Aneuploid colon cancer cells have a robust spindle checkpoint

Abstract

Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused by mismatch repair defects, the lesions responsible for CIN are unknown. The observation that CIN cells fail to undergo mitotic arrest following spindle damage suggested that mutations in spindle checkpoint genes may account for CIN. However, here we show that CIN cells do undergo mitotic arrest in response to spindle damage. Although the maximum mitotic index achieved by CIN lines is diminished relative to MIN lines, CIN cells clearly have a robust spindle checkpoint. Consistently, mutations in spindle checkpoint genes are rare in human tumours. In contrast, the adenomatous polyposis coli (APC) gene is frequently mutated in CIN cells. Significantly, we show here that expression of an APC mutant in MIN cells reduces the mitotic index following spindle damage to a level observed in CIN cells, suggesting that APC dysfunction may contribute to CIN.

Fig. 1. CIN cells undergo mitotic arrest following spindle damage. HeLa, HCT-116 (MIN) and HT-29 (CIN) cells were treated without or with 0.2 µg/ml nocodazole for 18 h. (A) Phase-contrast microscopy shows the majority of the cells in all lines round up following nocodazole treatment. (B) Immunofluorescence microscopy shows that in nocodazole, the majority of cells have condensed chromosomes (red) which are positive for phospho-histone H3 (green). (C) Flow cytometry shows that in the absence of nocodazole all three lines exhibit typical DNA content profiles (upper panels) with few MPM-2 positive cells (lower panels). In the presence of nocodazole all the lines exhibit large G₂/M peaks and the majority of the cells are MPM-2 positive indicating that they are mitotic.

Fig. 2. CIN lines exhibit a robust spindle checkpoint. The lines indicated were plated out and treated with nocodazole to prevent spindle assembly. (A) Plot of mitotic index every 6 h. (B) Mitotic index at 18 h with each value representing the average and standard error from four independent experiments. (C) Percentage of cells with DNA contents >4N after 48 h.

Fig. 3. Expression of N-APC compromises spindle checkpoint function. (A) HCT-116 cells stably transfected with control or N-APC vectors stained with anti-Myc antibodies and Hoechst dye. Only background staining is observed in the control line whereas extensive cytoplasmic staining is visible in the N-APC lines. (B) Stably transfected HCT-116 lines were treated with nocodazole to prevent spindle assembly. After 18 h the mitotic index was determined. Each value represents the average and standard error from three independent experiments. The mitotic index of lines expressing N-APC is reduced compared with the controls.

Fig. 4. N-APC localizes to the centrosome. (A–D) Transfected cells stained with anti-Myc antibodies and Hoechst as indicated. (A) HeLa and (B) BHK cell showing localization of N-APC to the cytoplasm and centrosome. (C) BHK cell showing localization of full length APC to the cytoplasm. (D) BHK cell showing co-localization of N-APC and cenexin. (E) DLD-1 cells stained with antibodies against APC and aurora a (Bischoff et al., 1998) showing co-localization to centrosomes. White arrows identify the centrosomes.