ROLE OF REL-RELATED FACTORS IN CONTROL OF C-MYC GENE-TRANSCRIPTION INRECEPTOR-MEDIATED APOPTOSIS OF THE MURINE B-CELL WEHI-231 LINE

Treatment of immature murine B lymphocytes with an antiserum against t heir surface immunoglobulin (sIg)M results in cell death via apoptosis . The WEHI 231 B cell line (IgM, kappa) has been used extensively as a model for this anti-Ig receptor-mediated apoptosis. Anti-sIg treatmen t of WEHI 231 cells causes an early, transient increase in the levels of c-myc messenger RNA and gene transcription, followed by a rapid dec line below control values. Given the evidence for a role of the c-myc gene in promoting apoptosis, we have characterized the nature and kine tics of changes in the binding of Rel-related factors, which modulate c-myc promoter activity. In exponentially growing WEHI 231 cells, mult iple Rel-related binding activities were detectable. The major binding species was identified as p50/c-Rel heterodimers; only minor amounts of nuclear factor kappa B (NF-kappa B) (p50/p65) were detectable. Cotr ansfection of an inhibitor of NF-kappa B (I kappa B)-alpha expression vector reduced c-myc-promoter/upstream/exon1-CAT reporter construct ac tivity, indicating the role of Rel factor binding in c-myc basal expre ssion in these cells. Treatment with anti-sIg resulted in a rapid tran sient increase in the rate of c-myc gene transcription and in the bind ing of Rel factors. At later times, formation of p50 homodimer complex es occurred. In cotransfection analysis, p65 and c-Rel expression pote ntly and modestly transactivated the c-myc promoter, respectively, whe reas, overexpression of the p50 subunit caused a significant drop in i ts activity. The role of activation of Rel-family binding was demonstr ated directly upon addition of the antioxidant pyrrolidinedithiocarbam ate, which inhibited the anti-sIg-mediated activation of the endogenou s c-myc gene. Similarly, induction after anti-sIg treatment of a trans fected c-myc promoter was abrogated upon cotransfection of an I kappa B-alpha expression vector. These results implicate the Rel-family in I g receptor-mediated signals controlling the activation of c-myr gene t ranscription in WEHI 231 cells, and suggest a role for this family in apoptosis of this line, which is mediated through a c-myc signaling pa thway.