A cancer/testis antigen CAGE is widely expressed in various cancer tissues and cancer cell lines but not in normal tissues except the testis. cyclin-dependent kinases and subsequently accelerating the G1 to S progression. Moreover increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even in a growth arrested state indicating a direct effect of CAGE on G1 cyclin expression. CAGE-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors and among the AP-1 members c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced retinoblastoma phosphorylation and subsequent E2F-1 nuclear translocation. In contrast Rabbit Polyclonal to BRI3B. small interfering RNA-mediated knockdown of CAGE suppressed the expression of G1 cyclins activation of AP-1 and E2F-1 and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclins D1 and E. gene displays testis-specific expression among normal tissues and cancer-specific expression among various human cancer tissues and cell lines particularly originated Isoliquiritin from gastric Isoliquiritin cervical Isoliquiritin lung liver kidney and colon cancers. Also like many C/T antigens the gene is localized to the X chromosome. Additionally CAGE expression was found to be epigenetically regulated depending on the methylation status of CpG sites of the gene the functional effect of CAGE overexpression on cell proliferation and tumor growth was assessed by an cell culture system and an xenograft tumor model respectively. Here we demonstrate the cell proliferation-stimulating activity of the C/T antigen CAGE and its function in promoting G1 progression in the cell cycle. These results may provide insight into the potential mechanism and role of many other C/T antigens in cancer development and progression. EXPERIMENTAL PROCEDURES Cell Culture Antibodies and Reagents Tet-On sublines of HeLa human cervical cancer cells obtained from Clontech Laboratories (Mountain View CA) were cultured in DMEM supplemented with 10% Tet system-approved fetal bovine serum Isoliquiritin (Clontech) 100 units/ml penicillin 100 μg/ml streptomycin and 200 μg/ml G418 in 5% CO2 at 37 °C. NIH3T3 mouse fibroblast cells were cultured in DMEM supplemented with 10% calf serum. Preparation of anti-CAGE antibody was described in a previous study (9). Monoclonal antibodies against cyclin D1 (M-20) cyclin E (HE12) cyclin A (BF683) cyclin B (GNS1) CDK4 (C-22) CDK2 (M2) Rb (IF8) p53 (DO-1) E2F-1 (KH95) p15 (K-18) p16 (N-20) p18 (18P118) p19 (DCS-100) p21 (F-5) p27 Isoliquiritin (F-8) JunB (210) JunD (329) c-Fos (D-1) FosB (C-11) Fra-1 (C-12) Fra-2 (L-15) and p65 (SC-109X) were purchased from Santa Cruz Biotechnology (Santa Cruz CA). Antibodies specific to phospho-RbSer-795 phospho-c-JunSer-63/Ser-73 and c-Jun were obtained from Cell Signaling Technology (Danvers MA) and an antibody to histone H3 was obtained from Upstate Chemicon (Temecula CA). All other reagents were from Sigma unless otherwise indicated. Generation of Stable Tetracycline-inducible CAGE Transfectant Clones of HeLa Cells Full-length CAGE cDNA was subcloned into the site downstream of a tetracycline-responsive transactivator-binding promoter of the pTRE2 vector (Clontech) and transfected into HeLa/Tet-On cells using Lipofectamine/PLUS reagent (Invitrogen). CAGE transfectant clones of HeLa/Tet-On cells were isolated by growing the cells in DMEM containing 10 Tet system-approved fetal bovine serum 400 μg/ml G418 and 200 μg/ml hygromycin. Stable CAGE transfectant clones cultured in the absence or presence of doxycycline (1 μg/ml) were characterized by RT-PCR and immunoblotting analyses for the CAGE expression level. Selected transfectant clones with the doxycycline-inducible gene were maintained with working concentrations of 200 μg/ml for G418 and 100 μg/ml for hygromycin. Clonogenic Soft Agar Assay Tetracycline-inducible CAGE stable transfectant clones of HeLa/Tet-On cells were suspended in DMEM.