Charles Rauch

Identification and characterization of a new member of the TNF family that induces apoptosis

A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.

Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.

To evaluate the utility of tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ–huCD95L in vivo, administration of either LZ–huTRAIL or LZ–muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ–huTRAIL actively suppressed growth of the TRAIL–sensitive human mammary adenocarcinoma cell line MDA–231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ–huTRAIL demonstrated clear areas of apoptotic necrosis within 9–12 hours of injection.

TRAIL‐R2: a novel apoptosis‐mediating receptor for TRAIL

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL‐R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL‐R2, by ligand‐based affinity purification and subsequent molecular cloning. TRAIL‐R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL‐R2 contains two extracellular cysteine‐rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell‐surface‐expressed TRAIL‐R2, and TRAIL‐induced apoptosis is inhibited by a TRAIL‐R2–Fc fusion protein. TRAIL‐R2 mRNA is widely expressed and the gene encoding TRAIL‐R2 is located on human chromosome 8p22‐21. Like TRAIL‐R1, TRAIL‐R2 engages a caspase‐dependent apoptotic pathway but, in contrast to TRAIL‐R1, TRAIL‐R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.

Identification of a ligand for the c-kit proto-oncogene

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.

FADD/MORT1 and Caspase-8 Are Recruited to TRAIL Receptors 1 and 2 and Are Essential for Apoptosis Mediated by TRAIL Receptor 2

Apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L) has been shown to exert important functions during various immunological processes. The involvement of the death adaptor proteins FADD/MORT1, TRADD, and RIP and the apoptosis-initiating caspases-8 and -10 in death signaling by the two death-inducing TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are controversial. Analysis of the native TRAIL death-inducing signaling complex (DISC) revealed ligand-dependent recruitment of FADD/MORT1 and caspase-8. Differential precipitation of ligand-stimulated TRAIL receptors demonstrated that FADD/MORT1 and caspase-8 were recruited to TRAIL-R1 and TRAIL-R2 independently of each other. FADD/MORT1- and caspase-8-deficient Jurkat cells expressing only TRAIL-R2 were resistant to TRAIL-induced apoptosis. Thus, FADD/MORT1 and caspase-8 are essential for apoptosis induction via TRAIL-R2.

A Poxvirus-Encoded Semaphorin Induces Cytokine Production from Monocytes and Binds to a Novel Cellular Semaphorin Receptor, VESPR

The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor indicated that it is a novel member of the plexin family and identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on, and induced cytokine production from, human monocytes. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs.