QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 November 2004
doi: 10.1242/jcs.01556

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tighe, A. Articles by Taylor, S. S. Search for Related Content PubMed PubMed Citation Articles by Tighe, A. Articles by Taylor, S. S.

Truncating APC mutations have dominant effects on proliferation, spindle checkpoint control, survival and chromosome stability

Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (47K): [in a new window]   Fig. 1. Generation of HCT-116 cells stably expressing N-terminal APC fragments. (A) Schematic of the APC protein (Mimori-Kiyosue and Tsukita, 2001) and the three N-terminal fragments used to create the stable cell lines, showing the binding sites for Asef (blue), ß-catenin/GSK3ß (pink, green), axin (orange), microtubules (purple), EB1 (yellow) and hDLG (black). (B) Protein extracts from tetracycline inducible 293 cells stably expressing myc-tagged N750 (lane 1), N1309 (lane 2), N1807 (lane 3) and full length APC (lane 4), blotted to detect myc-tagged proteins (left) and APC (right). The asterisk indicates a background band, while the arrow indicates endogenous full length APC. (C) Cloning efficiency of the cell lines indicated in either 10 µg/ml ZeocinTM (upper panel) or 200 µg/ml hygromycin B (middle panel). ß-Galactosidase activity exhibited by the cell lines is shown in the lower panel. (D) HCT-116-derived cells stably transfected with Myc-tagged control or N-APC vectors were stained with anti-Myc antibodies (red) and Hoechst dye (blue) to visualise the DNA. Only background staining is observed in the control line, whereas nuclear/cytoplasmic staining is visible in the N-APC lines. (E) Protein extracts from HCT-116 control (lane 1) and N750 cells (lane 2), and 293 N750 cells, uninduced (lane 3) or induced with tetracycline (lane 4) blotted to detect endogenous full length APC (upper panel) and Myc-tagged proteins (lower panel). The asterisk indicates a background band in the HCT-116 samples detected by the anti-myc antibody.

View larger version (28K): [in a new window]   Fig. 2. Expression of N-APC reduces the accumulation of mitotic cells in response to spindle damage. Stably transfected HCT-116 lines expressing N-terminal fragments of APC were treated with nocodazole to prevent spindle assembly. At the time points indicated cells were centrifuged onto slides and the mitotic index determined. (A) Bar chart of mitotic index after an 18-hour nocodazole incubation. Each value represents the mean and standard error from six observations. The asterisk indicates values that are significantly different (P<0.05) from the Myc control line as determined by an ANOVA analysis with Tukey-Kramer post test. (B) Line graph of mitotic index over a 48-hour time course. Cells expressing N-terminal APC fragments N750 and N1807 have a reduced mitotic index relative to controls.

View larger version (25K): [in a new window]   Fig. 3. Expression of N-APC compromises cellular proliferation. Cell proliferation over a period of 7 days was analysed using crystal violet staining. Line graphs of (A) stably transfected control and N-APC HCT-116 derived cell lines and (B) HeLa, MIN (HCT-116, DLD-1) and CIN (HT29, LoVo, SW480, SW837) cells. N750 and CIN cells grow slower than control and MIN cells respectively.

View larger version (28K): [in a new window]   Fig. 4. Expression of N-APC accelerates mitotic exit following release from a nocodazole block. Mitotic cells were isolated by selective detachment after a 13-hour nocodazole block, replated into medium containing 0.2 µg/ml nocodazole (solid symbols) or into nocodazole free medium (open symbols), and at the time points indicated, harvested and the mitotic index determined. (A) Line graphs plotting the mitotic index over time, showing that expression of N750 and N1309 accelerates mitotic exit. Values represent the mean and standard error derived from at least two independent experiments, in which at least 2000 cells were counted per time point per experiment. For clarity the data is shown on four separate graphs, with the Myc control data being replicated on each. (B) Bar graph plotting the mitotic index of HeLa, MIN (DLD-1, HCT-116, RKO) and CIN (HT29, LoVo, SW480, SW837) cells 4 hours after release from a 13 hours nocodazole block, showing that with the exception of HT29, CIN cells exit mitosis quicker than MIN cells. Values represent the mean and standard error from three observations with at least 1000 cells being counted.

View larger version (74K): [in a new window]   Fig. 5. Cells expressing N750 survive and proliferate following prolonged mitotic arrest. HCT-116 Myc control and N750 cells were incubated in the presence of either 0.02 µg/ml or 0.2 µg/ml nocodazole for 48 hours. On day 0 the nocodazole was washed away, fresh medium added and the cells incubated for up to 7 days. At the time points indicated, cells were analysed by phase-contrast microscopy and crystal violet staining. (A) Phase-contrast images of Myc control and N750 cells at day 0 and day 7, showing that 7 days after exposure to nocodazole Myc control cells appeared larger with many cytoplasmic vacuoles. In contrast, N750 cells appeared morphologically normal. Scale bar represents 200 µm. (B) Crystal violet stained plates of the cells in (A). (C) Quantification of the bound crystal violet, showing the change in cell number over time after 48 hours incubation in nocodazole.

View larger version (36K): [in a new window]   Fig. 6. N750 survivors contain chromosome aberrations and are highly aneuploid. Following exposure to nocodazole as shown in Fig. 5, metaphase spreads of Myc and N750 cells were prepared at the time points indicated and the chromosomes stained with Hoechst. (A) Histograms plotting the distribution of chromosome numbers at day 0, day 6 and day 36. (B) Examples of two chromosome spreads from N750 cells. Closer inspection revealed that spreads from the N750 cell line contained chromosomes with two constrictions (enlargements). The bar graph quantifies the percentage of metaphases containing chromosomes with two visible constrictions (Myc, n=84; N750, n=77). (C) Anaphase cells at day 6 showing anaphase bridges and lagging chromosomes in N750 cells. The bar graph quantifies the number of cells with abnormal anaphases (Myc, n=26; N750, n=29). (D) Chromosome spreads were prepared from the surviving Myc and N750 cells 36 days after release from 48 hours in 0.02 µg/ml nocodazole. Whereas almost all of the Myc control cells were near diploid, the N750 cells were highly aneuploid.

View larger version (37K): [in a new window]   Fig. 7. A clonal cell line expressing N750 induces chromosomal instability. The two clonal cell lines, pLP-Myc and pLP-N750, were analysed as described in the other figures. (A) Bar chart of mitotic index after incubation in 0.2 µg/ml nocodazole for 18 hours. (B) Line graph plotting mitotic index during a 48-hour exposure to 0.2 µg/ml nocodazole. (C) Line graph plotting change in mitotic index over time following release from a nocodazole block. Myc control cells (squares) and N750 cells (triangles) were released into medium containing either nocodazole (solid symbols) or drug free medium (open symbols). Cells expressing N750 exit mitosis faster than controls. (D) Metaphase spread showing the presence of rearranged chromosomes (arrowheads and enlargements) in the N750 line.

View larger version (41K): [in a new window]   Fig. 8. Endogenous N-APC localises to the centrosome during the early stages of mitosis. (A) Deconvolved image stacks of DLD-1 cells stained to detect APC (panel I, red in merged image), C-Nap1 (II, green in merged image) and DNA (blue in merged image). IV shows an enlargement of the centrosome boxed in III. V shows a 3D model of the centrosome in IV showing that the N-APC mutant localises to a region between the two centrosomes. (B) Deconvolved image stacks of DLD-1 cells stained to detect APC (red), aurora A (green) and DNA (blue). While N-APC can be detected at the centrosome in prophase and prometaphase (II and III), centrosomal localisation is not apparent in metaphase or anaphase (IV and V). Scale bars: 5 µm.

View larger version (30K): [in a new window]   Fig. 9. Expression of N-APC mutants reduces pole to pole distance. HCT-116 Myc control and N-APC cells were fixed in microtubule stabilising buffer and stained with antibodies against tubulin. Metaphase cells were then imaged by optical sectioning microscopy. (A) Projections of deconvolved image stacks showing representative mitotic spindles. Scale bar: 5 µm. (B) Bar graphs quantifying pole-pole distance. Values represent the mean and s.e.m. derived from at least six cells. (C) Bar graphs quantifying the tubulin density at the spindle midzone. Values represent the mean and s.e.m. derived from at least six cells.

View larger version (49K): [in a new window]   Fig. 10. N-APC mutants weaken kinetochore-microtubule interactions. HCT-116 Myc control and N-APC cells were stained to detect kinetochores (ACA, red), Bub1 or BubR1 (green), microtubules (green or blue) and the chromosomes (red) as indicated. Metaphase cells were then imaged by optical sectioning microscopy. (A,B) Projections of deconvolved image stacks showing that while inter-kinetochore distance is reduced in N-APC-expressing cells, kinetochore bound Bub1 and BubR1 is increased. Scale bars: 5 µm. (C) Bar graph quantifying the distance between sister kinetochores in metaphase cells. Values represent the mean and s.e.m. derived from at least 28 kinetochores in at least 4 cells. (D,E) Bar graphs quantifying the amount of Bub1 and BubR1 staining at metaphase kinetochores. Values represent the mean and s.e.m. derived from at least 68 kinetochores in at least four cells.