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Deleterious mutations in genes involved in the Fas apoptosis pathway lead to Autoimmune Lymphoproliferative Syndrome (ALPS). Demonstration of an apoptosis defect is critical for the diagnosis and study of ALPS. The traditional in vitro apoptosis assay, however, requires a week of experimental procedures. Here, we show that defects in Fas-induced apoptosis in PBMCs can be evaluated directly ex vivo using multicolor flow cytometry to analyze the apoptosis of effector memory T cells, a Fas-sensitive subset of PBMCs. This method allowed us to sensitively quantify defective apoptosis in ALPS patients within a few hours. Some ALPS patients (ALPS-sFAS) without germline mutations have somatic mutations in Fas specifically in double-negative αβ T cells (DNTs), an unusual lymphocyte population that is characteristically expanded in ALPS. Since DNTs have been notoriously difficult to culture, defective apoptosis has not been previously demonstrated for ALPS-sFAS patients. Using our novel ex vivo apoptosis assay, we measured Fas-induced apoptosis of DNTs for the first time and found that ALPS-sFAS patients had significant apoptosis defects in these cells compared to healthy controls. Hence, this rapid apoptosis assay can expedite the diagnosis of new ALPS patients, including those with somatic mutations, and facilitate clinical and molecular investigation of these diseases. [ABSTRACT FROM AUTHOR]

Recently, we identified Insulinoma-Glucagonoma clone 20 (IG20) that can render cells more susceptible to tumor necrosis factor-alpha (TNF-a)-induced apoptosis. In addition, it can slow cell proliferation, and enhance drug- and radiation-induced cell death. TNF-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in some cancer cells and render others susceptible to cotreatment with drugs and irradiation, with little or no effect on most normal cells. In this study, we investigated the potential of IG20 to enhance TRAIL-induced apoptosis and found that it can render cells more susceptible to TRAIL treatment through enhanced activation of caspases. Further, we showed that this effect can be suppressed by caspase inhibitors, p35 and CrmA, and a dominant-negative Fas-associated death domain-containing protein (DN-FADD). Results from colocalization and immunoprecipitation studies showed that IG20 can interact with TRAIL death receptors (DR), DR4 and DR5 and increase recruitment of FADD and caspase-8 into the TRAIL death-inducing signaling complex (DISC). These results indicate that IG20 is a novel protein that can enhance TRAIL-induced apoptosis by facilitating DISC formation.Oncogene (2004) 23, 6083-6094. doi:10.1038/sj.onc.1207804 Published online 21 June 2004 [ABSTRACT FROM AUTHOR]