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This cell line is sensitive to FasL or agonist antibody in the absence of cycloheximide

This cell line is sensitive to FasL or agonist antibody in the absence of cycloheximide. has identified membrane-bound receptors and their cognate ligands that together begin the program that ultimately leads to TAK-632 cell death (5). One class of receptors falls into the tumor necrosis factor (TNF) receptor superfamily of which the Fas receptor (also called CD95 or Apo-1) is usually a member TAK-632 (6, 7). This receptor has three cysteine-rich extracellular domains and an intracellular death domain required for signaling (8). Ligation of the receptor by its cognate ligand FasL (9) or an agonistic antibody (10) leads to the recruitment of an adapter molecule FADD/MORT-1 (11, 12). This conversation is mediated by a death domain name in FADD/MORT1 with the death domain name of Fas. Additionally FADD/MORT1 contains a death effector domain name that recruits the protease caspase-8 (also called FLICE, MACH, and Mch5) to this signaling complex (13C15). This zymogen, through proximity with other caspase-8 molecules, is usually cleaved, rendering it fully active, thus beginning a protease cascade that leads to cell death (16). A counterpoint to this death activation is usually inhibition of cell death. A number of molecules have been identified that act on various components in the pathway. These include a soluble form of Fas (17) and a secreted decoy receptor that binds and competes for the ligand (18). At the level of the receptor, adenoviral MYH10 proteins E3 10.4K/14.5K force endocytosis of Fas and other receptors, thus protecting infected cells and aiding the virus to complete its life cycle (19, 20). A group of proteins called FLIPs, which possess a death effector domain, compete for the recruitment of FADD/MORT1 and caspase-8 at the ligated receptor (21). An extension to this control is the molecule toso (22) that induces c-FLIP expression uniquely in hematopoietic lineages. Finally, a number of molecules have been identified called inhibitors of apoptosis proteins (23), a protein from the cowpox virus (crmA) (24), and adenovirus (E3C14.7K) (25), all of which inhibit caspase activity. The majority of these mechanisms inhibit the signal pathway downstream of the receptor and hence will inhibit the death signal from several receptors, i.e., Fas and TNF- receptor. In this study we report the cloning of a molecule that uniquely inhibits death mediated by Fas, but not TNF- receptor. The cloning relies on a technology for stable gene transfer of representative cDNA libraries to diverse cell types. The high transfer efficiencies enable genetic screens to identify cDNAs either by complementation of mutant cell lines or by virtue of ectopic expression. Materials and Methods TAK-632 cDNA and Library Construction. Total RNA was isolated from the human lung fibroblast cell line MRC-5 (ATCC CCL-171) (26), and poly(A)+ RNA was prepared and converted to double-stranded DNA by using the Superscript Choice system (GIBCO/BRL) according to the manufacturers instructions. Size-fractionated cDNA species ( 500 bp) was ligated into the retroviral vector pCLMFG.MCS (N.S., M.J.S., and I.M.V., unpublished work). The ligation was transformed into supercompetent DH10B (GIBCO/BRL), which generated a cDNA library of 2 106 individual clones. Retroviral Vector Generation. Fifteen micrograms of plasmid DNA prepared from the pooled cDNA clones was transfected with 5 g of an expression vector for vesicular stomatitis virus G protein into a cell line 293 gp/bsr (N.S. and I.M.V., unpublished work). Forty-eight hours later, supernatant made up of the cDNA viral vectors was recovered. The supernatant was used to infect 5 106 HeLa cells and left for 24 hr, after which the media were replaced with a second collection from the transfection for 24 hr. Selection for Resistant Cells. A mouse anti-human Fas mAb, clone CH-11 (10) (Kamiya Biomedical, Thousand Oaks, CA), was used at a final concentration of 500 g/ml. The media were changed every 3C4 days. After 1 week, the surviving clones were pooled into one plate, and selection was maintained for an additional 15 days, with a change of media every 3C4 days. After this time the surviving pool was expanded in the presence of selection, and genomic DNA was isolated. Rescue of Complementing cDNA. PCR was performed by using the TaqPlus Precision system (Stratagene). A cycle of 95C for 30.


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