Raymond G. Goodwin

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.

The Novel Receptor TRAIL-R4 Induces NF-κB and Protects against TRAIL-Mediated Apoptosis, yet Retains an Incomplete Death Domain

A fourth member of the emerging TRAIL receptor family, TRAIL-R4, has been cloned and characterized. TRAIL-R4 encodes a 386-amino acid protein with an extracellular domain showing 58%-70% identity to those of TRAIL-R1, TRAIL-R2, and TRAIL-R3. The signaling capacity of TRAIL-R4 is similar to that of TRAIL-R1 and TRAIL-R2 with respect to NF-kappaB activation, but differs in its inability to induce apoptosis. Yet TRAIL-R4 retains a C-terminal element containing one third of a consensus death domain motif. Transient overexpression of TRAIL-R4 in cells normally sensitive to TRAIL-mediated killing confers complete protection, suggesting that one function of TRAIL-R4 may be inhibition of TRAIL cytotoxicity. Like TRAIL-R1 and TRAIL-R2, this receptor shows widespread tissue expression. The human TRAIL-R4 gene has been mapped to chromosome 8p22-21, clustered with three other TRAIL receptors.

CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF

CD30 is a surface marker for neoplastic cells of Hodgkins lymphoma and shows sequence homology to members of the tumor necrosis factor (TNF) receptor superfamily. Using a chimeric probe consisting of the extracellular domain of CD30 fused to truncated immunoglobulin heavy chains, we expression cloned the cDNA cognate from the murine T cell clone 7B9. The encoded protein is a 239 amino acid type II membrane protein whose C-terminal domain shows significant homology to TNF alpha, TNF beta, and the CD40L. Cross-hybridization to an induced peripheral blood T cell cDNA library yielded the human homolog, which is 72% identical at the amino acid level. The recombinant human ligand enhances the proliferation of CD3-activated T cells yet induces differential responses, including cell death, in several CD30+ lymphoma-derived clones. The human and murine genes map to 9q33 and the proximal region of chromosome 4, respectively.

Cloning of the human and murine interleukin-7 receptors: Demonstration of a soluble form and homology to a new receptor superfamily

cDNA clones encoding the human and murine interleukin-7 (IL-7) receptor were isolated and expressed in COS-7 cells. Binding of radiolabeled IL-7 to the recombinant IL-7 receptors produced curvilinear Scatchard plots containing high and low affinity classes. These binding properties, as well as the molecular size of the cloned receptor, were comparable to the native forms of the IL-7 receptor. In addition, several cDNA clones were isolated that encode a secreted form of the human IL-7 receptor capable of binding IL-7 in solution. Analysis of the sequence of the IL-7 receptor revealed significant homology in the extracellular domain to several recently cloned cytokine receptors, demonstrating that the IL-7 receptor is a member of a new receptor superfamily.

The mouse fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster

The gene for the mouse Fas ligand was cloned and its chromosomal position determined. Fasl was tightly linked to gld (no crossovers in 567 meiotic events) on mouse chromosome 1 and closely linked with a novel member of the same TNF family of ligands, the Ox40 ligand (Ox40l, 1 crossover in 567 meiotic events). Southern blot analysis did not reveal any difference between the Fasl gene from gld and wild-type mice and levels of Fasl mRNA transcripts were similar in PMA and ionomycin induced wild-type and coisogenic gld T cells. Sequence analysis of the gld gene indicated a single amino acid change (Phe Leu) in the COOH terminal portion of this type II transmembrane protein, and COS cells transfected with Fasl cDNA from gld mice failed to induce apoptosis of Fas-expressing target cells. Thus, the data demonstrate that the gld phenotype is the result of a point mutation in the Fasl gene and that Fasl is part of a complex of ligands structurally related to TNF mapping within a small region of mouse chromosome 1.

Molecular and biological characterization of a ligand for CD27 defines a new family of cytokines with homology to tumor necrosis factor

CD27 is a surface antigen found on T and B cells that has homology to a family of molecules including the receptors for tumor necrosis factor (TNF) and nerve growth factor. A cDNA encoding a ligand for CD27 was isolated by a direct-expression cloning strategy using a fusion protein composed of the extracellular domain of CD27 linked to the constant domain of a human immunoglobulin G1 molecule as a probe. The predicted protein product is a type II transmembrane protein whose gene maps to 19p13 and that shows homology to TNF and the ligand for CD40. Biological characterization indicates that the cloned ligand induces the proliferation of costimulated T cells and enhances the generation of cytolytic T cells.

T2 open reading frame from the shope fibroma virus encodes a soluble form of the TNF receptor

A transcriptionally active open reading frame (T2) from Shope Fibroma Virus was recently shown to have striking sequence homology with members of a new superfamily of cell surface proteins, including a receptor for human tumor necrosis factor. Here we report that recombinant T2 protein expressed in COS cells is a soluble, secreted glycoprotein which specifically binds human TNF alpha and beta, and inhibits binding of these cytokines to native TNF receptors on cells. T2 binding of TNF is not inhibited by nerve growth factor, although the nerve growth factor receptor is also a member of the same family, nor by nine other recombinant cytokines. Further, the repeating domain structure of T2 most closely resembles that of the type I TNF receptor (p75) and is significantly different from other family members, including the type II TNF receptor (p55). Since T2 possesses a leader sequence but lacks a transmembrane domain, these results confirm the original suggestion (1) that T2 represents a soluble form of the type I TNF receptor which is secreted from virally infected cells, and whose function is to immunosuppress the host by abrogating the potentially destructive effects of TNF. This is the first such virally-encoded soluble cytokine receptor to be identified, and may represent a more general mechanism by which viruses subvert the host immune system.

Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor.

Clones encoding the type 1 (p80) and type 2 (p60) forms of the murine receptors for tumor necrosis factor (TNF) were isolated by cross-hybridization using probes derived from the cloned human TNF receptors. Each of the murine receptors shows strong sequence homology to the corresponding human receptor (approximately 65% amino acid identity) throughout the molecule but only modest homology, limited to ligand-binding domains, between themselves. The ligand-binding characteristics of the recombinant murine receptors mirror those of the human homologs: both receptor types bind TNF-alpha and -beta with multiple affinity classes, and the ligands cross-compete. Analysis of the murine transcripts encoding these receptors revealed the presence of RNAs for one or both forms of the receptors in all cells examined. It was also demonstrated that for both types of human TNF receptor, variably sized transcripts are observed in different cells. The murine cDNAs were further used to determine the chromosomal locations of the TNF receptor genes. They are not linked, in contrast to the ligands, and map to chromosomes 4 (type 1) and 6 (type 2).