Stk39 is DNA-hypermethylated, and its expression is repressed in TCL1-tg B-cell lymphomas. A: Genomic bisulfite sequencing of part of a Stk39 promoter region CpG island located between −203 and +52 from the major transcription start site (+1). Each row corresponds to the sequence of an individual clone of wild-type spleen (WT1), BLL1, or BLL3 cells (open circles, unmethylated CpG sites; closed circles, methylated sites). B: Stk39 expression in wild-type (1 to 4) spleens, premalignant TCL1-tg (1 to 4) spleens, or TCL1-tg B-cell lymphomas was determined by quantitative PCR. The mean Stk39 expression for each group is shown by a dashed line. Stk39 expression was normalized to 36b4 (Rplp0) and set to 1.0 for WT1. All samples were analyzed in triplicate, a two-tailed t-test was performed to determine statistical significance, and data are plotted as the mean ± SD. C: SPAK protein expression was evaluated by Western blot of whole spleen or sorted B- and non-B (NB)-cell fractions, from wild-type spleen (WT5), premalignant TCL1-tg (Tg5) spleen, or TCL1-tg B-cell lymphomas (DLBCL3, BLL4). MZL, marginal zone lymphoma.

Loss of SPAK protects B cells from apoptosis induced by DNA double-strand breaks. A: STK39 expression in Nalm6 B cells at 2 weeks after infection with empty-vector, RNAi1, or RNAi2 retroviruses was determined by quantitative PCR. STK39 expression was normalized to 36B4 (Rplp0) and set to 1.0 for parent Nalm6 cells. Samples were analyzed in triplicate, and data shown are representative of four independent experiments. B: SPAK, OSR1, and actin expression was evaluated by Western blot in Nalm6 cells from A. Densitometry units (DU) are displayed as intensity of SPAK or OSR1, normalized to actin. Data shown are representative of three independent experiments. C: At 24 hours after treatment with 0.25 or 0.5 μmol/L etoposide (Eto), control and RNAi1 Nalm6 cells were evaluated for apoptosis by flow cytometry. Each bar graph represents the percent annexin V⁺ cells in the treated sample minus that in an untreated sample control. Samples were analyzed in triplicate, and data shown are representative of eight independent experiments. *P < 0.05. D: Control and RNAi1 Nalm6 cells were treated with escalating doses of IR, rested for 18 hours, and evaluated for apoptosis as in C. Samples were analyzed in triplicate, and data shown are representative of eight independent experiments.*P < 0.05. E: SPAK expression was evaluated by Western blot in BCBL1 cells infected with empty-control or RNAi1 retroviruses after 2 weeks of puromycin selection. Data shown are representative of two independent experiments. F: At 24 hours after treatment with two doses of Eto, control and RNAi1 BCBL1 cells were evaluated for apoptosis by flow cytometry. Each bar graph represents the percent annexin V⁺ cells in the treated sample minus that in an untreated sample control. Samples were analyzed in triplicate, and data shown are representative of five independent experiments. *P < 0.05.

Loss of SPAK results in protection from DNA DSBs via reduced caspase activation. A: Cleavage of procaspase 3 (Pro) was assessed by Western blot after exposure to 0.5 μmol/L etoposide (Eto). Densitometry units (DU) are displayed as intensity of cleaved caspase 3 normalized to actin. B: Nalm6 cells were or were not preincubated with 20 μmol/L Q-VD-OPH for 1 hour and subjected to 0.5 μmol/L etoposide for 24 hours. Apoptosis was measured by flow cytometry. Percent annexin V⁺ is shown. Samples were analyzed in triplicate, and data shown are representative of two independent experiments. C: Apoptosis was measured, as in B, after a 1-hour preincubation with SP600125 and 24-hour incubation with 0.5 μmol/L etoposide. Samples were analyzed in triplicate, and data shown are representative of four independent experiments. *P < 0.05. DMSO, dimethylsulfoxide.

SPAK expression is repressed in human B-cell lymphomas. A: Immunohistochemistry for SPAK expression in a reactive tonsil GC (original magnification, ×5). Left panel is secondary HRP antibody-alone control, and right panel is incubated with anti-SPAK antiserum. B: Western blot for SPAK expression in naive (N), GC, and memory (M) B cells sorted from tonsil. C: SPAK immunohistochemistry in a marginal zone lymphoma expressing SPAK in 75 to 100% of tumor cells (original magnification, ×200). D: SPAK immunohistochemistry in a SPAK-negative small lymphocytic lymphoma (original magnification, ×200). E: Histogram of 42 human B-cell lymphomas showing cases with >50% or <50% SPAK-positive tumor cells by IHC. F: Summary table of immunohistochemical analysis for SPAK expression from E.