Antigen receptor‐initiated growth inhibition is blocked in CD45‐loss variants of a mature B lymphoma, with limited effects on apoptosis

Abstract

Role of CD45 in B cell antigen receptor (BcR)-mediated signaling events in mature B cells was examined using BAL-17 and its CD45-negative clones. In the CD45-negative clones, BcR stimulation induced tyrosine phosphorylation almost identical to the parental cells, with a few exceptions of reduced phosphorylation, especially of a protein of about 60 kDa. BcR-induced calcium responses were reduced in the CD45-negative clones, but the kinetics were similar to the parent. BcR stimulation led to growth inhibition in the parental cells, but signals for growth inhibition were completely blocked in the CD45-negative clones. Interestingly, the same stimulation induced low, but significant levels of apoptosis both in the parent and in the CD45-negative clones. Thus, in mature BAL-17 cells, CD45 subtly mediate early signaling events (tyrosine phosphorylation and Ca2+ mobilization), and is absolutely required for the signaling pathway leading to growth regulation, but has limited effects on apoptosis.

Dynamic regulation of Src-family kinases by CD45 in B cells

“…30,31 We further showed that, in WEHI-231 cells, CD45 constitutively inhibits Lyn by dephosphorylating both the COOH-terminal and autophosphorylation sites, but that BCR ligation induces phosphorylation of the 2 sites. 27, 28 We therefore suggested that the inhibitory activity of CD45 was somehow diminished upon BCR ligation.…”

“…27,29,31 These cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 M 2-mercaptoethanol (2-ME), 100 g/mL streptomycin, and 100 U/mL penicillin. CD45 exon 9 knock-out (KO) 38 and C57BL/6 (B6) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and Japan SLC (Hamamatsu, Japan), respectively, and used at 6 to 12 weeks of age.…”

Different sensitivity to receptor editing of B cells from mice hemizygous or homozygous for targeted Ig transgenes

“…The tyrosine phosphatase CD45 is required for efficient BCR signal transduction (23,24,(34)(35)(36)(37)(38). As a consequence, CD45 deficiency promotes survival and accumulation of self-reactive B cells in vivo (38).…”

CD45 Is Required for CD40-induced Inhibition of DNA Synthesis and Regulation of c-Jun NH2-terminal Kinase and p38 in BAL-17 B Cells

“…Cells-The WEHI-231 and BAL-17 murine B cell lines and the BAL-17-derived, CD45-deficient clone 44 were all described previously (35,36). All cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 50 M 2-mercaptoethanol, 100 g/ml streptomycin, and 100 units/ml penicillin (complete medium).…”

“…Proliferation Assay-Proliferation assay was performed as previously described (35,36 Electroporation-To introduce DNA into cells, 2 ϫ 10 7 cells were washed twice with phosphate-buffered saline and suspended with 0.4 ml of cytomix buffer (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 / KH 2 PO 4 , pH 7.6, 25 mM HEPES, pH 7.6, 2 mM EGTA, pH 7.6, 5 mM MgCl 2 , 2 mM ATP, and 5 mM glutathione) containing 40 g of DNA, and electroporated at 264 V, 950 microfarads. DNAs introduced were: the pcDLSR␣ expression vector containing Src-CD45 (a chimeric cDNA encoding the intracellular murine CD45 preceded by a short aminoterminal sequence from p60 c-src ) (38) or the pcDLSR␣ empty vector (a gift from Dr. Y. Minami, Kobe University, Kobe, Japan), and pEFII expression vector containing a gene encoding a hemagglutinin-tagged dominant-negative SEK1 mutant (SEK-AL) (39) (a gift of Dr. Jim Woodgett, Ontario Cancer Institute, Toronto, Canada), which had been excised from pcDNA3 Zeo, or the empty vector (a gift from Dr. G. Koretzky, University of Pennsylvania, Philadelphia, PA).…”