The product of the FAC gene does not directly inactivate the fas pathway. B-cell lines from normal volunteers and isogenic cell lines from children with FA-C were exposed to mitomycin C, or to agonistic anti-fas antibodies, and were analyzed for apoptosis using the TUNEL assay. Results are expressed as percent apoptotic cells (mean ± SD). (A) Mitomycin C hypersensitivity was noted in FAC mutant cells (HSC536N). HSC536N cells transduced with a retroviral vector expressing only the neomycin phosphotransferase gene (HSC536N/neo) were also hypersensitive. Transduction of normal FAC cDNA (HSC536N/FAC/neo) corrects mitomycin C–induced apoptosis in FAC cells. JY cells are EBV-transformed cells from a normal volunteer. (B) Exposure of the isogenic cells to agonistic anti-fas antibody induced substantial apoptosis (quantified 48 hours after exposure to the antibody) in all three of the isogenic sets (only one is shown here). (C) Flow cytometric analysis of the isogenic cells shown in (B) indicated that fas expression in HSC536N and HSC536N/neo was similar to that of normal EBV-transformed cells and the corrected FA-C cells (HSC536N FAC/neo). The group of low-intensity peaks in the histogram (on the left) represent binding of an isotypic control antibody with each of the four cell types; the high-intensity group on the right shows binding of an anti-fas antibody with the same cell lines. (D) Kinetics of Stat1 phosphorylation differed between isogenic lines. Shown is an immunoblot analysis of phosphorylated Stat1 0, 15, 30, and 60 minutes after exposure of the cell lines to IFN-γ. JY and the complemented FA-C cells (HSC536N fac ) demonstrate a normal inductive response that declines after 30 minutes. HSC536N neo and HSC536N cells show constitutive phosphorylation of Stat1, largely the β splice form, a minor inductive effect of IFN-γ on Stat1α, and persistence of the phosphoprotein for more than 60 minutes after exposure to IFN-γ.