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Genomic Instability of Human Mammary Epithelial Cells Overexpressing a Truncated Form of EMSY

Afshin Raouf, Lindsay Brown, Nikoleta Vrcelj, Karen To, Winnie Kwok, David Huntsman, Connie J. Eaves

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).


    ABSTRACT
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The EMSY gene encodes a protein that interacts with Brca2 and is amplified in some sporadic cases of human breast cancer. To examine whether overexpression of EMSY would mimic the chromosome instability phenotype that is associated with the loss of Brca2 function, we constructed a lentiviral vector (Lenti-EMSY/GFP) that encodes a truncated form of the Emsy protein, including its Brca2-interacting domain, and green fluorescent protein (GFP) and used it to transduce human telomerase-immortalized human breast epithelial (184-hTert) cells, which have a nearly normal karyotype. At passage 5 after transduction, 39 (26%) of 150 EMSY/GFP-transduced metaphase cells contained at least one structural chromosomal abnormality compared with 19 (13%) of 150 GFP-transduced metaphase cells (P = .003, chi-square test); at passage 10, the corresponding frequencies were 42% and 15%, respectively (P<.001). Mitomycin C also produced a severalfold higher frequency of chromosome breaks in the EMSY/GFP-transduced cells than in the control cells. These results support the hypothesis that EMSY overexpression can play a role in the genesis of human breast cancer.


The EMSY gene (1) encodes a protein whose amino-terminal region binds to the protein domain encoded by the third exon of the BRCA2 gene, which is deleted from the germline DNA of familial breast cancer kindreds (2). The amino-terminal region of the Emsy protein also contains separate domains that bind to the HP1{beta} and BS69 proteins (Fig. 1, A), which suggests that Emsy may also play a role in chromatin remodeling (1). In irradiated cells, Emsy has been found to colocalize with {gamma}-histone 2AX in response to DNA damage (1). BRCA2 gene mutations are not seen in sporadic breast cancer, but the EMSY gene is amplified in 13% of these cancers (1). In primary breast cancer samples and breast cancer cell lines, amplification of 11q13.5, the chromosomal region that contains the EMSY gene, is specifically correlated with an increase in levels of EMSY messenger RNA (mRNA) (3). Taken together, these findings have led to the suggestion that overexpression of the EMSY gene might be an alternative mechanism for suppressing Brca2 activity, which could lead to the emergence of malignant breast cell populations (46).



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Fig. 1. The lentiviral expression vector, Lenti-EMSY-GFP, and isolation of transduced 184-hTert cells. (A) Design of the vector. An 810-bp region of the EMSY cDNA encoding the Brca2-binding domain (B2) and the HP1{beta} (H) and BS69 (BS) binding domains was amplified by PCR using oligonucleotide primers EMSY-1–810-F (5'-GGCGCGCCCCACCATGCCTGTTGTGTGGCC-3'; forward primer) and EMSY-1–810-R (5'-TTAATTAATGTCTGTGTTGATGGTTTAG-3'; reverse primer) and cloned into the PCR-2.1-Topo vector (Invitrogen, Burlington, Ontario, Canada). The cloned insert was isolated by digestion of the plasmid with AscI and PacI, purified, and reinserted into the multiple cloning site (MCS) of the KA391 lentiviral vector. Vesicular stomatitis virus envelope-pseudotyped Lenti-EMSY/GFP or Lenti-GFP virus was produced in 293T cells, concentrated, and titered on HeLa cells as previously described (11). (B) Fluorescence-activated cell sorter analysis profiles of parental 184-hTert cells (left panel), EMSY/GFP-transduced cells analyzed 2 days after transduction (middle panel), and control GFP-transduced cells analyzed 2 days after transduction (right panel). Approximately 105 log phase 184-hTert cells were cultured overnight in six-well plates in regular growth medium (50% Dulbecco's minimal essential medium with 1000 mg/L glucose and 50% Ham's F12 medium plus 2.38 g/L HEPES, 10 ng/L epidermal growth factor, 0.25 ng/mL insulin, 10–6 M hydrocortisone, 1 µg/L transferrin, 2.6 ng/mL sodium selenite, 30 U/mL prolactin, 1 nM estradiol, 2.5 mg/mL isoproterenol, and 400 µg/mL G418), and the next day, the cells were incubated for 4 hours with 500 µL of medium containing 1.2 x 107 infectious units of Lenti-EMSY/GFP or Lenti-GFP virus, after which the medium was replaced with the normal growth medium for another 48 hours. Cells displaying the top 20% of fluorescence levels (arrows) were isolated using a FACSVantage flow cytometry system (Becton Dickinson, City, CA) and further propagated in six-well plates. PI = propidium iodide, FSC = forward light scattering characteristics. (C) Transcript levels in passage 5 EMSY/GFP- and control GFP-transduced 184-hTert cells from all three transduction experiments were quantified by real-time PCR (7500 Sequence Detection System, Applied Biosystems) using TaqMan probes and primers designed to specifically detect transcripts containing the 5' or 3' ends of EMSY. This was done to allow the full-length (endogenous) EMSY transcripts (which contained both 5' and 3' sequences) to be measured separately from the more prevalent transgene-derived transcripts (which contained 5' sequences but not 3' sequences). EMSY transcript levels in each sample were normalized relative to an internal reference control and then compared to similarly normalized values measured in GFP-transduced cells. Values shown are the mean fold difference (± standard deviation) between values from EMSY/GFP- versus control GFP-transduced cells. (D) Northern blot analysis of EMSY RNA expression in EMSY/GFP-transduced (+) or GFP-transduced (–) 184-hTert cells from three separate experiments (lanes 1–6). RNA was extracted at passage 2 after transduction, and 10 µg was size fractionated on an agarose gel. Four clones of EMSY/GFP-transduced cells from the first experiment were isolated at passage 3 and grown for five additional passages (lanes 7–10). EMSY mRNA levels were detected by using a 32P-labeled probe specific for the human EMSY cDNA.

 
BRCA2-null cells exhibit a chromosome instability phenotype that is characterized by the accumulation of structural chromosomal abnormalities in response to exposure to mitomycin C (79). In humans, loss of heterozygosity at the BRCA2 locus is associated with genomic instability early in breast cancer development and antedates the appearance of carcinoma in situ (10). We therefore hypothesized that forced overexpression of EMSY might cause a chromosome instability phenotype in human breast epithelial cells similar to that typical of BRCA2-null cells.

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{beta}- 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 7–10), 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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Fig. 2. EMSY-overexpressing cells accumulate structural chromosome abnormalities. (A) Karyotype of an EMSY-overexpressing cell showing numerous structural abnormalities, including deletions of 12p and 12q, a translocation (7;21), and a break (chromosome 7), and monosomy of chromosome 8 and chromosome 21 (arrows). (B) Mitomycin C-treated EMSY-overexpressing cells displaying triradial chromosomes (two examples shown, photographed at 100x magnification). (C) Interphase nuclei from two EMSY-overexpressing cells showing a variety of aneuploid features (photographed at 100x magnification). The nucleus on the left has an additional chromosome 1 (gold) and has lost one chromosome 8 (red). The nucleus on the right shows the loss of one chromosome 17 (aqua). Two copies of chromosome 11 (two green signals) were present in both nuclei. (D) Fluorescence in situ hybridization analysis (24 color) of chromosomes 5, 6, and 12 from three of the four clones studied (i, ii, iii) showing unique chromosomal abnormalities. Each chromosome was painted with a whole-chromosome painting probe that was labeled with a different fluorochrome or combination of fluorochromes.

 

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Table 1.  Summary of the frequency of structural and numerical chromosomal abnormalities seen in EMSY-overexpressing cells and control GFP-transduced cells analyzed at passages 5 and 10 after transduction*

 
A hallmark of BRCA2-deficient cells is their increased sensitivity to DNA crosslinking agents such as mitomycin C, which cause the cells to accumulate structural chromosomal abnormalities because of their decreased ability to repair double-stranded breaks in their DNA (79). We next examined whether treatment of the EMSY-overexpressing cells with mitomycin C would similarly enhance this chromosome instability phenotype. Accordingly, EMSY/GFP-transduced cells at passage 11 from two separate experiments were exposed to 0.4 µM mitomycin C (a preestablished, nontoxic concentration) for 16–18 hours and then harvested for cytogenetic analysis (20 metaphase cells examined per experiment). The results showed that the frequency of cells with chromosome breaks was statistically significantly higher in mitomycin C-treated EMSY-overexpressing cells than in mitomycin C-treated control GFP-transduced cells (29 versus 9 affected cells, respectively, out of 40 cells analyzed in each group; P<.001, chi-square test). Two examples of abnormalities seen in mitomycin C-treated EMSY-overexpressing cells are shown in Fig. 2, B. By contrast, in the absence of mitomycin C treatment, the frequency of cells with breaks was very low in both types of cells (2 of 40 EMSY-overexpressing cells affected and 0 of 40 control cells affected).

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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DH and CJE are senior coauthors of this work.

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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Manuscript received February 21, 2005; revised June 21, 2005; accepted June 24, 2005.



             
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