FADD is required for DR4-and DR5-mediated apoptosis - Lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated b y its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the sig naling molecules downstream of these receptors, however, remains highly con troversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-ind uced apoptosis. More recently, using FADD-deficient (-/-) mouse embryonic f ibroblasts, Yeh ct at (Yeh, W. C., Pompa, J. L., McCurrach, M. E., Shu, H.- B., Elia, A. J,, Shahinian, A, Ng, M., Wakeham, A, Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T,W. (1998) Science 279, 1954-1958) concluded that DR4 utilizes a FADD-independent apoptotic pa thway. The latter experiment, however, involved transient overexpression, w hich often leads to nonspecific aggregation of death domain-containing rece ptors. To address this issue in a more physiological setting, we stably tra nsfected mouse DR4/5, human DR4, or human DR5 into FADD(-/-) mouse embryoni c fibroblast cells. We showed that FADD(-/-) MEF cells stably transfected w ith TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast , TRAIL receptors stably transfected into heterozygous FADD(+/-) cells or F ADD(-/-) cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.