title: Regulation of TNFRSF6 (Fas) expression in ataxia telangiectasia cells by ionizing radiation.
abstract: Several studies have shown that ionizing radiation induces transcription of the TNFRSF6 (Fas) gene, leading to augmented TNFRSF6 protein levels at the surface of irradiated cells. We have examined TNFRSF6 expression in an apparently normal lymphocyte line and in a lymphocyte cell line derived from a patient with ataxia telangiectasia (AT) before and after exposure to radiation (0-10 Gy). Plasma membranes were isolated from normal lymphocytes and AT cells and subjected to Western blot analysis, using a TNFRSF6-specific monoclonal antibody to probe resolved proteins transferred onto nitrocellulose membranes. In both cell types, the presence of a 48-kDa band corresponding to the molecular mass of TNFRSF6 was revealed. Analysis of FITC-conjugated anti-TNFRSF6 antibody-stained normal lymphocytes and AT cells confirmed TNFRSF6 expression in both cell types. In MTT assays, AT cells treated with agonistic anti-TNFRSF6 Ab (CH.11) displayed a 25.9% decrease in cell viability, relative to cells treated with isotype-matched IgM Ab, suggesting the presence of a biologically active TNFRSF6 receptor at the AT cell surface. Exposure to cycloheximide (0-5 microg/ml), a metabolic inhibitor, enhanced sensitivity of AT cells to CH.11. Normal lymphocytes exhibited increased levels of apoptosis (approximately 34% cell death relative to cells treated with isotype-matched IgM Ab) when exposed to CH.11; however, the degree of cell death was not altered significantly with increasing concentrations of cycloheximide. When AT cells were exposed to 0.1, 0.5, 2 and 10 Gy, the activities of caspases 3 and 8 increased in a dose-dependent manner at 24 h postirradiation and reached a plateau by 72 h. A similar trend for activation of caspase 3 and 8 was observed in normal lymphocytes after irradiation. To assess the roles of TNFRSF6 and/or caspase 8 in radiation-induced cell death of AT and normal lymphocytes, and to determine whether hyper-radiosensitivity in AT cells is correlated with increased activity of these two components of the TNFRSF6 pathway, AT and normal lymphocytes were irradiated in the presence of ZB4, an anti-TNFRSF6 blocking antibody, and a caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was determined by Annexin V staining using flow cytometry. Incubation with ZB4 anti-TNFRSF6 antibody did not alter the fraction of apoptotic cells in either AT cells or normal lymphocytes treated with doses of radiation ranging from 0-10 Gy. In contrast, apoptosis was significantly reduced in both cell lines in the presence of Z-IETD-FMK when samples were exposed to low-dose (< or = 2 Gy) radiation. Relative to control samples (those not incubated with Z-IETD-FMK), no difference in the level of apoptosis was observed in AT or normal lymphocytes treated with 10 Gy. These data indicate that: (a) despite radiation-induced up-regulation of TNFRSF6 at the cell surface, the death-promoting receptor does not play a role in radiation-mediated cytotoxicity; (b) apoptosis in lymphocytes irradiated with low (< or = 2 Gy) but not high doses (>2 Gy) proceeds at least in part through activation of caspase 8; and (3) since blocking anti-TNFRSF6 antibody (ZB4) did not reduce levels of apoptosis in irradiated AT cells to those of normal lymphocytes, TNFRSF6 is unlikely to play a significant role in the hyper-radiosensitivity exhibited by cells having the AT phenotype.
authors.affiliation: Department of Medicine, Bridgeport Hospital, Yale University School of Medicine, Connecticut 06610, USA.
authors.forename: J
authors.initials: J
authors.lastname: Albanese
authors.forename: N
authors.initials: N
authors.lastname: Dainiak
mesh_terms: D017304:Annexin A5 / Q000096:biosynthesis; D017209:Apoptosis / Q000528:radiation effects; D001260:Ataxia Telangiectasia / Q000378:metabolism* / Q000473:pathology; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000528:radiation effects*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects*; D009842:Oligopeptides / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014965:X-Rays; D019014:fas Receptor / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D017304:Annexin A5; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011500:Protein Synthesis Inhibitors; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D019014:fas Receptor; D003513:Cycloheximide; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases