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Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells.

Kersseboom R, Middendorp S, Dingjan GM, Dahlenborg K, Reth M, Jumaa H, Hendriks RW - J. Exp. Med. (2003)

Bottom Line: Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk.Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor.To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Erasmus MC Rotterdam, PO Box 1738, NL-3000 DR Rotterdam, Netherlands.

ABSTRACT
Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.

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Analysis of IL-7 driven BM cultures from Btk and SLP-65 mutant mice. (A) Proliferative response to 100 U/ml IL-7, as determined by [3H]thymidine incorporation after 5 d of culture. Bars represent mean cpm and SEM of triplicate cultures. (B) Forward scatter (FSC) values and expression profiles of IgM, IgD, SLC, and cytoplasmic κ L chain of IL-7 driven BM cultures from the indicated mice. Data are displayed as histogram overlays of B220+ cells, either cultured under proliferating conditions (with 100 U/ml IL-7 for 7 d, thin lines) or under differentiating conditions (after 5 d of culture with IL-7 and subsequently without IL-7 for 2 d, bold lines). The percentages shown represent the fractions of the cells that are within the indicated marker under the two different culture conditions. (C) Percentage of surface IgM+ cells within the fraction of small FSC B220+ cells after 7 d of culture in the presence of the indicated concentrations of IL-7. Data are representative of four mice per group.
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fig2: Analysis of IL-7 driven BM cultures from Btk and SLP-65 mutant mice. (A) Proliferative response to 100 U/ml IL-7, as determined by [3H]thymidine incorporation after 5 d of culture. Bars represent mean cpm and SEM of triplicate cultures. (B) Forward scatter (FSC) values and expression profiles of IgM, IgD, SLC, and cytoplasmic κ L chain of IL-7 driven BM cultures from the indicated mice. Data are displayed as histogram overlays of B220+ cells, either cultured under proliferating conditions (with 100 U/ml IL-7 for 7 d, thin lines) or under differentiating conditions (after 5 d of culture with IL-7 and subsequently without IL-7 for 2 d, bold lines). The percentages shown represent the fractions of the cells that are within the indicated marker under the two different culture conditions. (C) Percentage of surface IgM+ cells within the fraction of small FSC B220+ cells after 7 d of culture in the presence of the indicated concentrations of IL-7. Data are representative of four mice per group.

Mentions: Cytoplasmic μ+ pre-B cells undergo rapid cell division in response to IL-7 in vitro, whereby subsequent removal of IL-7 strongly induces exit from cell cycle and further differentiation into surface IgM+ B cells (25). We previously reported that Btk and SLP-65 single mutant pre-B cells manifest an enhanced proliferative response to IL-7 (14, 16). When we compared total BM cells from Btk and SLP-65 single mutants and the double mutant, these three groups of mice showed similar [3H]thymidine incorporation values after 5 d of culture in the presence of 100 U/ml IL-7 (Fig. 2 A). When total BM cell suspensions from WT mice were cultured in the presence of IL-7 for 7 d, the majority of cells consisted of B220+ cytoplasmic μ+ pre-B cells that were surface μ− or μlow, while a significant fraction (∼20–30%) performed productive κ L chain rearrangements and matured to surface IgM+IgD− or IgM+IgD+ B cell stages (Fig. 2 B, thin lines). In contrast, the IL-7 driven BM cultures from Btk and SLP-65 single or double mutant mice consisted almost exclusively of large μ+ SLC+ pre-B cells which did not express κ L chains in their cytoplasm (Fig. 2 B, thin lines).


Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells.

Kersseboom R, Middendorp S, Dingjan GM, Dahlenborg K, Reth M, Jumaa H, Hendriks RW - J. Exp. Med. (2003)

Analysis of IL-7 driven BM cultures from Btk and SLP-65 mutant mice. (A) Proliferative response to 100 U/ml IL-7, as determined by [3H]thymidine incorporation after 5 d of culture. Bars represent mean cpm and SEM of triplicate cultures. (B) Forward scatter (FSC) values and expression profiles of IgM, IgD, SLC, and cytoplasmic κ L chain of IL-7 driven BM cultures from the indicated mice. Data are displayed as histogram overlays of B220+ cells, either cultured under proliferating conditions (with 100 U/ml IL-7 for 7 d, thin lines) or under differentiating conditions (after 5 d of culture with IL-7 and subsequently without IL-7 for 2 d, bold lines). The percentages shown represent the fractions of the cells that are within the indicated marker under the two different culture conditions. (C) Percentage of surface IgM+ cells within the fraction of small FSC B220+ cells after 7 d of culture in the presence of the indicated concentrations of IL-7. Data are representative of four mice per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196076&req=5

fig2: Analysis of IL-7 driven BM cultures from Btk and SLP-65 mutant mice. (A) Proliferative response to 100 U/ml IL-7, as determined by [3H]thymidine incorporation after 5 d of culture. Bars represent mean cpm and SEM of triplicate cultures. (B) Forward scatter (FSC) values and expression profiles of IgM, IgD, SLC, and cytoplasmic κ L chain of IL-7 driven BM cultures from the indicated mice. Data are displayed as histogram overlays of B220+ cells, either cultured under proliferating conditions (with 100 U/ml IL-7 for 7 d, thin lines) or under differentiating conditions (after 5 d of culture with IL-7 and subsequently without IL-7 for 2 d, bold lines). The percentages shown represent the fractions of the cells that are within the indicated marker under the two different culture conditions. (C) Percentage of surface IgM+ cells within the fraction of small FSC B220+ cells after 7 d of culture in the presence of the indicated concentrations of IL-7. Data are representative of four mice per group.
Mentions: Cytoplasmic μ+ pre-B cells undergo rapid cell division in response to IL-7 in vitro, whereby subsequent removal of IL-7 strongly induces exit from cell cycle and further differentiation into surface IgM+ B cells (25). We previously reported that Btk and SLP-65 single mutant pre-B cells manifest an enhanced proliferative response to IL-7 (14, 16). When we compared total BM cells from Btk and SLP-65 single mutants and the double mutant, these three groups of mice showed similar [3H]thymidine incorporation values after 5 d of culture in the presence of 100 U/ml IL-7 (Fig. 2 A). When total BM cell suspensions from WT mice were cultured in the presence of IL-7 for 7 d, the majority of cells consisted of B220+ cytoplasmic μ+ pre-B cells that were surface μ− or μlow, while a significant fraction (∼20–30%) performed productive κ L chain rearrangements and matured to surface IgM+IgD− or IgM+IgD+ B cell stages (Fig. 2 B, thin lines). In contrast, the IL-7 driven BM cultures from Btk and SLP-65 single or double mutant mice consisted almost exclusively of large μ+ SLC+ pre-B cells which did not express κ L chains in their cytoplasm (Fig. 2 B, thin lines).

Bottom Line: Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk.Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor.To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Erasmus MC Rotterdam, PO Box 1738, NL-3000 DR Rotterdam, Netherlands.

ABSTRACT
Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.

Show MeSH
Related in: MedlinePlus