Deregulated Aurora-B induced tetraploidy promotes tumorigenesis - PubMed (original) (raw)
Deregulated Aurora-B induced tetraploidy promotes tumorigenesis
Hao G Nguyen et al. FASEB J. 2009 Aug.
Abstract
High expression of Aurora-B has been observed in various cancers, and inhibition of this kinase has been shown to halt cellular proliferation. However, the mechanism of effect of Aurora-B on cellular transformation has not been fully explored. Here, we show that overexpression of Aurora-B in murine epithelial cells promotes generation of tetraploids. In search of a related mechanism, spectral karyotyping was carried out, showing premature chromatid separation (PCS). Of interest, PCS is a hallmark of Robert's syndrome, which also involves cellular polyploidy and aneuploidy. Sorted tetraploid Aurora-B-overexpressing cells promoted significant mammary epithelial cancers when injected into nude mice, as compared to injection of nonfractionated cells, suggesting that tetraploidy is an important mediator of Aurora-B-induced tumorigenesis. Comparative chromosome hybridization performed on DNA derived from tetraploid cell-induced tumors indicates amplifications and deletions of regions throughout the genome, which include tumor-promoting or tumor-suppressing genes, respectively. Thus, sustained expression of Aurora-B induces tetraploidy, which, in turn, facilitates genomic instability and tumor development in a xenograft model.
Figures
Figure 1.
Stable Aurora-B induces PCS in NmuMG cells, in both 2N and 4N cells. A) Mitosis carrying 2N range of chromosomes (42, XX with trisomies of no. 15 and 19), which show the phenomenon of total PCS, characterized by the separated and splayed chromatids and a discernable centromere involving all chromosomes (also known as C-anaphase cells). This is demonstrated by FISH, using BAC RP23-51O13 for p53 status (top left), SKY (top middle), and DAPI banding (top right). Because of the mitotic event leading to PCS, each chromatid of no. 11 chromosome is recognized to carry an individual FISH signal of p53. B) As clearly shown by SKY (bottom panel), the total number of chromatids is 84. C) Mitosis carrying 4N range of chromosomes (84, XXXX), as demonstrated by FISH using BAC RP23-51O13 for p53 status (top left), also showed PCS in all chromosomes, SKY (upper right) and DAPI banding (bottom left). Total number of p53 signals (i.e., 8) is demonstrated to coincide with the total number of chromatids of no. 11 chromosome, which clearly show total PCS events. Long arrows indicate p53 gene location. Chromosomal rearrangements were also noted in the SKY analysis, as indicated by short arrows in A, bottom panel.
Figure 2.
Normal p53 gene distribution is observed in both diploid and tetraploid A-box mutated NmuMG cells. Normal NmuMG cells or stably overexpressing nondegradable mutant Aurora-B (A-box-mutated) were cultured in DMEM medium with 10% FBS and treated with colcemid (inhibitor of tubulin polymerization, 15 ng/ml) for 18 h, and chromosomes were prepared by acetic acid/methanol fixation and subjected to SKY, FISH, and DAPI banding analysis. A) Mitosis carrying 2N normal (42, XX with trisomies of chromosomes no. 15 and 19), as demonstrated by FISH using BAC RP23–51O13 for p53 status (top left), SKY (top middle), and DAPI banding (top right). SKY (bottom panel) demonstrates that p53 is located on chromosome 11B3, consistent with the data published on the NCBI site (http://genome.ucsc.edu/cgi-bin/hgGateway? clade=vertebrate&org=Mouse&db=0&hgsid=69124056). On the basis of the intensity of FISH signals, we conclude that each cell carrying 2N range of chromosomes contains 2 copies of p53 genes per cell, or 4 copies of p53 genes per mitosis following duplication of the gene through the prior S phase. B) Mitosis carrying 4N range of chromosomes (84, XXXX), as demonstrated by FISH using BAC RP23–51O13 for p53 status, SKY, and DAPI banding. Number of p53 signals coincides with copy number of chromosome no. 11.
Figure 3.
In vivo tumorigenicity of NmuMG cells stably expressing wild-type Aurora-B or Aurora-B mutant. A) Ploidy analysis of NmuMG cells, A-box-Aurora-B mutant stable transfectant before (top) and after sorting (bottom) by FACS. In the latter case, an equal number of the sorted cells was subjected to FACS analysis. Histograms are representative of 3 independent experiments. Histograms of sorted cells with 2N, 4N, and 8N DNA content are superimposed. Overlapping regions (dark green) represent cells with ploidy content other than that indicated. B) Nude mice (nu/nu) were subcutaneously injected into mammary fat pad with pooled cells overexpressing wild-type Aurora-B (top panel) or stable A-Box-Aurora-B mutant (bottom panel) or sorted tetraploid cells sorted as in panel A (tumors not depicted) at a concentration of 2 × 106 cells/mouse (∼27 g total weight). Tumors were surgically removed after 8–10 wk postinjection and visualized using a Zeiss Stemi SV6 dissecting microscope, as described in Materials and Methods. Mouse 5 did not develop a tumor. C) Average tumor weights (g). Error bars =
sd
. Values of P < 0.05 indicate a statistically significant difference between groups using 1-way ANOVA test. D) Tumor histology shows random and blinded H&E-stained paraffin-embedded tissue sections that were sent to pathology for independent interpretation. Tumors from 4N cells show grade 3 adenocarcinoma, poorly differentiated neoplastic epithelial cells with significant nuclear atypical and high mitotic activities. There are irregular borders and evidence of tumor invading the adjacent layers.
Figure 4.
Tumors from tetraploid cells showed various degrees of amplification and deletions, detected by array CGH. Tumors were harvested from sites injected with 4N cells and subjected to standard DNA extraction method. Purified DNA was sent to Roswell Park Cancer Institute for array CGH using the mouse 6.5K RPCI-23/-24 BAC array. Array CGH was performed as a sex-mismatch to provide an internal hybridization control for chromosome X and Y copy number differences. A) Data set for the whole genome. B) Typical data set from chromosome 1 with signal intensity of ±0.1 represents normal range. Signals >+0.1 represent amplification regions of the chromosome and <−0.1 represent regions of gene deletion. C) Typical pattern of signal that indicates whole chromosome amplification, including trisomies or tetrasomies. Trisomies of chromosomes 15 and 19 are shown.
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