Induction of Cell Death by RELT, a Recently Identified TNFR

Tumor Necrosis Factor Receptor (TNFR) family members induce a variety of cellular responses including apoptotic cell death and are implicated in a variety of human diseases including cancer, inflammation and periodontal disease. RELT (Receptor Expressed in Lymphoid Tissues) is a recently identified member of this family whose function is largely unknown. Previous results suggest that RELT induces cell death in human epithelial cells. Objectives: To define the portion of the RELT protein required to induce cell death in human epithelial cells. Methods: The ability of different RELT deletion mutant constructs to induce cell death in human epithelial cells was examined. Mutagenesis of the RELT gene using site-directed mutagenesis followed by transformation of mutated plasmid constructs into E. coli cells was performed. Plasmid DNA was isolated from E. coli clones and automated DNA sequencing was utilized to verify creation of mutant constructs. Transient transfection of HEK-293 cells was used to examine the effect of expressing mutant RELT constructs in a human epithelial cell line. Cell death was assayed by both X-gal staining and TUNEL staining to detect fragmented DNA. Expression of wild-type RELT and Caspase-8 were used as positive controls, expression of empty plasmid vector was used as a negative control. Western blots were used to confirm expression of mutant protein constructs. Results: Four intracellular deletion mutants of RELT were successfully created that were used in combination with previous collection of RELT deletion mutants. The expression of most proteins was verified. The mutant protein constructs retained varying abilities to induce apoptosis morphology and DNA fragmentation in HEK cell line. Conclusion: Expression of RELT in HEK-293 cells induces apoptotic like morphology and DNA fragmentation supporting previous results. Preliminary results suggest that there may not be one single intracellular domain of RELT responsible for inducing cell death. Support: NIH/NIDCR U24 DE016502