Widespread genomic instability mediated by a pathway involving glycoprotein Ib alpha and Aurora B kinase

Abstract

c-Myc (Myc) oncoprotein induction of genomic instability (GI) contributes to its initial transforming function and subsequent tumor cell evolution. We describe here a pathway by which Myc, via its target protein glycoprotein Ibalpha (GpIb alpha), mediates GI. Proteomic profiling revealed that the serine/threonine kinase Aurora B is down-regulated by GpIb alpha in p53-deficient primary human fibroblasts. The phenotypes of Aurora B deficiency are strikingly reminiscent of Myc or GpIb alpha overexpression and include double-stranded DNA breaks, altered nuclear size and morphology, chromatin bridges, cleavage furrow regression, and tetraploidy. During mitosis, GpIb alpha and Aurora B redistribute to the cleavage furrow along with other cleavage furrow proteins. GpIb alpha overexpression at levels comparable with those seen in some tumor cells causes the dispersal of these proteins but not Aurora B, resulting in furrow regression and cytokinesis failure. Aurora B normalization redirects the mislocalized furrow proteins to their proper location, corrects the cleavage furrow abnormalities, and restores genomic stability. Aurora B thus appears necessary for a previously unrecognized function in guiding and positioning a number of key proteins, including GpIb alpha to the cleavage furrow. These findings underscore the importance of maintaining a delicate balance among cleavage furrow-associated proteins during mitosis. Suppression of Aurora B via GpIb alpha provides a unifying and mechanistic explanation for several types of Myc-mediated GI.

FIGURE 1.

Detection of Aurora B. A, endogenous Aurora B levels. Lysates from the four indicated human telomerase reverse transcriptase-immortalized HFF cell lines (see supplemental Fig. 1) were immunoblotted for Aurora B expression. Note the down-regulation of Aurora B in both GpIbα overexpressing cell lines, thus confirming the results depicted in supplemental Fig. 2. B, shp53+GpIbα cells were transduced with a lentiviral vector encoding V5 epitope-tagged Aurora B or the control empty vector. The resultant blasticidin-resistant clones were pooled and used for immunoblotting. The top panel shows the results obtained with an anti V5 mAb, and the middle panel shows repeat immunoblotting with the anti-Aurora B mAb used in A. Note that transduction with the Aurora B-encoding lentiviral vector restored Aurora B to a level equal to or only modestly exceeding that of the endogenous protein in vector control cells (compare first and last lanes). C, immunolocalization of Aurora B. The indicated cell lines were grown on glass coverslips and then fixed and immunostained for Aurora B using the antibodies described under “Experimental Procedures.” Note the common and generally nuclear localization of Aurora B in all cell lines and the weaker signal in those lines expressing GpIbα.

FIGURE 2.

Re-expression of Aurora B is associated with a suppression of DSBs. A, DSBs assessed by immunostaining for γ-H2AX. Coverslips containing each of the indicated cell types were fixed and stained with a mAb directed against γ-H2AX (red), a surrogate indicator of DSBs (31, 60). Nuclear counterstaining (blue) was performed with DAPI as described previously (37). Typical examples of positive nuclei are shown here. B, quantification of DSBs as determined by immunostaining for γ-H2AX. At least 100 cells from each of three different fields were observed, and the fraction of DSBs was quantified (37). The average percentage of positive cells ± S.E. is depicted. The p values were determined by Student's unpaired two-tailed t test. Note that the restoration of Aurora B in shp53+GpIbα cells significantly reduced the fraction of cells with DSBs. C, the above findings were corroborated by staining of separate coverslips for an independent marker of DSBs, Ser(P)¹⁹⁸¹-ATM (30). D, similar quantification performed with Ser(P)¹⁹⁸¹-ATM immunostaining.

FIGURE 3.

Reduced incidence of abnormal nuclei following restoration of Aurora B. A, representative microscopic fields of HFF cell lines. Note examples of cells with micronuclei (arrows, first and second panels), ≥2 nuclei (third panel), and dysmorphic and/or abnormally sized nuclei (fourth and fifth panels). Also note the presence of chromatin bridges (sixth panel) (see supplemental Fig. S3 and Fig. 2a for additional examples. B, quantification of nuclear defects from A. The nuclei from at least 200 cells from three different coverslips were examined. The bars indicated S.E.

FIGURE 4.

Inhibition of pseudo-tetraploidy/aneuploidy induction by restoration of Aurora B. Flow cytometric profiles of propidium iodide-stained nuclei from each of the indicated cell lines were obtained on either log phase populations or following a 16-h exposure to colcemid as described previously (39). Propidium iodide staining was performed on isolated nuclei to eliminate the contributions of cells with multiple nuclei. Typical results are shown from a total of three to five individual experiments performed with each cell line.

FIGURE 5.

Normalization of mislocalized cleavage furrow proteins and cytokinesis failure following restoration of Aurora B. A, localization of endogenous GpIbα, actin, filamin, RhoA, and Aurora B at the cleavage furrow in mitotic shp53 cells. Typical examples are depicted. Each of the indicated endogenous proteins was detected by immunostaining with appropriate monoclonal antibodies as described previously.³ The patterns of staining are indistinguishable from those seen in vector cells (not shown). B, mislocalization of GpIbα, actin, filamin, and RhoA in shp53+GpIbα cells but retention of Aurora B at the cleavage furrow. C, normalization of all proteins at the cleavage furrow of shp53+GpIbα+Aurora B cells. D, quantification of mitotic cells with mislocalization of the above cleavage furrow-associated proteins. At least 100 mitotic cells from each group were evaluated following immunostaining. The percentage of cells with mislocalized proteins was determined on mitotic cells with obvious cleavage furrows. The error bars represent S.D. The p values are based on a two-tailed Student's t test in a comparison with shp53+GpIbα+vector cells. Note that the localization of endogenous Aurora B did not appreciably change among the different cell lines. E, cytokinesis failure is corrected by Aurora B restoration. The percentage of cells undergoing cytokinesis failure in each of the indicated cell lines was determined by live cell differential interference contrast microscopy as described previously.³ A total of 91 shp53 cells, 53 shp53+Gp1bα+vector cells, and 76 shp53+Gp1bα+Aurora B cells were scored.

FIGURE 6.

Chromatin bridges in shp53+GpIbα cells are reduced by restoration of Aurora B. A, representative images of chromatin bridges associated with co-localization of Aurora B. shp53+GpIbα cells were immunostained to localize endogenous Aurora B and then counterstained with DAPI. B, quantification of mitotic nuclei with chromatin bridges. Each of the indicated cell lines was stained with DAPI, and the percentage of cells with chromatin bridges as a percentage of all mitotic nuclei was determined. Note the normalization of GpIbα-induced bridging defects following the restoration of Aurora B.

FIGURE 7.

Model for GI mediated by the Myc → MT-MC1 → GpIbα pathway. MT-MC1 is a direct target of Myc and GpIbα is up-regulated both by Myc and MT-MC1 (32, 34). The induction of DSBs by Myc occurs through both reactive oxygen species-dependent (dotted arrow) and -independent pathways, which, in the latter case, involve GpIbα (, , and Fig. 2). Concurrently, GpIbα down-regulates Aurora B, which, as described in the current work, also suppresses DSBs (Fig. 2). Aberrant repair of DSBs via nonhomologous end joining can lead to chromatin bridges (13, 69, 70, 74, 75), resulting in delayed cleavage furor formation and abscission. The delay would normally persist until chromatin bridges had been resolved or until lagging chromosomes had further migrated away from the cleavage plane. Aurora B deficiency, possibly in combination with an overwhelming number of chromatin bridges, would prevent a timely resolution of these defects, leading to eventual cleavage furrow regression, cytokinesis failure, and the development of either true tetraploidy or multinucleation (Figs. 4 and 5).