Human biliary glycoprotein (BGP)1 is a member of the CEA gene family, a gene cluster on chromosome 19, including CEA, BGP, nonspecific cross-reacting antigen, and pregnancy-specific glycoprotein (1-5). BGP was first described as a CEA cross-reacting substance in bile (6) and was localized to the cell surface of bile canaliculi in the liver and gallbladder mucosa (7). Molecular cloning studies on BGP (8, 9) revealed that it exists in alternatively spliced isoforms from a single gene (9). BGP has a transmembrane domain and, depending on alternative splicing, a long (71 amino acids) or short (10 amino acids) cytoplasmic domain. The long form can be phosphorylated by protein kinases (10) after activation by antibody binding (11) We have previously shown that type II interferon, interferon-␥ (IFN-␥), is capable of up-regulating both the BGP and CEA genes, but by different mechanisms (22). CEA is slowly induced by IFN-␥ over a period of 24 -72 h, requiring new protein synthesis, while BGP is rapidly induced from 4 -24 h, without significant inhibition by cycloheximide (22). Rapid induction of gene expression by IFN-␥ is known to be mediated by GAS (for a review, see Ref. 23), found in a variety of genes, including interferon regulatory factor-1 (IRF-1). IRF-1, in turn, can activate a large number of genes by binding to the interferon-stimulated response element (ISRE). ISRE was previously identified for genes activated by type I interferons, IFN-␣ and IFN-␤. Thus, gene activation by IRF-1 is a major crossover pathway for gene activation by type I and II interferons. The mRNA for IRF-1 has a short half-life (30 min) (24) and is rapidly down-regulated by IRF-2 by competing for the same ISRE-binding site (25,26). Because of their potent effects on cell growth, IRF-1 has been termed a tumor suppressor gene and IRF-2 an oncogene (27).In this report, we show that the BGP promoter has an ISRE that is specifically activated by IRF-1 after treatment of colorectal or HeLa (cervical) cells with IFN-␥. IRF-1 mRNA is superinduced in these cells by cycloheximide, and a major species of BGP mRNA is inhibited by cycloheximide. We also show that both USF and Sp1 bind to another footprint in the BGP promoter and that an Sp1-like protein is induced by IFN-␥ in HT-29 cells. MATERIALS AND METHODS Cell Culture and TreatmentThe colon carcinoma cell lines HT-29 and SW403 and the cervix carcinoma cell line HeLa were obtained from American Type Culture Collection. The cells (0.5 ϫ 10 6 cells/ml) were suspended in 20 ml of medium in 75-cm 2 flasks, allowed to reach semiconfluency (3 days), and