APOPTOSIS THROUGH CD95 (FAS APO-1), BUT NOT A CD40/CD95 CHIMERIC RECEPTOR, IS INHIBITED BY PHORBOL-12-MYRISTATE-13-ACETATE/

CD95 (Fas/APO-1)-mediated apoptosis appears to be regulated by positiv e and negative signaling molecules, A human CD40/CD95 chimeric recepto r was stably transfected into CD95-expressing human Jurkat T cells, an d signaling through native and chimeric CD95 was compared in the same cell type to assess contributions of the CD95 extracellular and intrac ellular domains, Apoptosis was induced in these transfectants by solub le CD40 ligand and also by the anti-CD40 monoclonal antibodies (mAb) M 2 and M3, The M2 mAb was more effective than M3 in these transfectants , In contrast to apoptosis mediated through native CD95, CD40/CD95-med iated apoptosis was not inhibited by phorbol-12-myristate-13-acetate ( PMA), The apoptotic response to the anti-CD40 mAb M3, but not M2, was enhanced by PMA and dibutyryl cyclic adenosine monophosphate (db-cAMP) , which also increased mRNA levels and surface expression of CD40/CD95 , The enhancing effects of PMA, but not those of db-cAMP, were sensiti ve to cycloheximide, The M2 and M3 mAbs appeared to have virtually ide ntical binding affinities but, when added to cells together, M3 inhibi ted M2-induced apoptosis, These mAbs may bind neighboring epitopes, bu t M2 induces a more effective signaling-competent conformation upon th e chimeric receptor, These data suggest that dimerization, however onl y in a signaling-competent conformation, was sufficient to induce apop tosis, When expressed as a chimera with the CD40 extracellular domain, the CD95 intracellular domain was not inhibited by protein kinase C ( PKC)-dependent pathways, suggesting that the CD95 extracellular domain is required for association with a molecule that inhibits the apoptot ic signal.