Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF- related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel o f human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4), Immobilized anti-TRAIL-Ri or -R2 mAbs were c ytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to r ecombinant TRAIL were also resistant to these mAbs and only became sensitiv e when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced d eath was characterized by the activation of intracellular caspases, which c ould be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zV AD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone ( zIETD-fmk), When used in solution, one of the anti-TRAIL-R2 mAbs was capabl e of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cel ls and prevented TRAIL-induced death of these cells, whereas two of the ant i-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R 1:Fc. Furthermore, use of the blocking anti-TRAIL-R2, mAb allowed us to dem onstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 we re necessary and sufficient to mediate cell death. In contrast, the express ion of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in de termining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.