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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.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.

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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Characterization of pre-B cell tumors by flow cytometry. (A) Dot plots for cytoplasmic SLC and μ H chain in gated B220+ cells from tumor samples from the indicated mice, grown for 1 to 3 wk in the presence of IL-7. (B) Flow cytometric analysis of lacZ expression in gated CD19+B220+ pre-B lymphoma cells in a lymph node from a Btk+/−SLP-65−/− mouse. (C) Phenotype of two pre-B cell lymphoma cultures, showing variable expression of cytoplasmic κ L chain, CD43, and CD2. Cell suspensions were stained for the indicated markers in combination with B220, and the results are displayed as dot plots of gated B220+ cells. (D) Phenotype of two separate tumor cell suspensions derived from BM and mesenteric lymph node from a single mouse, which were cultured in the presence of Il-7 for 7 d.
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fig5: Characterization of pre-B cell tumors by flow cytometry. (A) Dot plots for cytoplasmic SLC and μ H chain in gated B220+ cells from tumor samples from the indicated mice, grown for 1 to 3 wk in the presence of IL-7. (B) Flow cytometric analysis of lacZ expression in gated CD19+B220+ pre-B lymphoma cells in a lymph node from a Btk+/−SLP-65−/− mouse. (C) Phenotype of two pre-B cell lymphoma cultures, showing variable expression of cytoplasmic κ L chain, CD43, and CD2. Cell suspensions were stained for the indicated markers in combination with B220, and the results are displayed as dot plots of gated B220+ cells. (D) Phenotype of two separate tumor cell suspensions derived from BM and mesenteric lymph node from a single mouse, which were cultured in the presence of Il-7 for 7 d.

Mentions: All pre-B cell tumors characterized expressed high levels of SLC and Ig μ H chain in their cytoplasm, irrespective of the Btk genotype of the SLP-65−/− mice (Fig. 5 A). The two Btk+/− SLP-65−/− mice present in our panel of mutant mice also rapidly developed pre-B cell tumors. Because of the presence of a LacZ reporter in the targeted Btk allele, we could evaluate the X-chromosome inactivation status of the lymphoma cells (20). As a result of the process of random X-chromosome inactivation in Btk+/− heterozygous females, each X chromosome is active in about half of the pre-B cells. Similar to findings in lymphoma cells from male Btk−SLP-65−/− mice, we observed that the majority of lymphoma cells from Btk+/−SLP-65−/− mice were LacZ+. These results indicate that the Btk+/− lymphoma cells carried the disrupted Btk allele on the active X chromosome and therefore were functionally Btk-deficient (Fig. 5 B).


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)

Characterization of pre-B cell tumors by flow cytometry. (A) Dot plots for cytoplasmic SLC and μ H chain in gated B220+ cells from tumor samples from the indicated mice, grown for 1 to 3 wk in the presence of IL-7. (B) Flow cytometric analysis of lacZ expression in gated CD19+B220+ pre-B lymphoma cells in a lymph node from a Btk+/−SLP-65−/− mouse. (C) Phenotype of two pre-B cell lymphoma cultures, showing variable expression of cytoplasmic κ L chain, CD43, and CD2. Cell suspensions were stained for the indicated markers in combination with B220, and the results are displayed as dot plots of gated B220+ cells. (D) Phenotype of two separate tumor cell suspensions derived from BM and mesenteric lymph node from a single mouse, which were cultured in the presence of Il-7 for 7 d.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Characterization of pre-B cell tumors by flow cytometry. (A) Dot plots for cytoplasmic SLC and μ H chain in gated B220+ cells from tumor samples from the indicated mice, grown for 1 to 3 wk in the presence of IL-7. (B) Flow cytometric analysis of lacZ expression in gated CD19+B220+ pre-B lymphoma cells in a lymph node from a Btk+/−SLP-65−/− mouse. (C) Phenotype of two pre-B cell lymphoma cultures, showing variable expression of cytoplasmic κ L chain, CD43, and CD2. Cell suspensions were stained for the indicated markers in combination with B220, and the results are displayed as dot plots of gated B220+ cells. (D) Phenotype of two separate tumor cell suspensions derived from BM and mesenteric lymph node from a single mouse, which were cultured in the presence of Il-7 for 7 d.
Mentions: All pre-B cell tumors characterized expressed high levels of SLC and Ig μ H chain in their cytoplasm, irrespective of the Btk genotype of the SLP-65−/− mice (Fig. 5 A). The two Btk+/− SLP-65−/− mice present in our panel of mutant mice also rapidly developed pre-B cell tumors. Because of the presence of a LacZ reporter in the targeted Btk allele, we could evaluate the X-chromosome inactivation status of the lymphoma cells (20). As a result of the process of random X-chromosome inactivation in Btk+/− heterozygous females, each X chromosome is active in about half of the pre-B cells. Similar to findings in lymphoma cells from male Btk−SLP-65−/− mice, we observed that the majority of lymphoma cells from Btk+/−SLP-65−/− mice were LacZ+. These results indicate that the Btk+/− lymphoma cells carried the disrupted Btk allele on the active X chromosome and therefore were functionally Btk-deficient (Fig. 5 B).

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.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.

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