Potentiation of Fas-Mediated Apoptosis of Murine Granulosa Cells by Interferon-γ, Tumor Necrosis Factor-α, and CycloheximideThis work was supported by NIH Grant HD-32535

• Published in: Endocrinology (Philadelphia) 1998-12, Vol.139 (12), p.4860-4869
• Main Authors: Quirk, Susan M., Porter, Dale A., Huber, Sarah C., Cowan, Robert G.
• Format: Article
• Language: English
• Subjects: Annexin V; Antibodies; Antigens; Apoptosis; CD95 antigen; Cell death; Cell surface receptors; Cycloheximide; Cytokines; Cytotoxicity; DNA fragmentation; Fas antigen; FasL protein; Follicles; Gene expression; Granulosa cells; Immunocytochemistry; Immunoglobulin G; Inhibitors; Interferon; Monoclonal antibodies; Mortality; Pretreatment; Protein biosynthesis; Protein folding; Protein synthesis; Proteins; Receptors; Toxicity; Tumor necrosis factor receptors; Tumor necrosis factor-TNF; Tumor necrosis factor-α; γ-Interferon
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.139.12.6353

Abstract The Fas antigen is a transmembrane receptor belonging to the tumor necrosis factor-α (TNF) receptor family that, when activated by Fas ligand or agonistic antibodies, induces death by apoptosis. Although the presence of Fas antigen in ovarian tissues has been demonstrated, little is known about whether Fas antigen is functional in the ovary. This report shows that murine granulosa cells are initially resistant to antibody-induced Fas-mediated apoptosis, but will undergo apoptosis when cotreated with TNF and interferon-γ (IFN) or cycloheximide (CX). Granulosa cells were obtained from follicles of 23-day-old mice 2 days after injection of PMSG. Twenty-four hours after plating, cells were pretreated with either 0 or 200 U/ml IFN, which has been shown to induce Fas antigen expression and is required for Fas-mediated killing in many cell types. At 48 h, cells were treated with 2 μg/ml control IgG, 2 μg/ml anti-Fas antigen antibody (Fas mAb), 10 ng/ml TNF, or Fas mAb and TNF. Cytotoxicity (percent killing) relative to control IgG was determined at 72 h by counting granulosa cells after trypsinization. In the absence of IFN, no cytotoxicity was observed. In the presence of IFN, neither TNF or Fas mAb alone was cytotoxic, but the combination of Fas mAb and TNF resulted in 25% killing (P < 0.05). Fas antigen messenger RNA (mRNA) was detectable in cultures not treated with cytokines and was increased 5-fold by TNF, 2-fold by IFN, and 17-fold by the combination of IFN and TNF. To test whether the presence of a labile inhibitor(s) of Fas-mediated killing in granulosa cells is the cause of resistance to Fas mAb, the protein synthesis inhibitor CX was used. Experiments were performed as described above, except that cells were treated with 0.5 μg/ml CX in conjunction with other treatments at 48 h. Fas mAb treatment in the presence of CX induced 25% cell death without IFN pretreatment and 38% with IFN (P < 0.05). TNF treatment in the presence of CX had no effect alone, but potentiated the effects of Fas mAb, resulting in 56% killing in the absence of IFN and 86% killing in the presence of IFN (P < 0.05). Cells stained positively for DNA fragmentation and annexin V binding, features characteristic of apoptosis. Because initial experiments showed that treatment with TNF alone increased Fas mRNA levels, the effect of pretreating cells for 24 h with TNF before treatment with Fas mAb was tested. Pretreatment with TNF or IFN alone did not promote Fas mAb-mediated killi