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Abnormal Cytokinesis in Cells Deficient in the Breast Cancer Susceptibility Protein BRCA2

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Science  29 Oct 2004:
Vol. 306, Issue 5697, pp. 876-879
DOI: 10.1126/science.1102574

Abstract

Germ-line mutations inactivating BRCA2 predispose to cancer. BRCA2-deficient cells exhibit alterations in chromosome number (aneuploidy), as well as structurally aberrant chromosomes. Here, we show that BRCA2 deficiency impairs the completion of cell division by cytokinesis. BRCA2 inactivation in murine embryo fibroblasts (MEFs) and HeLa cells by targeted gene disruption or RNA interference delays and prevents cell cleavage. Impeded cell separation is accompanied by abnormalities in myosin II organization during the late stages in cytokinesis. BRCA2 may have a role in regulating these events, as it localizes to the cytokinetic midbody. Our findings thus link cytokinetic abnormalities to a hereditary cancer syndrome characterized by chromosomal instability and may help to explain why BRCA2-deficient tumors are frequently aneuploid.

Inherited mutations affecting the BRCA2 tumor suppressor predispose to breast, ovarian, and other epithelial cancers with high penetrance (1). BRCA2-deficient cells accumulate gross chromosomal rearrangements, including translocations and large deletions during cell division (25), anomalies that are attributed to the control by BRCA2 of the RAD51 enzyme in reactions for DNA repair and recombination, during the S phase of the cell cycle (6). However, BRCA2 inactivation also triggers alterations in chromosome number (35) and abnormalities, such as centrosome amplification (4), that might arise from distinct roles during other cell-cycle phases. For instance, inhibition of BRCA2 by antibody microinjection is reported to delay the transition from G2 to M phase (7), a period when BRCA2 is phosphorylated by the mitotic kinase, Plk1 (8, 9).

To investigate possible functions of BRCA2 during mitosis, we monitored cell division by serial time-lapse imaging in murine embryo fibroblast (MEF) cultures homozygous for a targeted mutation (Brca2Tr) that truncates and inactivates Brca2 (3, 10). MEF cultures isolated from littermate embryos with the Brca2+/+ or Brca2Tr/+ genotype at an identical passage in culture served as controls. A frequency distribution is shown in Fig. 1A for the time taken for cells to progress from anaphase onset (when chromosome segregation becomes visible), to complete daughter cell separation. Although in Brca2+/+ cells (controls), this takes about 35 min, it is slightly prolonged (median, 45 min) in Brca2Tr/+ cells and is severely extended in Brca2Tr/Tr cells (median, 90 min). Binucleate cells, the product of incomplete cell division, occur frequently after Brca2 inactivation (Fig. 1B). A representative series of time-lapse images (fig. S1) shows delayed progression through cytokinesis, culminating in nuclear division without cell separation, and generating a binucleate cell. The frequency of binucleates increases during the passage of Brca2Tr/Tr cells in culture (supporting online text), but is not significantly changed by different culture densities (from 0.4 to 1.2 × 105 cells per ml) or by the addition of Matrigel extracellular matrix (11).

Fig. 1.

BRCA2 inactivation delays and prevents cytokinesis. (A) Frequency distribution of the time taken after anaphase to complete cytokinesis. Brca2Tr/Tr MEFs during their second passage in culture were compared with Brca2Tr/+ and Brca2+/+ cells from litter-mate embryos. Mitosis was monitored in living cells by serial time-lapse imaging (12). Live cells were visualized by bright-field microscopy every 5 min from anaphase onset until completion of cell separation, or for up to 6 hours. The percentage of cells on the vertical axis is plotted against time taken to complete cytokinesis. Cells that failed to complete cytokinesis within 6 hours are enumerated under “Fail to divide.” Results shown are typical of at least three independent experiments, using three distinct MEF cultures for each genotype. (B) Cells that failed to divide after 6 hours were considered in two groups: the percentage of those that remain in mitosis without completing cytokinesis (“Not completed cytokinesis”) and those that complete nuclear division but not cytokinesis (“Binucleate cells, no cytokinesis”). (C) BRCA2 depletion by siRNA delays cytokinesis and provokes abnormal divisions. HeLa cells treated with control or BRCA2 siRNAs (12) (fig. S2) were monitored by serial time-lapse imaging as above.

Cytokinetic abnormalities also apparently occur in vivo. Binucleation is more than 30 times as frequent (2.7 ± 0.7%) in cells freshly isolated from day 13 to 14 mutant embryos than in wild-type controls (0.08 ± 0.01%). Indeed, although some Brca2Tr/Tr embryos survive (with growth retardation and developmental defects) until later stages in development, up to 50% are lost early during embryogenesis (10).

Cytokinesis is also delayed or prevented when BRCA2 is depleted by RNA interference using short, interfering (si)RNAs (12) (fig. S2). The period from anaphase onset to completion of cell division is significantly extended in HeLa cells treated with BRCA2 siRNA (median, 112 min) when compared with controls (median, 60 min); again, this delay is associated with failure to divide (Fig. 1C).

Assembly and activation of the actomyosin contractile ring are key events during cytokinesis (13, 14), brought about by the organization of actin and type II myosin in the ingressing cleavage furrow. During cleavage (fig. S3), myosin II normally concentrates at the site of furrow formation [Fig. 2A, arrows in (a)]. This is not seen in more than 50% of cells treated with BRCA2 siRNA [Fig. 2, A (b) and B]. Moreover, during the abscission of daughter cells in telophase, the normal accumulation of myosin II as a band at each cell edge [Fig. 2A (c), enlarged image] is undetectable in many cells after BRCA2 depletion [Fig. 2, A (d) and B].

Fig. 2.

Myosin II mislocalization after BRCA2 inactivation. (A) Myosin II distribution during cleavage and abscission in HeLa cells treated with control or BRCA2 siRNAs. Representative images of cells co-stained (12) with DAPI and antibodies against myosin II or tubulin are shown. Arrows mark myosin II accumulation at the site of furrow formation in (a). The areas boxed in white in (c) and (d) show the midbody and are enlarged below. (B) Frequency of abnormal myosin II organization in cells undergoing cleavage or abscission steps during cytokinesis. The number of stained cells analyzed in each sample is indicated by n. (C) Abnormal abscission in Capan-1 cells. Staining for DNA, myosin II, and tubulin was performed as described above. A representative image is shown. The area boxed in white is enlarged to the right. Scale bars, 5 μm.

A significant accumulation of Brca2Tr/Tr MEFs in abscission suggests delayed progression through late stages in cytokinesis. Brca2Tr/Tr MEFs in abscission compose 64 ± 10% of all cells in stages from anaphase onset to cell separation (n = 158 in three experiments), compared with 36 ± 7% of wild-type controls [(n = 86), P = 0.04 by the two-tailed t test]. Cells treated with BRCA2 siRNA also tend to accumulate in abscission [56 ± 6% of all cells from anaphase onset to cell separation (n = 191 cells in three experiments), compared with 47 ± 3% of control siRNA [(n = 193), P = 0.1]. Incomplete BRCA2 depletion by siRNA (fig. S2) may explain why this effect is not more pronounced.

Similar abnormalities in cytokinesis occur in an epithelial cancer cell line, Capan-1, isolated from a patient carrying the non-functional BRCA2 6174delT mutation (15). Asynchronous cultures contain many binucleate cells [mean frequency, 12 ± 2% (n = 500)], and show abnormal myosin II organization and midbody morphology during abscission (Fig. 2C).

The intracellular localization of BRCA2 during cytokinesis is consistent with its possible participation in these events. A key group of proteins implicated in cytokinesis (1619), including the inner centromere protein (INCENP), Aurora B kinase, and survivin, localize to central structures during cell separation. BRCA2 follows a similar but not identical pattern. In human cells labeled with the DNA dye DAPI (4′,6′-diamidino-2-phenylindole) and with antibodies against BRCA2 (12) and Aurora B, BRCA2 colocalizes with Aurora B in central structures during the elongation stage of cytokinesis (arrow in Fig. 3D), and notably, both proteins accumulate in the midbody during late cleavage and abscission (Fig. 3, B and C).

Fig. 3.

Localization of BRCA2 to cytokinetic structures. The panels show typical HeLa cells at the elongation, late cleavage, and abscission steps in cytokinesis. Staining was with DAPI and mouse monoclonal antibodies against Aurora B and BRCA2 (12) (fig. S4). Arrows mark BRCA2 staining in (D). In the Merge images (A to C), co-localization of the red and green channels appears as yellow-green areas. (D to L) Individual images from which Merge panels were assembled. Scale bars, 5 μm.

Collectively, our findings suggest that BRCA2 may regulate the fidelity of late stages in cytokinesis but is not an essential component of the machinery for cell separation. Thus, BRCA2-deficient cells experience considerable delays in cytokinesis, but many, nevertheless, go on to complete cell division. Abnormal organization of myosin II in the contractile ring occurs during cleavage and abscission. BRCA2 may have a role in regulating these events, as it migrates from central structures during the elongation phase of cytokinesis to the cytokinetic midbody during cleavage and abscission.

BRCA2 is required for the repair of DNA double-strand breaks by recombination (20), and BRCA2-deficient cells spontaneously acquire DNA breaks and structurally aberrant chromosomes during the S phase (35). Attempts to segregate malformed chromosomes, if they lag on the central spindle, might prolong or prevent cytokinesis. But it is difficult to explain delayed abscission, abnormal myosin II organization, or the mitotic localization pattern of BRCA2 on this basis.

Besides structurally aberrant chromosomes, primary cultures of BRCA2-deficient cells accumulate with 4N and greater DNA content during successive passage, consistent with exit from mitosis without cytokinesis (3). Also, aneuploidy often occurs in cancers from BRCA2 mutation carriers (2) and might, as in sporadic cancers, predict poor clinical outcomes. Our findings suggest that both these phenotypes may arise from the inactivation of previously unrecognized functions of BRCA2 in cytokinesis, which is a possible link between cytokinetic abnormalities and the pathogenesis of a human genetic disease associated with chromosomal instability and cancer predisposition.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1102574/DC1

Materials and Methods

SOM Text

Figs. S1 to S4

References and Notes

References and Notes

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