Up-regulation of cyclin D1 promoter activity and the protein expression by β-catenin. (A) The 293 cells were transfected with 0.4 μg of cyclin D1 reporter (−1745CD1LUC) along with increasing amounts of the wild-type human β-catenin expression plasmid. (B) Cells were transfected with 0.4 μg of cyclin D1 reporter (−1745CD1LUC) with 1.0 μg of β-catenin expression plasmid and 1.0 μg of different negative β-catenin/Tcf regulators or with control pcDNA3 plasmid. (C) Cyclin D1 reporters (0.4 μg) (−1745CD1LUC, −163CD1LUC, and its mutant, 163CD1LUCm, which had an AT to GC change at nucleotides −75 and −74) and 1.5 μg of β-catenin expression plasmid were transfected into 293 cells. (D) The whole cell lysate of 293 cells, 293 cells transfected with empty vector, and two constitutively-activated β-catenin stable transfectants were separated by SDS/PAGE and analyzed by Western blotting with antibodies that recognized myc-tagged β-catenin, cyclin D1 (purchased from NeoMarkers), and α-actin (purchased from Oncogene).

Correlation between cyclin D1 expression and β-catenin/Tcf4 activity in breast cancer cell lines. (A) Cell lysates from different breast cell lines were separated by SDS/PAGE and analyzed by Western blotting with antibodies, which recognized cyclin D1 and α-actin (Top). The relative β-catenin/Tcf4 activity in different cell lines were determined by the TOP/FOP luciferase activities in each cells (Middle). The density of cyclin D1 bands were quantitated (by NIH image, an analyzing software) and plotted with β-catenin/Tcf4 activity (TOP/FOP) with the r = 0.967 by linear regression. Also, the DNA-binding activities of β-catenin/Tcf4 were determined by gel-shift assay as described previously (Bottom) (4). Lanes 1–8 show the β-catenin/Tcf4-binding activity in indicated cell lines. Lanes 9–12 are the controls to demonstrate the specific binding. Lane 9 and 10, the same as lane 8 except 60-fold excess of wild-type (lane 9) or the mutant (lane 10) cold oligonucleotide was added; lane 11, nuclear extract was from the 293 vector control line; lane 12, nuclear extract was from 293 β-catenin stable line. (B) MCF-7 cells (Top) or HBL100 cells (Bottom) were cotransfected with cyclin D1 reporter (−1745CD1LUC) with different negative β-catenin/Tcf regulators or with the control pcDNA3 plasmid. The absolute luciferase activity of cyclin D1 reporter alone in MCF-7 cells was ≈7-fold higher than that in HBL100 cells.

Correlation between activated β-catenin and cyclin D1 overexpression and their association with poor patient survival rate. (A) Breast cancer tissue stained with β-catenin antibody (a, cytoplasm/nucleus; b, membrane) and cyclin D1 antibody (c, overexpression; d, negative). The right panels (e–h) showed the respective negative controls for a-d using PBS instead of primary antibodies. (B) Kaplan-Meier analysis for survival correlated with the subcellular localization of β-catenin (Top) and cyclin D1 expression (Bottom). The medium of follow-up of patients was 48 months.