Affiliations of authors: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada (AR, KT, WK, CJE); Genetic Pathology Evaluation Center, Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver General Hospital, and the University of British Columbia, Vancouver, British Columbia, Canada (LB, NV, DH); Prostate Center, Vancouver General Hospital, Vancouver, British Columbia, Canada (LB, NV, DH); Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada (CJE)
Correspondence to: Connie J. Eaves, PhD, Terry Fox Laboratory, 675 West 10th Ave., Vancouver, BC V5Z 1L3, Canada (e-mail: ceaves{at}bccrc.ca) or David Huntsman, MD, Department of Pathology, British Columbia Cancer Agency, 600 West 10th Ave., Vancouver, BC V5Z 4E6, Canada (e-mail: dhhuntsman{at}bccancer.bc.ca).
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ABSTRACT |
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To test this hypothesis, we isolated a 810-bp 5' fragment from the EMSY complementary DNA (cDNA) that encodes the Brca2-interacting domain as well as the HP1- and BS69-binding domains and inserted this fragment into a lentiviral vector that already contained an internal ribosomal entry site element and the cDNA for enhanced green fluorescent protein (eGFP) (11) (Fig. 1, A). We then used this vector or the control lentiviral vector expressing GFP only (Lenti-GFP) to infect log-phase 184-hTert cells, a telomerase-immortalized line of human breast epithelial cells (12) generously supplied by S. Dunn, Department of Pediatrics and Experimental Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Two days later, we used a fluorescence-activated cell sorter to select EMSY/GFP- and control GFP-transduced cells that displayed high levels of green fluorescence (Fig. 1, B), and the selected cells were cultured further for up to five passages. By passage 5, the EMSY/GFP-transduced cells from three independent transduction experiments expressed 68- to 300-fold higher levels of EMSY mRNA that contained sequences from the 5' end of the EMSY gene compared with control GFP-transduced cells (Fig. 1, C). In the same EMSY/GFP-transduced cells, expression of the endogenous EMSY gene (i.e., EMSY mRNA that contained sequences from the 3' end of the gene) (Fig. 1, C) or genes encoding Brca1, Brca2, p21, or p53 (data not shown) was not different from the expression of any of these genes in the GFP-transduced control cells. The level of 5' EMSY transcripts in the transduced cells continued to increase with further cell passage (data not shown), suggesting overgrowth of the cultures by cells that expressed the highest levels of the EMSY transgene.
The parental 184-hTert cells had a nearly diploid karyotype (48, XX, +20, +20) and were chromosomally stable. By contrast, in three separate experiments, cells that overexpressed mRNA from the EMSY transgene rapidly accumulated structural chromosomal abnormalities. These abnormalities included deletions, translocations, marker chromosomes, chromosome fragments, and dicentric chromosomes (Fig. 2, A), none of which were seen in metaphases from control GFP-transduced cells. For example, at passage 5 after transduction, 39 (26%) of 150 metaphases of EMSY/GFP-transduced cells contained at least one structural chromosomal abnormality compared with 19 (13%) of 150 metaphases from the GFP-transduced cells (P = .003, chi-square test; 50 metaphase cells of each type examined in each of three experiments) (Table 1). The highest frequency of cells with structural chromosomal abnormalities (32%) was seen in cells that expressed the highest levels of the EMSY transgene (Experiment 1). Conversely, cells that expressed lower levels of the EMSY transgene contained fewer cells with structural chromosomal abnormalities (22% in Experiment 2 and 24% in Experiment 3). By passage 10 after transduction, the frequency of structural chromosomal abnormalities in the EMSY-overexpressing cells had increased further to 44%, 48%, and 34% in Experiments 1, 2, and 3, respectively, but remained low (18%, 12%, and 14%, respectively) in the corresponding control cells (P<.001, chi-square test) (Table 1). We also analyzed EMSY/GFP-transduced and control GFP-transduced cells in interphase for chromosome copy number changes at four loci using a commercially available kit (Breast Aneusomy Multi-Color Probe Set; Vysis, Downers Grove, IL). This analysis showed that the frequency of aneuploid cells in the EMSY-overexpressing cells was statistically significantly higher than that in the control GFP-overexpressing cells at passage 5 (76 aneuploid cells per 300 cells analyzed versus 33 aneuploid cells per 300 cells analyzed, P<.001, chi-square test) and at passage 10 (88 aneuploid cells per 300 cells analyzed versus 34 aneuploid cells per 300 cells analyzed, P<.001, chi-square test). Examples of these aneuploid cells are shown in Fig. 2C. We also isolated four clones from EMSY/GFP-transduced cells at passage 3 from Experiment 1 (Fig. 1, D, lanes 710), confirmed their separate origins by integration site analysis of the EMSY transgene (data not shown), and then analyzed 15 metaphases from each clone using 24-color fluorescence in situ hybridization. All four clones contained cells that had a variety of complex structural as well as numerical chromosomal aberrations, i.e., karyotypes ranging from near tetraploid to near diploid, with multiple deletions and translocations. Examples of these abnormalities in three of the clones are shown in Fig. 2, D. EMSY/GFP-transduced cell populations had a statistically significantly higher frequency of aneuploid or polyploid cells than GFP-transduced cell populations at passage 10 (P = .002, chi-square test) (Table 1) but not at passage 5 (P>.05, chi-square test). Taken together, these findings indicate that cells that express elevated levels of the 5' end of EMSY display a genomic instability phenotype that is consistent, specific, stable, and probably dependent on the level of EMSY transgene mRNA expression.
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Thus, overexpression of a 5' fragment of EMSY induces a chromosome instability phenotype in human breast epithelial cells that is similar to that of BRCA2-deficient cells. Although this observation has yet to be extended to other cell lines, the data provide new experimental evidence to support the hypothesis that elevated levels of Emsy may play a role in sporadic breast oncogenesis by deregulating genomic stability. There are several potential mechanisms by which such a phenotype might be produced, including effects of Emsy overexpression on the fidelity of DNA replication and/or repair of double-stranded breaks through interference with the normal role of Brca2 and its ability to bind Rad51 (13). EMSY-overexpressing cancers, like those with a BRCA2-null genotype, might then be expected to have an enhanced sensitivity to agents (other than mitomycin C) that selectively damage BRCA2-null cells (14). Alternatively, overexpression of the truncated EMSY cDNA used in this study might produce a BRCA-null phenotype through a dominant-negative mechanism. Future experiments in which the full-length Emsy protein is overexpressed will be important to resolve these possibilities. It will also be of interest to determine whether the truncated form of Emsy expressed in the present studies affects the half-life or activity of Brca2, or its other binding partners. It should be noted that the levels of EMSY expression obtained in the experimental model described here are more than 10-fold higher than those documented in naturally arising tumors that take years to develop (3), but also allow a pronounced genomic instability phenotype to be produced within a few weeks. This model should thus provide a useful system for future delineation of the mechanisms by which the effects of more subtle changes in EMSY overexpression may be mediated.
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NOTES |
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This work was supported by grants from Genome Canada, the Lohn Foundation, and the Canadian Institutes of Health Research (CIHR). A. Raouf was supported by postdoctoral fellowships from the British Columbia Breast Cancer Foundation and the CIHR. D. Huntsman is a Michael Smith Foundation for Health Research Scholar.
We thank Luke Hughes-Davies (University of Cambridge, United Kingdom) for the EMSY cDNA and helpful comments, Philippe Leboulch (Harvard Medical School, Boston, MA) for the KA391 lentiviral vector, Sandra Dunn (Department of Pediatrics and Experimental Medicine, University of British Columbia, Vancouver, Canada) for the 184-hTert cells, Melinda Miller and Glen Edin for technical assistance, John Bentley for statistical analysis, and Adrienne Wanhill for assistance in manuscript preparation.
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REFERENCES |
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Manuscript received February 21, 2005; revised June 21, 2005; accepted June 24, 2005.
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