Chapter 22 Methods of study of tumor necrosis factor-related ligands in apoptosis

• Published in: Methods in Cell Biology Vol. 66; pp. 499 - 526
• Main Authors: Rooney, Isabelle A., Benedict, Chris A., Norris, Paula S., Ware, Carl F.
• Format: Book Chapter
• Language: English
• Published: Elsevier Science & Technology 2001
• ISBN: 0125441657; 9780125441650
• ISSN: 0091-679X
• DOI: 10.1016/S0091-679X(01)66023-0

This chapter describes production and characterization of standard reagents used in tumor necrosis factor (TNF) research, and discusses application of these tools to study the proapoptotic effects of TNF-related ligands on cells and their modulation by viruses. The tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily currently contains more than 20 ligand-receptor systems. TNF-related ligands are type II transmembrane proteins that generally form trimers. These multivalent ligands initiate signal transduction by aggregation of their specific cell surface receptors, which are type I transmembrane glycoproteins. The chapter discusses that direct measurement of apoptotic potential is performed by examining the effect of a ligand in cell culture models, either expressed on cell membranes or produced in soluble form. Therefore, an important preliminary step in the characterization of a new TNF family ligand is cloning and expression of the ligand in a form that allows accurate quantification and standardization of apoptotic responses. Key reagents include: (1) soluble recombinant TNF family ligands; (2) cells expressing full-length, membrane-bound ligands; and (3) soluble, recombinant forms of TNF receptor proteins.