Spry2 is DNA-hypermethylated, and its expression is repressed in TCL1-tg B-cell lymphomas. (A) Spry2 RLGS locus (arrow) is shown for WT mouse spleen and 3 TCL1-tg B- cell tumors. Comparisons to the WT spot intensity scored BLL3 as unmethylated/partially methylated (> 50% of WT) and BLL1a and MZL1 as DNA hypermethylated (< 50% of WT) for the surveyed NotI site.^14,40 (B) GBS of a CpG-rich region located between −728 and −484 in the mouse Spry2 promoter. Each row represents the sequence of an individual clone of WT spleen, MZL2, or BLL1a cells. ○, represents unmethylated CpG sites; ●, methylated sites; squares, 2 CpG loci within the Not1 site. (C) Spry2 expression in WT or TCL1-tg B-cell lymphomas was determined by quantitative PCR. Average Spry2 expression for each group (WT, hypermethylated, and unmethylated/partially methylated lymphomas) is shown by a dashed line. Spry2 expression was normalized to 36b4 (Rplp0) expression. DNA methylation status for WT or TCL1-tg B-cell lymphomas was also assessed by MSP. The top and bottom bands correspond to PCR products amplified by primers specific for methylated (M) or unmethylated (U) CpG sites after bisulfite conversion.

Spry2 is DNA hypermethylated, and its expression is repressed in mouse B-cell lymphoma lines but can be reactivated by treatment with a demethylating agent. (A) GBS of a CpG-rich region located between −728 and −484 in the Spry2 promoter in mouse B-cell lines 3-1, Wehi, Bal 117, and A20. Each row represents the sequence of an individual clone. ○ represents unmethylated CpG sites; ●, methylated sites; and squares, 2 CpG loci within the Not1 site. (B) Spry2 expression was assayed by quantitative PCR in 3-1, Wehi, Bal 117, and A20 mouse B-cell lines. N.D. indicates not detected. Spry2 expression is normalized to 36b4 (Rplp0) expression. DNA methylation status for each tumor cell line was also assessed by MSP. The top and bottom bands correspond to PCR products amplified by primers specific for methylated (M) or unmethylated (U) CpG sites after bisulfite conversion. (C) Spry2 expression assayed in triplicate without and with 5-aza-dC and/or 100 nm TSA treatment.

DNA methylation of the SPRY2 promoter is associated with reduced SPRY2 expression in patient lymphoma samples. (A) SPRY2 expression normalized to GAPDH expression was assessed by quantitative PCR in 7 DLBCLs and 1 BL, and the DNA methylation status for each tumor was assessed by MSP. The top and bottom bands correspond to PCR products amplified by primers specific for methylated (M) or unmethylated (U) CpG sites after bisulfite conversion. Estimated percentage of tumor cells in each sample is shown in parentheses. (B) GBS of a CpG-rich region located between −163 and +25 in the SPRY2 promoter in DLBCL5, DLBCL7, and BL1 patient samples. Each row represents the sequence of an individual clone. ○ represents unmethylated CpG sites; ●, methylated CpG sites.

SPRY2 is DNA hypermethylated, and its expression is repressed but can be reactivated by treatment with a demethylating agent in human B-cell lymphoma lines. (A) SPRY2 expression normalized to GAPDH expression was assessed by quantitative PCR in human tonsil B cells (B-cell sort), EBV-immortalized PBL, and a panel of human B-cell lymphoma lines. N.D. indicates not detected. DNA methylation status for sorted B cells, PBLs, and each tumor cell line was assessed by MSP. The top and bottom bands correspond to PCR products amplified by primers specific for methylated (M) or unmethylated (U) CpG sites after bisulfite conversion. (B) GBS of a CpG-rich region located between −163 and +25 in the SPRY2 promoter in human tonsil B cells (B-cell sort) and human B-cell lines Nalm-6, 2F7, P3HR1, and BL41. Each row represents the sequence of an individual clone. ○ represents unmethylated CpG sites; ●, methylated CpG sites. (C) SPRY2 expression normalized to GAPDH expression was assayed in triplicate by quantitative PCR without and with 5-aza-dC and/or 100 nm TSA treatment.

Induction of SPRY2 protein expression by CD40/BCR costimulation in B cells is ERK1/2 and TCL1 dependent. (A) SPRY2 protein expression in sorted WT B cells (pooled from 3 mice) stimulated with anti-CD40 Ab for 24 hours followed by costimulation with anti-IgM Ab for the indicated times. Freshly isolated splenic B cells before CD40 or BCR stimulations are shown in lane 1. The number beneath each lane shows the SPRY2 protein level normalized to the ACTIN level, as determined by densitometry. (B) SPRY1 and SPRY2 protein expression in WT B cells. Freshly isolated splenic B cells (pooled from 2 mice) before (−) or after stimulation (+) with anti-CD40 for 24 hours, followed by anti-IgM for 24 hours. Human embryonic kidney (HEK) 293T cells overexpressing SPRY1 or SPRY2 were used as positive controls (data not shown). (C) SPRY2 protein expression in WT B cells pooled from 2 mice costimulated as in panel B without (−) or with (+) U0126, a MEK1/2 inhibitor. (D) CD19-sorted human tonsil B cells were stimulated for the indicated times with phorbol 12-myristate 13-acetate and ionomycin, followed by Western blot. (E) Nalm-6 cells were infected with a retrovirus expressing a constitutively active form of MEK1 (CA-MEK1) or an empty vector control, followed by stable cell line establishment and Western blot. (F) ERK1/2 phosphorylation time course in WT (wt) and nonmalignant TCL1-tg (tg) splenic B cells costimulated by CD40/BCR without (−) or with (+) U0126. (G) SPRY2 protein expression in nonmalignant TCL1-tg (tg) or WT (wt) splenic B cells costimulated for 18 hours as in panel A. B cells were pooled from 2 TCL1-tg or 2 WT mouse spleens. The number beneath each lane shows the SPRY2 protein level normalized to the ACTIN level, as determined by densitometry. The mean and SD from this and a repeat experiment are plotted (right), normalized to the expression level for WT uninduced B cells. Data are representative of 2 (C-E), 3 (A,B,F), or 4 (G) independent experiments.

SPRY2 overexpression decreases B-cell viability and reduces ERK1/2 signaling activity. (A) Nalm-6 cells infected with control (vector) or SPRY2-expressing retroviruses (SPRY2) were evaluated for viability by MTT assay over a 4-day time course. The data are from triplicate samples normalized to day 0 absorbance at 570 nm, which is set to 1.0. (B) Nalm-6 cells infected with control (vector) or SPRY2-expressing retroviruses (SPRY2) were evaluated for apoptosis by annexin V–FITC and propidium iodide staining with flow cytometry. (C) Nalm-6 cells infected with a control (−) or SPRY2-expressing (+) retrovirus were evaluated for ERK1/2 phosphorylation after anti-IgM stimulation. Data are representative of 3 (B,C) or 4 (A) independent experiments.

Inactivation of Spry2 increases ERK1/2-dependent B-cell proliferation and survival. (A) Freshly isolated Spry2-null or WT splenic B cells from sex-matched littermates were assessed for ERK1/2 phosphorylation by Western blot. (B) Spry2-null or WT B cells were assayed for CD69 (ERK1/2-dependent) and CD25 (ERK1/2-independent) expression after 16 hours of costimulation with anti-CD40 and anti-IgM Abs. The mean fluorescence intensity for each B-cell activation marker from 3 separate experiments as measured by flow cytometry after costimulation is shown. (C) ³H-thymidine incorporation by Spry2-null or WT splenic B cells from sex-matched littermates, stimulated with serum alone or with serum plus anti-CD40 plus anti-IgM costimulation in the presence or absence of the MEK 1/2 inhibitor, U0126. ³H-thymidine was added to the medium at 44 hours of incubation for a final 16 hours of labeling (60-hour total culture time). (D) Spry2-null or WT splenic B cells from sex-matched littermates were anti-CD40 plus anti-IgM costimulated and evaluated for apoptosis by annexin V–FITC and propidium iodide staining with flow cytometry. (E) Percentage of CD19⁺B220⁺ B cells determined by flow cytometry from the bone marrows of 7 poly-dI:dC-injected Mx1-Cre × Spry2^fl/fl (Spry2-null), 6 poly-dI:dC-injected Spry2^fl/fl (WT), and 3 1× PBS, (pH 7.4)-injected Mx1-Cre × Spry2^fl/fl mice (WT). (F) Model illustrating SPRY2-mediated regulation of the ERK signal transduction pathway in mature peripheral B cells. The diagram on the left (WT) shows that BCR/CD40 signaling results in activation of ERK1/2, which in turn stimulates B-cell proliferation. However, it also induces expression of SPRY2 protein, which subsequently represses ERK1/2 pathway signaling and prevents excessive cell proliferation. The diagram on the right (Spry2-null) illustrates the consequences of loss of SPRY2 function, by either dense promoter DNA hypermethylation or gene knockout: repression of ERK1/2 pathway signaling does not occur, resulting in increased B-cell proliferation. TCL1 also enhances the ERK signaling pathway in response to BCR stimulation in mature peripheral B cells, which results in an increase in Spry2 protein expression. Data are representative of 2 (A), 3 (C), or 4 (B,D) independent experiments.