulation (1 h) were analyzed by qRT-PCR from 7 patients’ samples (UPN2, 3, 4, 5, 6, 7 and 8). Fold increase of mRNA level were calculated compared with unstimulated cells in all experiments. All measurements were done in duplicate and the mean is provided.
Mentions: BCR-induced expressions of c-MYC and EGR-1 were then confirmed by kinetic experiments in MCL cell lines (Granta-519 and HBL-2) (Figure 1A and B) and in MCL patients’ samples (Figure 1C). For MCL cell lines, basal levels of EGR-1 mRNA was rapidly increased within 30 min upon BCR ligation, peaked at 1 h and gradually returned to basal level within 3 to 6 hours. Similarly, EGR-1 protein levels increased upon anti-IgM stimulation and returned to basal level within 6 h (Figure 1B). A similar increase was observed for primary cells (UPN7 and UPN10) with EGR-1 proteins still detectable at 6 hours (Figure 1C). C-MYC expression was significantly induced upon BCR engagement in patients’ cells only (Figure 1A for Granta-519 and HBL-2 cells, Figure 1C for UPN7). The pattern of c-MYC mRNA induction differed from that of EGR-1 and displayed a constant increase at least up to 3 h associated with an increase of c-MYC protein (Figure 1C). We next evaluated the impact of BCR stimulation on a series of 7 patients’ samples (Figure 1D). Upon 1 h of anti-IgM stimulation, EGR-1 mRNA expression was highly upregulated in 4 out of 7 cases and clearly with a major extent as compared to induction of c-MYC (median fold increase: 30.1 and 2.98 for EGR-1 and c-MYC respectively). No correlation was observed between IGHV mutational status and intensity of BCR-induced responses (not shown).

Bottom Line: Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1.Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival.Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM, UMR U978, Adaptateur de Signalisation en Hématologie, F-93000, Bobigny, France. fanny.baran-marszak@avc.aphp.fr.

ABSTRACT

Background: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL.

Methods: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry.

Results: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

Conclusions: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

No MeSH data available.


Related in: MedlinePlus