Lymphoma models for B cell activation and tolerance. III. Cell cycle dependence for negative signalling of WEHI-231 B lymphoma cells by anti-mu.

Abstract

It has become increasingly apparent that B lymphomas provide clonal models for antigen-specific lymphocytes and for the analysis of both positive and negative signalling in B cells. In addition, anti-Ig reagents have been used to mimic antigen/tolerogen in their interaction with B cell receptors at a polyclonal level (1-3). We have been studying the effects of anti-Ig on a group of unique B cell lymphomas as models for either tolerogenic or immunogenic signalling (4-6). One such line, WEHI-231, has been shown (7, 8) to be readily inhibited in its growth by anti-Ig reagents. We confirmed the sensitivity of this line to growth inhibition by anti-u and anti-K reagents, and determined its kinetics, specificity, and site of the block in the cell cycle at the G1/S interface (4). In this report, we have enriched WEHI-231 lymphoma cells at various points in G1 to S and analyzed the effects of anti-# on progression through S phase. Our data suggest that critical events occur early in GI and are uniquely sensitive to modulation by anti-~. These studies now will allow sensitive molecular approaches towards an understanding of B cell signalling, as well as methods to regulate lymphoma growth per se. Materials and MethodsCell Lines and Their Maintenance. WEHI-231 B lymphoma cells arose in a (BALB/c x NZB) F~ mouse after mineral oil induction (9). Clone 28, obtained from Dr. Noel Warner (Becton Dickinson Research Laboratories, Mountain View, CA), has been maintained in our laboratory for several years in DMEM supplemented with 5-10% FCS, 100 U/ml penicillin, 100 #g/ml streptomycin, 2 mM L-glutamine, and 5 x 10 -5 M 2-ME (4, 5). It is grown as a single cell suspension with occasional aggregates, and is split twice weekly. As a standard test fbr anti-# sensitivity, 104 WEHI 231 cells were cultured in a total volume of 0.2 ml with various concentrations of goat anti-/~ or other antisera for 48 h, at which time they were pulsed with 1 ~Ci of tritiated thymidine. 18 h later they were harvested on glass fiber filters and assayed by liquid scintillation spectrometry.

Activation-induced cell death in B lymphocytes

“…Furthermore, cells are sensitive to anti-IgM treatment only when in early G1 phase [3]. As one would predict, in these growth arrested cells (such as anti-IgM-treated WEHI-231 or CH31 cells), pRb is in its active, growth suppressive form, i.e.…”

“…An enormous amount of data in this field comes from in vitro models for B cell tolerance using either human or murine B-lymphoma cells of immature phenotype. WEHI-231, CH31 and CH33 cells, for example, are prototypical of such immature murine B-lymphoma cells in which mIgM crosslinking results in G1 growth arrest [2], [3] and subsequent apoptosis [4], [5]. In this review, signal transduction pathways initiated by BCR crosslinking will be discussed.…”

Role of cyclin A and p27 in anti-IgM induced G1 growth arrest of murine B-cell lymphomas.

“…That is, cross-linking of surface IgM receptors on WEHI-231 cells induces cell cycle blockade, followed by programmed cell death (Benhamou et al, 1990;Hasbold and Klaus, 1990). Despite transition through DNA synthesis and mitosis (Scott et al, 1986). This change in the sensitivity of cells to the growth-suppressing effects of anti-IgM does not depend on early membrane events because the level of expression of sIgM and calcium mobilization upon IgM cross-linking remain the same in different phases of the cell cycle (Scott et al, 1987).…”

Involvement of cell cycle progression in survival signaling through CD40 in the B-lymphocyte line WEHI-231

“…In contrast, overexpression of Bcl-xL induces the survival of BCR-ligated WEHI-231 cells even in the presence of cell cycle When B-cell lines such as WEHI-231 undergo BCR ligationinduced apoptosis, cell cycle is arrested prior to apoptosis. [21][22][23][24] Since restoration of cell cycle progression is involved in CD40-mediated abrogation of apoptosis in BCR-ligated WEHI-231 cells, cell cycle arrest induced by BCR ligation may be involved in BCR-mediated apoptosis in these cells. Involvement of cell cycle arrest in BCR-mediated apoptosis was previously suggested by Sonnenshine and colleagues.…”

“…This is also supported by the finding that CD40 ligation induces proliferation as well as survival of BCR-ligated WEHI-231 cells, 3 which otherwise undergo cell cycle arrest in the G1 phase prior to apoptosis. [21][22][23][24] Since overexpression of antiapoptotic members of the Bcl-2 family, Bcl-xL and A1, blocks apoptosis but not cell cycle arrest in these cells, 15,17 survival does not restore the cell cycling of BCR-ligated WEHI-231 cells. Cell cycle progression of BCR-ligated B cells by CD40 ligation thus cannot be attributed to its antiapoptotic activity, but is induced by its cell cycle-promoting function, probably through reduction of p27 kip1 and induction of CDKs.…”