Conceived and designed the experiments: YW NO KLN. Performed the experiments: YW KLN. Analyzed the data: YW KLN. Contributed reagents/materials/analysis tools: RJC NO. Wrote the paper: YW KLN. Participated in manuscript revision: RJC NO.
The authors have declared that no competing interests exist.
Truncating mutations in the tumor suppressor gene
We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo IIα in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo IIα isoform and lacking topo IIβ did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo IIα. Of note, although M3-APC can bind β-catenin, the G2 arrest did not correlate with β-catenin expression or activity, similar to what was seen with M2-APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2-APC region.
Together, our data establish that the 20-amino acid repeat region of APC interacts with topo IIα to enhance its activity
Mutation of the tumor suppressor
Among the multiple functions of APC identified is cell cycle regulation
Besides being an enzyme that catalyzes DNA topology changes
Previously, we found full-length endogenous APC interacts with endogenous topo IIα but not with topo IIβ
Previously, we identified an interaction between endogenous APC and topo IIα
(A) Schematic diagram of APC protein showing location of M2 and M3-APC fused to the C-terminus of EGFP and used in all cell studies. (B) A GFP antibody was used to immunoprecipitate (IP) EGFP from HCT116βw cells expressing either EGFP-M2-APC, EGFP-M3-APC or EGFP. Immunoblots (IB) were probed using antibodies indicated to the left of the gel. Ten percent input equals 10 µg total protein. Topo IIα co-precipitates with both M2-APC and M3-APC, but not with EGFP. Topo IIβ does not co-precipitate with M2- or M3-APC, but β-catenin does co-precipitate with both. Blots probed for α-tubulin served as a loading control for the input samples and a negative control for the immunoprecipitations. (*) marks migration of antibody heavy chain. Representative blots from three independent experiments are shown.
Given that exogenous M2- and M3-APC each interact with endogenous topo IIα, we tested whether both APC fragments would also influence two different reactions catalyzed by topo IIα. Purified non-overlapping recombinant M2- and M3-APC fragments each enhanced the ability of purified topo IIα to resolve highly catenated kinetoplast DNA into decatenated mini DNA circles
(A) Purified recombinant human topo IIα (0.12 µM) could slightly decatenate catenated DNA (catDNA) (lane 4). Addition of increasing amounts (0.12, 0.24, and 0.6 µM) of purified recombinant M2-APC (amino acid 1000–1326, lanes 5–7) or non-overlapping M3-APC (amino acid 1330–2058, lanes 8–10) resulted in progressively enhanced topo IIα DNA decatenation activity. M2-, or M3-APC (0.6 µM) alone did not display decatenation activity in the absence of topo IIα(lanes 2 and 3, respectively). (B) Using a higher concentration of topo IIα (0.18 µM) that displays slightly more activity in the absence of other proteins, the addition of M2- and M3-APC (0.18 µM) enhances topo IIα activity (lanes 7 and 8, respectively). In contrast, BSA (0.18 µM) did not enhance the DNA decatenation activity of topo IIα (lane 6). cat DNA, catenated kinetoplast DNA (kDNA); decat DNA, decatenated kDNA. (C) Purified recombinant human topo IIα (0.35 µM) could slightly relax supercoiled pBR322 plasmid DNA (lane 4). Addition of increasing amounts (0.35, 0.70, and 1.35 µM) of purified recombinant M2-APC (lanes 5–7) or M3-APC (lanes 8–10) resulted in progressively relaxed plasmids as indicated by slower migrating bands. M2-, or M3-APC (0.70 µM) did not display relaxation activity in the absence of topo IIα (lanes 2 and 3, respectively). (D) Under conditions where topo IIα displayed moderate plasmid relaxation activity, even in the absence of other proteins (lane 2), addition of BSA (0.35 µM) did not enhance this activity (lane 3), whereas addition of either M2- or M3-APC did (note reduction in faster migrating, highly supercoiled forms of DNA in lanes 5 and 7 compared to lane 2). (A–D) Representative assays from at least four independent experiments are shown.
M2- and M3-APC each bind endogenous topo IIα in cells (
(A) Histograms showing representative FACS displays of cell cycle distribution assessed by Hoechst blue staining at 24, 48, and 72 hours post-transfection with expression constructs for EGFP fused M2- or M3-APC, or EGFP alone. Only EGFP-positive cells are displayed. (B) Bar graphs show FACS-based cell cycle distribution at 24, 48, and 72 hours post-transfection as the average of three independent experiments. Error bars represent standard deviation. When compared to cells expressing only EGFP, by 72 hours post-transfection, the fraction of M2-APC expressing cells in G2/M increased by 2.4-fold, and the S phase decreased by 77%; the fraction of M3-APC expressing cells in G2/M increased by 2-fold, and the S phase decreased by 31%. (C) Live cell scoring for mitotic indices of 100 EGFP-positive cells 24 hours and 48 hours post-transfection. Three independent experiments revealed no change in the mitotic population when cells expressed M2 (24 hours, 2±1,
M2-APC expression elicits cell accumulation in the G2 phase rather than in mitosis
Tumor suppressor p53 participates in various pathways that regulate the G2/M transition (reviewed in
Graph shows the G2/M population from HL-60 and HL-60/MX2 cells expressing EGFP-M2- or M3-APC for 48 hours. Parental HL60 cells exhibit a 2-fold increase in G2/M when expressing M2-APC and a 4-fold increase when expressing M3-APC. A student's t-test demonstrated that these increases in G2/M were significant for M2 (* p = 0.02) and M3 (** p = 0.03), compared to cells expressing only EGFP. HL60/MX2 cells exhibit a decrease in G2/M when expressing M2- or M3-APC. However, these differences were not significant: M2 (
Based on our data, we hypothesize that exogenous M2- and M3-APC each interact with endogenous topo IIα, resulting in a p53-independent cell cycle arrest in G2. Ideally, to demonstrate that this APC-mediated cell cycle regulation is dependent upon topo IIα, we would eliminate topo IIα from the analyzed cells. Unfortunately, topo IIα is an essential protein and its perturbation typically results in cell cycle delay followed by cell death
The majority of somatic
(A) SW480 cells with endogenous truncated APC and expressing M2-, M3-APC or EGFP alone have similar cell cycle distributions. Histograms showing FACS analysis of EGFP-positive cells at 48 hours post-transfection. (B) Bar graphs show the average cell cycle distribution of SW480 cells expressing EGFP, M2 or M3-APC from three independent experiments. Error bars represent standard deviation.
Cell Line | APC mutation 1 |
APC mutation 2 |
Inclusion of M2/M3 in APC truncation | Karyotype | Reference |
C106 | 1238 | 1490 | M2 | 79 | |
C70 | 1309 | LOH | M2 | 115–130 | |
C84 | 1451 | 2843 | M2 | 56 | |
C99 | 1367 | LOH | M2 | 52 | |
CaCo/Caco2/TC7 | 1367 | LOH | M2 | 96 | |
CoLo205 | 1554 | 2843 | M2 | 68–75 | |
COLO320 | 810 | LOH | None | 45–58, 53 | |
DLD-1/HCT15 | 1417 | LOH | M2 | 44–47 | |
GP2D | 1444 | LOH | M2 | 45–47 | |
HT29 | 853 | 1555 | M2 | 69–73 | |
HT55 | 1131 | 1308 | M2 | 80 | |
LoVo | 1114 | 1429 | M2 | 47–57 | |
LS1034 | 1309 | LOH | most of M2 | 77 | |
LS411 | 789 | 1556 | M2 | 70–76 | |
SKCO1 | 1317 | 1443 | M2 | 70–80 | |
SW1417 | 1450 | LOH | M2 | 66–71 | |
SW403 | 1197 | 1278 | most of M2 | 60–65; 68 | |
SW480 | 1368 | LOH | M2 | 54–58 | |
SW620 | 1338 | N/D | M2 | 45–57 | |
SW837 | 1450 | LOH | M2 | 38–40 | |
SW948 | 1114 | 1429 | M2 | 67 | |
T84 | 1488 | LOH | M2 | 47–57 | |
VACO4A | 1354 | LOH | M2 | 60–65 | |
VACO5 | 1419 | 1554 | M2 | 43–47 | |
HCA7 | 2843 | 2843 | N/A | 42–43 | |
HCT116 | 2843 | 2843 | N/A | 43–46 | |
LS174T | 2843 | 2843 | N/A | 47,45, 46–47 | |
RKO |
2843 | 2843 | N/A | 45–47 | |
SW48 | 2843 | 2843 | N/A | 46–47 | |
LS180 | 2843 | 2843 | N/A | 45 |
APC status from
APC status from
N/D = Not detected N/A = Not applicable.
A major function of the central region of tumor suppressor APC is to target, β-catenin for proteasome-mediated destruction. We have previously reported that expression of M2-APC does not alter β-catenin localization, level or activity and concluded that the G2 arrest triggered by M2-APC is not likely mediated by β-catenin
(A) HCT116βm cells that express only stabilized β-catenin show accumulation in G2/M when expressing M2- or M3-APC. Histograms showing representative FACS displays of cell cycle distribution assessed by Hoechst blue staining at 48 hours post-transfection. Only EGFP-positive cells are displayed. (B) Bar graphs show FACS-based cell cycle distribution from three independent experiments. Error bars represent standard deviation. When compared to cells expressing only EGFP, by 48 hours post-transfection, the fraction of M2 or M3-APC expressing cells in G2/M increased by 1.7-and 1.6-fold, respectively. (C) Expression of M2-APC does not alter β-catenin activity in HCT116βw (β-cat wt) or HCT116βm (β-cat mut) cells; however, expression of M3-APC leads to distinct changes of β-catenin activity in the two cell lines. Luciferase activities were determined 48 hours post-transfection and activity of the β-catenin reporter construct (TOP-flash) was normalized against both pRL-TK Renilla activity and FOP-flash reporter activity. p values for HCT116βm (β-cat mut) cells are
Although derived from human colon cancer tissue, the HCT116 cell line retains a stable diploid karyotype. Not only did HCT116 cells expressing either M2- or M3-APC show progressive G2 arrest, but they displayed a significant increase in aneuploidy (
Aneuploid cells were quantified by FACS analysis as shown in
In this study, we identified a novel topo IIα binding domain (M3) in the central region of APC that enhances both decatenation and relaxation activities of purified topo IIα. Cells expressing M2- or M3-APC accumulated in the G2 phase of the cell cycle and showed increased aneuploidy; however, this result was not observed in cells with endogenous truncated APC missing part of the M3 domain. The G2 arrest was also independent of p53 but was dependent on topo IIα. Our data indicate the central region of APC interacts with topo IIα and thereby regulates G2-M cell cycle progression.
Topo IIα and topo IIβ are 75% identical in protein sequence and share some functional similarity in promoting DNA topology changes. However, successful generation of cell lines and mouse models completely lacking topo IIβ
We previously reported that expression of M2-APC in HCT116βw cells leads to abnormal nuclear morphology and G2 cell cycle arrest
Although both M2- and M3-APC bind topo IIα and trigger G2 cell cycle arrest when expressed in HCT116βw, HCT116βm, or HL60 cells, the cellular response to the two fragments is not identical. Altered nuclear morphology was observed 24 hours after expression of M2-APC
Over 60% of FAP polyps display aneuploidy
We propose that expression of M2- or M3-APC causes altered topo IIα activity, thus activating the G2 decatenation checkpoint, which leads to G2 arrest. Aneuploid cells would result from altered topo IIα activity in the small percentage of mitotic cells that escape the G2 decatenation checkpoint. The G2 decatenation checkpoint is vital for cell cycle control and genomic integrity. Cells lacking the G2 decatenation checkpoint become aneuploid
HCT116βw (containing one wild-type allele of β-catenin) and HCT116βm (containing one mutant allele of β-catenin) cells (a gift from Dr. Bert Vogelstein) and SW480 (ATCC) were grown in McCoy's 5A medium (Gibco) supplemented with 10% FBS (Hyclone). HL60 cells (ATCC) were grown in Iscove's Modified Dulbecco's Medium (ATCC) supplemented with 20% FBS (Hyclone). HL60/MX2 (ATCC) were grown in RPMI 1640 medium (Cellgro) supplemented with 10% FBS. Expression constructs for APC fragments fused to EGFP were kindly provided by Dr. Naoki Watanabe and have been described previously
HCT116βw cells were transfected using Lipofectamine2000 reagent according to the manufacturer's protocol (Invitrogen). Transfection efficiencies estimated by FACs analysis were, on average, 48% for EGFP-M2-APC and 62% for EGFP-M3-APC. Estimated relative levels of M2-APC, M3-APC and full-length endogenous APC in whole cell lysates were 1.6∶0.7∶1. Immunoprecipitation (IP) and immunoblots (IB) were performed using anti-GFP pAb (Invitrogen) as described
Cells grown on plastic were treated with trypsin to obtain a single cell suspension. A total of 2 µg of EGFP, EGFP fused M2-, or M3-APC expression plasmid were electroporated using Nucleofector I (Amaxa) according to the manufacturer's protocol. Electroporation programs used were: HCT116βw (program D-32), HCT116βm (program D-32), HL60 (program T-19), and HL60/MX2 (program X-03). SW480 cells were transfected with Metafectine (Scientifix, Australia). Forty-eight hours post-transfection, single cells in suspension were stained with 0.5 µg/ml Hoechst 33342 (Invitrogen) for 30 minutes at 37°C. FACS analysis was performed using both UV and 488 nm lasers on a 5-laser BD LSRII flow cytometry (BD Bioscience). Ten thousand EGFP-positive cells were collected for each sample. Data were analyzed using BD FACSDiva Software (BD Bioscience) and plotted using WinMDI 2.9.
Recombinant S-tag fused M2-APC (amino acid 1000–1326) and M3-APC (amino acid 1330–2058) were generated as described
Cells transfected with EGFP or EGFP-fused M3-APC were fixed with 4% paraformaldehyde, and immunostaining was performed using anti-phospho-histone H3 (1∶500, Upstate) as described
HCT116βw and HCT116βm cells grown in 24-well plates were co-transfected using Metafectine reagent (Scientifix, Australia) with 2 µg of the EGFP-M2-APC, EGFP-M3-APC or EGFP expression construct, 100 ng of the TCF-reporter construct SuperTOP-flash or FOPflash (Upstate Biotechnology, Lake Placid, NY), and 50 ng of the pRL-TK Renilla luciferase construct (Promega, WI) as a control to normalize for transfection efficiency. After 48 hours, cells were harvested and luciferase activities were determined using the Dual-Luciferase® assay system (Promega) and a Turner Designs TD-20/20 luminometer. SuperTOP-flash and FOPflash luciferase activities were first normalized by pRL-TK Renilla luciferase, and then the normalized SuperTOP-flash luciferase activity was divided by normalized FOPflash luciferase activity to calculate relative β-catenin activity.
Cell cycle distribution in HCT116 βw cells expressing GFP, M2-APC, or M3-APC. Transfected cells were stained with Hoechst blue, and the cell cycle distribution G0/G1 (2N), S (between 2N and 4N), and G2/M (4N) was determined by FACS at three time points post-transfection. For each transfection, 10,000 GFP-positive cells were analyzed. Table shows the average from three independent experiments.
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Mitotic indices of HCT116βw cells expressing GFP, M2-APC, or M3-APC. Live GFP, M2-APC, and M3-APC expressing cells at 48 hours post-transfection were stained with Hochest blue. Mitotic cells were counted according to DNA morphology from 100 randomly selected GFP positive cells. Table shows the average from three independent experiments. p values were calculated by comparing M2 or M3-APC expressing cells to GFP expressing cells using student t test.
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Cell cycle distribution of parental HL60 and HL60/MX2 cells expressing GFP, M2-APC, or M3-APC. Cell cycle distributions of GFP, M2-APC, and M3-APC expressing cells at 48 hours post-transfection. For each transfection, 10,000 GFP-positive cells were analyzed. Table shows the average from three independent experiments.
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Cell cycle distribution of SW480 cells expressing GFP, M2-APC, or M3-APC. Cell cycle distributions of GFP, M2-APC, and M3-APC expressing cells at 48 hours post-transfection. For each transfection, 10,000 GFP-positive cells were analyzed. Table shows the average from three independent experiments. For aneupoid cells, p values for M2-APC is 0.16, and for M3-APC is 0.09.
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Cell cycle distribution of HCT116βm cells expressing GFP, M2-APC, or M3-APC. Cell cycle distributions of GFP, M2-APC, and M3-APC expressing cells at 48 hours post-transfection. For each transfection, 10,000 GFP-positive cells were analyzed. Table shows the average from three independent experiments.
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We thank Yoshiaki Azuma (University of Kansas) for technical assistance with purification of recombinant M2- and M3-APC proteins, Michael Dohn (Vanderbilt University) for critical reading of the manuscript, Kozo Kaibuchi (Nagoya University, Japan) for providing expression constructs for APC fragments fused to EGFP, Bert Vogelstein (The Johns Hopkins University) for providing the HCT116βw and HCT116βm cell lines (mut ko, β-cat wt/- and wt ko, β-cat -/mut, respectively), Jo Ann Byl (Vanderbilt University) for providing recombinant topo IIα proteins, and James Higginbotham (Vanderbilt University) for technical support with FACS-based cell cycle analysis. Topo IIα antisera used for this project was generously provided by Joseph A. Holden, in whose memory this work is dedicated.