MODULATION OF E2F ACTIVITY VIA SIGNALING THROUGH SURFACE IGM AND CD40RECEPTORS IN WEHI-231 B LYMPHOMA-CELLS

Stimulation of the phenotypically immature B cell lymphoma WEHI-231 wi th anti-IgM induces G(1) arrest followed by apoptotic cell death, whic h can be reversed by stimulation via the CD40 receptor. Here, we show that cells expressing bcl-x(L) (WEHI-bcl-x(L)) arrest at G(0)/G(1) fol lowing culture with anti-IgM but do not undergo apoptosis. These arres ted cells can be induced to reenter the cell cycle by ligation of CD40 . We have therefore used these cells as a model to study the regulatio n of the transcription factor E2F, which is critically involved in tra nsit through the cell cycle. We found that anti-IgM treatment induces the appearance of an inhibitory DNA binding complex containing the pRB -related pocket protein p130 together with E2F and a concomitant decre ase in ''free'' E2F, consisting of E2F1 and its partner DP1; these eff ects were reversed following stimulation via CD40. These changes in fr ee E2F levels were regulated by changes in E2F1 gene transcription, wh ich is at least partly a result of control of E2F1 promoter activity t hrough its E2F binding sites. Transient transfection experiments showe d that either E2F1 or the viral oncoprotein E1A, which sequesters pock et proteins, including p130, overcame anti-IgM-induced cell cycle arre st in WEHI-bcl-x(L). Taken together, these results indicate that WEHI- 231 sIgM ligation induces the accumulation of hypophosphorylated p130 with consequent inhibition of E2F1 gene transcription and cell cycle a rrest. Conversely, ligation of CD40 causes hyperphosphorylation of p13 0, thereby releasing the repression of E2F1 and other E2F-regulated ge nes, enabling the cells to reenter the cycle. These results, therefore , provide novel insights into the mechanisms whereby antigen receptors on immature B cells deliver inhibitory signals (leading to negative s election of self-reactive B cells) and how these signals can be modula ted by positive signals generated via CD40.