Colonic tumorigenesis in BubR1+/-ApcMin/+ compound mutant mice is linked to premature separation of sister chromatids and enhanced genomic instability

Abstract

Faithful chromosome segregation is essential for the maintenance of genetic stability during cell division and it is at least partly monitored by the spindle checkpoint, a surveillance mechanism preventing the cell from prematurely entering anaphase. The adenomatous polyposis coli (Apc) gene also plays an important role in regulating genomic stability, as mutations of Apc cause aneuploidy. Here we show that whereas Apc(Min)(/+) mice developed many adenomatous polyps, mostly in the small intestine, by 3 mo of age; BubR1(+/-)Apc(Min)(/+) compound mutant mice developed 10 times more colonic tumors than Apc(Min)(/+) mice. The colonic tumors in BubR1(+/-)Apc(Min)(/+) mice were in higher grades than those observed in Apc(Min)(/+) mice. Consistently, BubR1(+/-)Apc(Min)(/+) murine embryonic fibroblasts (MEFs) contained more beta-catenin and proliferated at a faster rate than WT or BubR1(+/-) MEFs. Moreover, BubR1(+/-)Apc(Min)(/+) MEFs slipped through mitosis in the presence of nocodazole and exhibited a higher rate of genomic instability than that of WT or BubR1(+/-) or Apc(Min)(/+) MEFs, accompanied by premature separation of sister chromatids. Together, our studies suggest that BubR1 and Apc functionally interact in regulating metaphase-anaphase transition, deregulation of which may play a key role in genomic instability and development and progression of colorectal cancer.

Fig. 1.

Haploinsufficiency of BubR1 and Apc results in an increased formation of colonic tumor polyps. (A) Genomic DNA samples isolated from mouse tails were subjected to genotyping by PCR using primers that detect both WT (Wt) and mutant alleles of Apc or BubR1. (B) Dissection micrographs of representative colons from WT and BubR1^+/–Apc^Min/+ mice at 3 mo of age. Arrows denote the colonic tumor masses. (C and D) Mice of various genotypes at 3 mo of age were killed. Intestines from each mouse were examined under a dissection microscope for tumor polyps. Average numbers of tumor masses in colon (C) and the small intestine (D) from WT, BubR1^+/–, Apc^Min/+, and BubR1^+/–Apc^Min/+ mice are shown. No visible intestinal tumor masses were detected in WT or BubR1^+/– mice. (E) Hematoxylin/eosin-stained sections of tumor masses from Apc^Min/+ (Upper) and BubR1^+/–Apc^Min/+ (Lower) mice. (×400.) (F) Sections of colonic tumors from BubR1^+/–Apc^Min/+ mice were subjected to immunohistochemical studies after staining with IgGs to proliferating cell nuclear antigen. (×400.) Typical malignant tissues (Upper) and normal colon tissues adjacent to the adenocarcinomas (Lower) are presented.

Fig. 2.

BubR1^+/–Apc^Min/+ MEFs proliferate at an accelerated rate. (A) Paired MEFs were lysed and an equal amount of cell lysates were blotted for Apc, BubR1, or β-actin. Arrow NS denotes a nonspecific cross-reactive band. (B) Equal amount of cell lysates from MEFs of various genotypes were blotted for β-catenin and β-actin. (C) Sections of normal colons from mice of various genotypes were subjected to immunohistochemical studies after staining with IgGs to cyclin D1. A typical adenocarcinoma section from BubR1^+/–Apc^Min/+ mice that was stained with cyclin D1 is also presented. (D) MEFs of various genotypes were subjected to cell proliferation assays using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The data are summarized from three independent experiments.

Fig. 3.

Analysis of BubR1 expression and apoptosis in small intestines by immunohistochemistry. (A) Sections of paraffin-embedded small intestine and colon from WT mice were stained with antibody to BubR1. Representative images at various magnifications are presented. Samples from at least three mice were examined. (B) Sections of paraffin-embedded small intestine and colon samples from BubR1^+/–Apc^Min/+ mice were examined for apoptosis by using a TUNEL kit. Representative images from three independent samples are presented.

Fig. 4.

Enhanced genomic instability in MEFs with compound mutations in BubR1 and Apc. (A) MEFs of various genotypes cultured on chamber slides were treated with nocodazole for the indicated times. At the end of the treatment, cells were fixed, stained with DAPI, and examined for the mitotic index. (B) A typical DAPI-stained cell with micronuclei. Arrows point to the micronuclei. (Bar, 2 μm.) (C) Percentage of micronuclei in MEFs of various genotypes. *, The difference between this group and WT or BubR^+/–Apc^Min/+ is statistically significant (P < 0.05); **, the difference between this group and the rest of the groups is statistically significant (P < 0.05). (D) The percentage of metaphase chromosomal counts from MEFs of various genotypes. Individual chromosomal counts were divided into three groups, namely, cells with a normal karyotype (diploid), a low chromosomal count (<diploid), and a high chromosomal count (>diploid). (E) Metaphase spreads from Wt MEFs (Left) and BubR1^+/–Apc^Min/+ MEFs (Center and Right). Arrows point to some separated sister chromatids.