Volume 123, Issue 3, Pages 751-763 (September 2002) The APC tumor suppressor controls entry into S-phase through its ability to regulate the cyclin D/RB pathway  Christopher D. Heinen, Kathleen Heppner Goss, James R. Cornelius, George F. Babcock, Erik S. Knudsen, Timothy Kowalik, Joanna Groden  Gastroenterology  Volume 123, Issue 3, Pages 751-763 (September 2002) DOI: 10.1053/gast.2002.35382 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 GFP-APC down-regulates endogenous, cytoplasmic β-catenin. SW480 cells were transfected with GFP or GFP-APC, stained with an anti–β-catenin antibody, and examined by fluorescent microscopy. Original magnification 400×. In control transfections with GFP (green cells, top left), all cells displayed high levels of β-catenin (red and yellow cells, top middle and right); however, in a cell transfected with GFP-APC (green cell, middle left), the β-catenin staining disappeared (arrow, middle and middle right), demonstrating that transfected APC down-regulates endogenous β-catenin. Cells cotransfected with GFP-APC and a constitutively active β-catenin expression plasmid (S37A) (green cell, bottom left) stained positive for β-catenin (arrow, bottom middle and right), suggesting that the mutant version of β-catenin was not down-regulated by APC. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 GFP-APC transfection increases the G1 population as determined by flow cytometry. SW480 cells transfected with GFP or GFP-APC were labeled with PI and analyzed by flow cytometry. (A) Cell cycle histograms were generated using cells with high GFP fluorescence. Comparing the histogram for cells transfected with GFP-APC vs. GFP revealed an increase in the G1 peak from cells transfected with GFP-APC with a subsequent decrease in S- and G2/M-phase. (B) The approximate percentages of cells in each of the major phases of the cell cycle were calculated using the Multicycle software package. The average difference in the percentage of cells (Y-axis) in each phase of the cell cycle (X-axis) between GFP-APC transfections and GFP transfections from 10 different experiments is graphed above. Percentages of cells in the three phases from GFP transfections were set to zero, and the bars represent the changes for GFP-APC transfections. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 GFP-APC transfection inhibits S-phase entry. SW480 cells transfected with GFP or GFP-APC were cultured in medium containing BrdU for 16 hours. The cells were stained with an anti-BrdU antibody and examined by fluorescent microscopy. Original magnification was 400×. (A) In control transfections with GFP (top three panels), the transfected cells (top left) displayed bright red nuclei when labeled with an anti-BrdU antibody (top middle and right); however, in the GFP-APC–transfected cell (green cell, bottom left), the nucleus remained unstained (arrow, bottom middle and right), indicating a failure to enter S-phase and incorporate BrdU. (B) The percentage of BrdU-positive cells was calculated for counts of 150–200 GFP-positive cells. The average percentage of BrdU positive cells (Y-axis) for 4 separate experiments is graphed. Each bar represents BrdU incorporation for the cells transfected as indicated on the X-axis. Transfection of GFP-APC decreased BrdU-positive cells by 63% compared with nontransfected or GFP-transfected cells. (C) Lysates from SW480 cells transfected with GFP or GFP-APC were examined by Western blotting with an anti–cyclin A antibody. An anti-actin antibody was used to equilibrate loading for each lane. Fluorescent-activated cell sorting was used to collect only transfected cells. Lysates from GFP-APC- transfected cells contained dramatically less cyclin A than lysates from GFP-transfected cells, consistent with the failure of GFP-APC–transfected cells to enter S-phase. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Cotransfection of a constitutively active β-catenin partially abrogates the growth arrest of GFP-APC–transfected cells. SW480 cells cotransfected with GFP-APC, and the indicated amounts of S37A were analyzed for BrdU incorporation (Y-axis). Each bar represents BrdU incorporation for the cells transfected as indicated on the X-axis. The addition of the indicated amounts of S37A with GFP-APC increased BrdU incorporation. This recovery of BrdU incorporation, although substantially above the levels of GFP-APC and the indicated empty DNA (pBabe vector) cotransfections, did not reach the level of that obtained in GFP and S37A cotransfections. Each experiment was performed three times. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 APC-induced G1 arrest results from the inhibition of β-catenin–mediated transcription activity. (A) β-catenin–mediated transcription was measured using a reporter construct containing three novel TCF binding sites upstream of a minimal c-Fos promoter driving luciferase (pTOPFLASH). pTOPFLASH was cotransfected into SW480 cells with GFP or GFP-APC, with or without S37A. Luciferase values were measured from lysates of the transfected cells. These values were corrected for cell number and transfection efficiency by cotransfecting pβ-gal and assaying for β-galactosidase activity, and for nonspecific transcription using values from parallel transfections with a mutant version of the reporter (pFOPFLASH). β-catenin–mediated transcription (X-axis) for each cotransfection (indicated on Y-axis) is reported as a percentage of the control (GFP cotransfection). GFP-APC cotransfection resulted in a 5-fold decrease in β-catenin–mediated transcription compared with GFP. Cotransfection of S37A and GFP-APC resulted in a less than 2-fold difference in β-catenin–mediated transcription compared with GFP. Each experiment was performed three times. (B) SW480 cells cotransfected with GFP-APC, S37A, and a dominant-negative version of human LEF-1 (ΔN67) were analyzed for BrdU incorporation (Y-axis). Each bar represents BrdU incorporation for the cells transfected as indicated on the X-axis. The addition of dominant-negative LEF-1 to GFP-APC and S37A-transfected cells decreased BrdU incorporation compared with GFP-APC and S37A-transfected cells. This decrease was similar to the level seen in GFP-APC and empty pBabe vector (DNA) cotransfections. Cotransfection of dominant-negative LEF-1 with GFP decreased BrdU incorporation compared with GFP and S37A cotransfections, but not to the extent observed in GFP-APC transfections. Each experiment was performed three times. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Cotransfection of E1a or E2F-1, -2, or -3 partially abrogates the growth arrest of GFP-APC–transfected cells. (A) Cotransfection of SW480 cells with E1a and GFP-APC, shown on the X-axis, increased BrdU incorporation (Y-axis) compared with GFP-APC and empty pBabe vector (DNA) cotransfections. This increase was similar to the increase observed with cotransfections of GFP-APC and S37A. A mutant E1a (E1aΔ) failed to reverse the growth arrest when cotransfected with GFP-APC. (B) Cotransfection of SW480 cells with E2F-1, -2, or -3 and GFP-APC, shown on the X-axis, increased BrdU incorporation (Y-axis) compared with GFP-APC and empty pBabe vector (DNA) cotransfections. This increase was similar to E1a cotransfections. (C) Cotransfection of either E2F-1, -2, or -3 and GFP-APC and S37A, shown on the X-axis, did not increase BrdU incorporation (Y-axis) compared with cotransfections of GFP-APC with E2F-1, -2, or -3, or GFP-APC and S37A. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 GFP-APC transfection arrests cells in G1 by inhibiting phosphorylation of RB. (A) Lysates generated from SW480 cells transfected with GFP or GFP-APC were examined by Western blotting with an anti-RB antibody. Fluorescent-activated cell sorting was used to collect transfected cells. Lysates from GFP-APC–transfected cells contained decreased hyperphosphorylated RB and subsequently increased hypophosphorylated RB compared with lysates from GFP- transfected cells. RBP, hyperphosphorylated RB; RBO, hypophosphorylated RB. Lysates generated from the prostatic adenocarcinoma cell line LNCaP cultured with or without androgen were used as controls.34 (B) Lysates generated from SW480 cells transfected with GFP or GFP-APC were examined by Western blotting with an anti–cyclin D1 antibody. An anti-actin antibody was used to equilibrate loading. Fluorescent-activated cell sorting was used to collect transfected cells. Lysates from GFP-APC–transfected cells contained 4.5-fold less cyclin D1 than lysates from GFP-transfected cells. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Cotransfection of cyclin D1 and c-MYC partially abrogates the growth arrest of GFP-APC–transfected cells. (A) Cotransfection of SW480 cells with cyclin D1 and GFP-APC (X-axis) increased BrdU incorporation (Y-axis) compared with GFP-APC and empty pBabe vector (DNA) cotransfections. This increase was similar to the increase observed with cotransfections of GFP-APC and oncogenic ß-catenin (S37A). Cotransfection of SW480 cells with c-MYC and GFP-APC (X-axis) increased BrdU incorporation (Y-axis) compared with GFP-APC and empty pBabe vector (DNA) cotransfections, although not to the extent of GFP-APC and cyclin D1 cotransfections. Cotransfection of cyclin D1, c-MYC, and GFP-APC (X-axis) also increased BrdU incorporation (Y-axis) compared with cotransfections of GFP-APC alone. These levels were similar to those observed in cotransfections of GFP-APC and cyclin D1 only. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 APC down-regulation of β-catenin functions upstream of RB in the cyclin D/RB pathway to prevent entry into S-phase. APC arrests SW480 colon cancer cells in G1 by inhibiting β-catenin–mediated gene transcription. This arrest is overcome by overexpressing a constitutively active β-catenin, E2F-1, -2, -3, or E1a and is characterized by reduced RB phosphorylation. The ability of APC to down-regulate β-catenin and function in the cyclin D/RB pathway may involve β-catenin effects on the gene activity of cyclin D1 and c-MYC.8,9 Therefore, mutation of APC would interfere with the dynamics of the G1 regulatory machinery, providing a mechanism by which most colon tumors disrupt G1/S progression. However, the possibility remains that APC functions additionally to arrest some cells in G1, independently of the cyclin D/RB pathway. Gastroenterology 2002 123, 751-763DOI: (10.1053/gast.2002.35382) Copyright © 2002 American Gastroenterological Association Terms and Conditions