Characterization of the human FLICE-inhibitory protein locus and comparison of the anti-apoptotic activity of four different FLIP isoforms

Death receptor-mediated apoptosis is involved in the regulation of immune r esponses and in the maintenance of immunological tolerance. FLICE-inhibitor y proteins (FLIPs) are important modulators of death receptor-mediated apop tosis. To date, the FLIP family encompasses multiple members, of which some are reported to be antiapoptotic and others pro-apoptotic. This led us to investigate the activity of several FLIP proteins in vitro. Concomitant wit h the cloning of various FLIP isoforms, a new and unexpected member of the FLIP family, denoted FLIPR, was isolated from the human Burkitt lymphoma B- cell line Raji. During the characterization of FLIPR, the genomic sequence of human FLIP was found in the NCBI GenBank. This enabled us to present the complete exon-intron constellation of the human FLIP gene and the generati on of all known human FLIP isoforms by alternative splicing. We show that t he human FLIP gene with a size of approximately 48 kb, consists of at least 14 exons and can give rise to 11 distinct isoforms by alternative splicing . When studying the activity of some of these isoforms, including FLIPR, th ey all efficiently inhibited Fas-mediated apoptosis in A20 B lymphoma cells by impeding caspase-8, -3 and -7 activity as well as poly(ADP-ribose) poly merase (PARP) cleavage.