Molecular Human Reproduction, Vol. 6, No. 9, 821-827,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Genetic abnormalities detected by comparative genomic hybridization in a human endometriosis-derived cell line
1 INSERM U507, Hôpital Necker, Paris, 2 Service de Gynécologie Obstétrique, Hôpital Beaujon, Clichy, 3 Service de Cytogénétique, Hôpital St Vincent de Paul, Paris and 4 INSERM U184, IGBMC, Illkirch, France
Abstract
Comparative genomic hybridization (CGH) was used in parallel with fluorescence in-situ hybridization (FISH) and conventional karyotyping to perform a genome-wide survey of DNA gains and losses in the endometriosis-derived permanent cell line, FbEM-1. The cytogenetic analysis showed a complex karyotype with numerical changes and multiple chromosome aberrations, including the der(1) complement marker exhibiting a large homogenous staining region (HSR). The chromosomal rearrangement interpreted as der(5) t(5;6)(q34;p11) was found in the majority of the metaphases indicating a clonal abnormality. Repeated CGH experiments demonstrated over-representation of chromosomes 1, 2, 3, 5, 6p, 7, 16, 17q, 20, 21q and 22q, while chromosomes 6q, 9, 11p, 12, 13q, 18 and X were under-represented. Using DNA from the original endometriotic tissues, including a peritoneal implant and ovarian endometrioma, CGH analysis revealed loss of DNA copy number on 1p, 22q and chromosome X, while gain was found on chromosomal arms 6p and 17q. FISH analysis confirmed that the gain at 17q includes amplification of the proto-oncogene HER-2/neu in 16% of the FbEM-1 nuclei and in 12% of cells from the primary ovarian endometrioma tissue. These findings demonstrate that FbEM-1 cells share certain molecular cytogenetic features with the original tissue and suggest that chromosomal instability is important in the development of endometriosis.
cell line/comparative genomic hybridization (CGH)/cytogenetics/endometriosis/FISH
Introduction
Endometriosis is a common cause of undefined infertility and pelvic pain in 10–15% of women of reproductive age. However, several studies suggest that endometriosis has a multidimensional aetiology, including hereditary, hormonal and immunological factors (Olive and Schwartz, 1993
; Brinton et al., 1997). This disease is characterized by monoclonal cell growth and can exhibit features of malignant behaviour including local invasion and metastasis (Gaetje et al., 1995; Jimbo et al., 1997; Sharpe-Timms, 1997).
The molecular basis of endometriosis include over-expression of c-myc and ERBB2 proto-oncogenes (Bergqvist et al., 1991; Schenken et al., 1991), and defects in aromatase, metalloproteinases (Noble et al., 1996; Sharpe-Timms et al., 1998) and growth factor expression (Huang and Yeh, 1994; Oral and Arici, 1996). To date, the genetic events underlying endometriotic cell expansion remain incompletely understood. However, recent molecular allelotyping and multicolour FISH studies have detected somatic genetic changes expressed as loss of heterozygosity (LOH) and aneuploidy of several chromosomes in a series of endometriotic samples (Jiang et al., 1996; Shin et al., 1997).
Knowledge of the mechanisms controlling endometriotic cell growth and differentiation is also limited, and this can be explained in part by the difficulties in harvesting and growing pure endometriotic cells in vitro (Ryan et al., 1994). In this context, chromosomal studies have not reported recurrent cytogenetic aberrations in primary cultures of endometriotic tissues (Dangel et al., 1994). In a previous study, we reported the establishment of a human endometriosis derived permanent cell line, FbEM-1, showing multiple chromosomal aberrations (Bouquet de Jolinière et al., 1997).
A recently developed molecular cytogenetic method called comparative genomic hybridization (CGH) is complementary to allelotyping and allows screening of the entire genome for chromosomal gains and/or losses (Kallioniemi et al., 1992a). Regions showing an increased copy number (gain or amplification) may harbour dominant oncogenes, whereas regions with a decreased copy number (loss) may contain tumour suppressor genes. The CGH approach has been successfully applied in a number of solid tumours (Bardi et al., 1995; Sonoda et al., 1997) and epithelial cell lines (Lastowska et al., 1998; Nupponen et al., 1998).
In this study, it was of interest to establish the cytogenetic characteristics of the endometriosis-derived FbEM-1 cell line regarding DNA copy number in comparison with the original lesion by the CGH method. The application of microdissection procedure facilitated the evaluation of somatic genomic changes of distinct endometriotic tissue areas. Based on the CGH profile showing gain of material on chromosome 17q, the gene copy level of HER-2/neu oncogene located on 17q11-21 was assessed by FISH strategy.
Materials and methods
FbEM-1 cell line
The human endometriosis-derived permanent cell line FbEM-1 was established from both a peritoneal implant and an ovarian endometrioma from a patient with gonadotrophin-releasing hormone (GnRH) agonist-resistant endometriosis categorized as IV according to the American Society for Reproductive Medicine (formerly American Fertility Society, 1985) classification as previously described (Bouquet de Jolinière et al., 1997). From the primary culture, this new cell line has been stably maintained for over 100 passages and the population doubling time has not varied for >2 years in culture. These cells grow in suspension, appear as a heterogeneous round or polygonal cell, exhibit pleomorphism and are immunoreactive for cytokeratins, vimentin and human leukocyte antigen I. The cultured cells are negative for various haematopoietic markers including the lymphoid cell antigens CD3, CD20 and CD45. In addition, the FbEM-1 cells were found to stain positively in periodic acid Schiff's (PAS) solution.
Endometriosis tissue samples
Histology of the large peritoneal implant and the ovarian endometrioma, used for establishment of the FbEM-1 cell line, was confirmed and selected areas were manually microdissected under an inverted microscope (Isola et al., 1994). Unstained sections (30–60) were used, and regions with a high density of endometriotic epithelial cells were separated from interstitial cells. Briefly, sections (20 µm) were cut on cryostat and fixed in 70, 85, and 100% ethanol for 5 min each. Every five sections, a 5 µm section was cut and stained with haematoxylin and eosin (H&E) to guide the microdissection procedure.
DNA extraction
High molecular weight DNA from the original peritoneal endometriotic tissue, from the ovarian endometrioma wall lining cells and from the FbEM-1 cell line (5 x 106 cells) was extracted with phenol–chloroform–isoamylalcohol (25:24:1 v/v). The concentration of DNA obtained from the total number of sections per sample (30–60) ranged between 5–35 µg. Normal reference DNA was extracted by the same procedure, using peripheral blood lymphocytes from a healthy donor.
Karyotype analysis
Metaphase chromosomes from different in-vitro passages of FbEM-1 cells were prepared according to standard protocols. R-bands by heating using Giemsa stain (RHG-banding), were obtained after incubation of the slides with metaphases in Earle's medium at 86°C for 1 h followed by Giemsa staining (Verma and Lubs, 1976). For cells at passages 5 and 57, chromosomes of 120 mitoses were counted and 40 metaphases were photographed and analysed (Mitelman, 1995).
Comparative gnomic hybridization (CGH)
CGH was performed essentially as described (du Manoir et al., 1993). Briefly, the test DNAs (FbEM-1 cellular and endometriotic tissue DNA) and normal reference DNA were differentially labelled by standard nick translation using biotin-16-dUTP and digoxigenin-11-dUTP (Boehringer Mannheim, Mannheim, Germany) respectively. Equal amounts (600 ng) of labelled test DNA, and normal reference DNA were co-precipitated with 25 µg of unlabelled human Cot-1 DNA (repetitive sequences enriched to total human DNA; Gibco BRL). The labelled probe DNA was resuspended in 10 µl hybridization mixture composed of 50% formamide, 2x SSC, saline sodium citrate, (1x SSC = 0.15 mol/l NaCl/0.015 mol/l sodium citrate, pH 7) and 10% dextran sulphate. After denaturation, the labelled DNA probes were co-hybridized to normal human metaphase spreads prepared by phytohaemaglutinin-stimulated peripheral blood lymphocyte culture. The hybridization was carried out at 37°C for 3–4 days. The slides were washed three times at 45°C for 5 min each in 50% formamide/2x SSC, followed by three washes at 45°C in 2x SSC and one wash in 0.1x SSC for 10 min. Biotinylated DNA sequences were visualized by fluorescein isothyocyanate (FITC)-conjugated avidin (Vector Laboratories, Burlingame, CA, USA) whereas digoxigenin-labelled sequences were detected using mouse anti-digoxin and goat anti-mouse tetramethyl rhodamine isothyosyanate (TRITC)-coupled antibodies (Sigma, France) as reported (du Manoir et al., 1993). Chromosome preparations were counterstained with 4'-6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma).
Digital image analysis
The images were captured using cooled charge-coupled device camera (Photometrics, Tucson, TX, USA) attached to a DMRB type fluorescence microscope (Leica, Bensheim, Germany). Fluorescence ratio profiles for each chromosome were calculated using an appropriate software program. For each hybridization the data from 10–12 representations of each chromosome were combined to obtain the mean of 95% confidence interval for that ratio. Gains or losses of chromosomes or chromosomal regions were detected on the basis of the ratio profiles deviating from the green to red balance value of 1.0 within 0.8–1.2 limits. The threshold values were determined by CGH experiments as reported previously (du Manoir et al., 1993). The centromeric and heterochromatic regions, the short arm of acrocentric chromosomes, and the telomeric regions were not included in the interpretation of gains and losses. Chromosome Y was also excluded from the analysis because the reference and test DNAs were of female origin.
Fluorescence in-situ hybridization (FISH)
Double colour FISH with painting probes for chromosomes 1, 5, 6 and 17 (Oncor Inc, Gaithersburg, MD, USA) was undertaken to validate the CGH results. Hybridization and washing conditions were performed according to the manufacturer's instructions for the Oncor chromosomes in-situ system for single gene copy probe or whole chromosomes painting detection (Oncor). According to these procedures, chromosomal spreads and cells on slides were denatured in 70% formamide/2x SSC at 70°C for 2 min and dehydrated in ethanol. Hybridization with denatured probes homogenized in 50% formamide/10% dextran sulphate was performed overnight at 37°C under a coverslip in a moist chamber. Digoxigenin-labelled probes were visualized by incubation with rhodamine (red colour; Boehringer Mannheim) and biotinylated probes were detected by fluorescein–avidin (green colour; Vector Laboratories, Burlingame, CA, USA). Chromosomes were counterstained with DAPI to permit the identification of each chromosome.
FISH with HER-2/neu probe
Changes affecting chromosome 17 and the HER-2/neu gene locus were determined by using a biotin-labelled chromosome-17-specific pericentromeric 
-satellite repeat DNA probe and a digoxigenin-labelled HER-2/neu gene-specific cosmid probe specific for 17q11.2–12 (Oncor). Hybridization was conducted overnight at 37°C and detected with a series of antibodies following the probe manufacturer's instructions (Oncor). The satellite and the HER-2/neu probes were hybridized in parallel, to FbEM-1 metaphases, tissue touch preparations of endometrioma wall lining cells and peripheral blood lymphocytes from a healthy donor were used as an internal control. Copy number of centromere 17 and HER-2/neu signals were counted for ~200 nuclei per smear and the ratio of the mean number of HER-2/neu signals statistically established.
Statistical analysis for FISH
Frequency tables were analysed with Fisher's exact test. Non-normal distributions between groups were compared using the Mann–Whitney test. All P values are two tailed.
Results
Cytogenetic analysis
Chromosomal analysis of FbEM-1 cell line was performed at different in-vitro passages using the RHG-banding method. The representative hypotriploid karyotype from one of the late passages (57) is shown in Figure 1 revealing complex structural changes with a modal number of 66 chromosomes. Four copies of chromosomes 1 and 20 were detected in 27 of 37 examined metaphases, whereas chromosomes 3, 11, 17, 21 and 22 were present in three copies. Remarkably, one or two copies of a chromosome complement interpreted as der(5) t(5;6)(q34;p11) was present in 85% of the analysed metaphases. This marker was found in the early passages of the FbEM-1 cells (passage 5) as well as in passage 57 with the same regularity and was considered as a clonal abnormality. The modal chromosome number remained stable between passages 5 and 57, suggesting that the karyotype did not change during the in-vitro propagation of FbEM-1 cells. Additional distinct marker chromosomes (10–12) were a frequent finding in ~70% of the studied cells. A homogeneous staining region (HSR) located on one large marker chromosome (labelled a), considered as der(1), was present in >70% of the FbEM-1 cells. The origin of the other marker chromosomes (including those designated b, c and d) was not definitely established (Figure 1).
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Comparative genomic hybridization analysis
CGH was performed with DNA extracted from the early passages of the FbEM-1 cell line and from microdissected different areas of the primary endometriotic tissues. The CGH profiles obtained after hybridization with FbEM-1 cellular DNA, indicate that chromosomal gains were more commonly observed than were chromosomal losses as estimated from the fluorescence ratio along the axis of each chromosome from 10 metaphase spreads. A summary of the copy number changes by chromosome is depicted in Figure 2
. As shown, the FbEM-1 cell line exhibits genetic imbalances expressed as over-representation on chromosomes 1, 2, 3, 5, 7, 16, 17, 20, 21 and 22 while loss of DNA sequences is observed on chromosomes 6q, 9, 11p, 12, 13q, 18 and chromosome X.
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Significant increase in DNA sequences copy number was detected at 1q, 5p, 6p and 17q chromosomal arms corresponding to amplifications. The strongest green hybridization signal was localized on the long arm of chromosome 1 and on the short arm of chromosome 6 encompassing the region at 6p24-ter.
To assess whether the DNA copy changes seen in FbEM-1 cells share similarity with genetic changes in the original tissues, separate CGH analyses using DNA from distinct areas of the peritoneal implant and ovarian endometrioma were carried out. Overall, the CGH data indicate that the total number of chromosomal aberrations in these tissues was significantly lower in comparison with the changes found in FbEM-1 cells. The chromosome imbalances included mostly loss of DNA sequences located on 1p (minimal commonly lost region 1p34-ter), 22q and chromosome X. Conversely, gains on chromosomal arms 6p and 17q were detected (Figure 3). Remarkably, identical CGH profiles were obtained using DNAs from the peritoneal implant and the ovarian endometrioma tissues (not shown).
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FISH analysis
The results obtained by the CGH were further extended by FISH analysis. Using whole chromosome 1 painting probe, three copies of chromosome 1 and three marker chromosomes containing segments from this chromosome were detected in >70% of the FbEM-1 metaphases examined (not shown). Dual colour FISH using paints for chromosomes 5 and 6, confirmed the translocation interpreted as der(5) t(5;6)(q34;p11) by the banding analysis. In addition, signals from chromosomes 5 and 6 were detected on three different marker chromosomes one of which consisted of alternating segments of the two chromosomes resulting in a `harlequin' appearance (Figure 4A
). Using chromosome 17-specific paint, two normal copies of chromosome 17 were observed in addition to translocated material on two marker chromosomes in >46% of the FbEM-1 metaphases (Figure 4B).
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Since HER-2/neu was one of the target genes located on 17q, FISH with HER-2/neu single locus probe was carried out using FbEM-1 cells, tissue touch preparations from the ovarian endometrioma tissue and normal lymphocyte nuclei as controls. On separate 17 centromere FISH, two signals per nucleus were observed in >90% of normal lymphocytes. The level of HER-2/neu gene copy number relative to chromosome 17 centromere probe was estimated by double-colour FISH on >200 FbEM-1 nuclei in comparison to the same number of interphase nuclei from the primary lesion. The range of chromosome 17 centromere numbers were 0.5–3.2 with >85% of nuclei averaging two chromosomes 17 in the FbEM-1 cells. Similar percentages for chromosome 17 centromere were found in the imprint preparations from different sites of the original tissue. Of FbEM-1 cells, ~38 ± 5.1% had a HER2/neu to chromosomes 17 ratio of >2. The number of HER-2/neu signals per cell was 11–21 (Figure 4C
) in >16% of the FbEM-1 cells. Comparatively, in ~12% of the autologous endometrioma wall lining cells, the observed HER-2/neu copy number ranged between 8–12 (Figure 4D). Remarkably, FISH analysis of FbEM-1 cells metaphase spreads indicated that the HER-2/neu gene clustered mostly on chromosomes other than 17. The number of HER-2/neu copies in a typical cluster varied from 4 to 10, and the number of clusters per metaphase ranged from 1 to 7 (data not shown). Discussion
The present study demonstrates the high complexity of the genetic processes occurring in the endometriosis-derived cell line FbEM-1. Karyotyping on various chromosomes revealed significant aberrations, including the der(5) t(5;6)(q34;p11) clonal abnormality and the der(1) marker chromosome containing a large HSR. Notably, this clonal rearrangement was confirmed by FISH in the majority of the FbEM-1 metaphase spreads from both early (5) and late (>57) passages. It is possible that a rearranged 5q region has been present from the outset, and that the der(5) t(5;6)(q34;p11) has been a consistent feature that was involved in the pathogenesis of the original endometriotic lesion. However, further cytogenetic and molecular genetic studies of endometriotic cells in primary culture and cell lines including the invasive endometriotic cell line EEC145T (Starzinski-Powitz et al., 1998), are needed to confirm whether the described aberrations are specifically linked to endometriosis.
Overall, the results of the CGH analysis revealed that DNA sequence copy gains predominated over copy number losses in FbEM-1cells. In addition, four distinct amplified regions were located on chromosomal arms 1q, 5p, 6p and 17q. Comparatively, a lower number of DNA copy aberrations were prevalent in the original endometriotic tissue. A large deletion of 1p arm, loss of the entire arm of chromosome 22q and the entire chromosome X were noticeable changes in both peritoneal implants and ovarian endometriotic tissue. Overlapping CGH profiles between the FbEM cells and the primary lesion were observed only for the 6p and 17q chromosomal arms. This discordance may be explained by the presence in the original tissue of histological foci with various developmental stages of the same disease. In this regard, tissue diversity has been a traditional pitfall impeding somatic genetic studies conducted particularly on ovarian tumours (Watson et al., 1998). Indeed, the endometriotic lesions often contain areas of histologically differing grades, including hyperplasia and severe cytological atypia (Czernobilsky and Morris, 1979). Moreover, appearance of adenocarcinoma in the background of endometriosis has been reported by several authors (Mostoufzadeh et al., 1980; Heaps et al., 1990; Vercellini et al., 1993). With regard to tissue diversity, a marked focal heterogeneity in the expression of oestrogen and progesterone receptors was also reported in endometriotic samples collected from different sites within the same patient (Howell et al., 1994). Thus, it is possible that the FbEM-1 cells arose from an endometriotic focus undergoing neoplastic transformation.
In accordance with the CGH profiles of FbEM-1 cells, the significant genetic gain found by the CGH on 1q, was related to the chromosome 1 aneusomy found in >70% of the FbEM-1 metaphases by banding analysis and confirmed by the FISH method. It is tempting to speculate that several potential candidate genes located on 1q may play a role in endometriotic cell growth and differentiation including the TRK, SKI proto-oncogenes and transforming growth factor ß (TGFß2). Conversely, loss of DNA sequences found on the p arm of chromosome 1 in the primary lesion, may suggest involvement of tumour suppressor genes located at this segment (White et al., 1997).
Furthermore, the CGH profile indicates that the entire p arm of chromosome 5 is also amplified in FbEM-1 cells. Amplification of 5p region has been described in various tumours, including endometrioid cancer (Pere et al., 1998) and carcinoma of the uterine cervix (Heselmayer et al., 1997). One relevant candidate gene mapped to 5p13 is SKP2, which encodes a protein associated with cyclin A CDK2. The protein has been shown to be essential for cellular entry into the S phase (Zhang et al., 1995).
Another notable finding in our study is the detection by CGH of a high copy number amplification on 6p24 in both FbEM-1 cells and the original lesion. One target gene, NRASL3, which belongs to the RAS proto-oncogene superfamily, might be amplified in this region. Other potential candidate genes within the over-represented segment may include the metalloendopeptidase meprin gene, MEP1A (Jiang et al., 1995), the PIM1 oncogene (Ziegler et al., 1990) and the TNF- and VEGFA genes both located on 6p21.3 region (Honchel et al., 1996; Vincenti et al., 1996). Concurrently with the gain on 6p, loss of the terminal part of 6q arm in FbEM-1 DNA was estimated from the CGH profile. Previous studies suggested that a suppressor gene located on 6q arm is involved in development of ovarian epithelial tumours. (Cliby et al., 1993; Osborne et al., 1994). Moreover, Tibiletti et al. (1996) reported that cytogenetic deletions involving the q arm of chromosome 6 were very common in early ovarian tumours of all histological types and suggested that this may be one of the earliest chromosomal aberrations in the pathogenesis of ovarian neoplasia.
Other copy number losses indicative of chromosomes where candidate tumour suppressor genes reside were mapped to chromosomes 9, 11, 12 and 13q in FbEM-1 cells and to the q arm of chromosome 22 in the original tissue. The loss of material on chromosome 9 is likely to involve copy number loss of the p16 (MTS1, CDKN2A) and p15 (CDKN2B) genes located at 9p12, both of which are cell cycle regulators (Igaki et al., 1995). One candidate for the tumour suppressor gene on the 22q is neurofibromatosis type 2 (NF2) gene at 22q12 (Ruttledge et al., 1994). Finally, deletion of the entire chromosome X in the cell line and the primary tissue found by CGH is of particular interest in endometriosis because of the presumed presence on the p arm of chromosome X of genes with tumour suppressor characteristics (Choi et al., 1997; Timmer et al., 1999).
Among the amplified regions, significant gain of DNA sequence copy number was observed by CGH analysis on the long arm of chromosome 17 in the cell line and in the original lesion. A candidate locus at the amplified region is the HER-2/neu (c-erbB-2 gene), known to be amplified and over-expressed in ovarian (Zheng et al., 1991) and breast tumours (Kallioniemi et al., 1992b), and in breast carcinoma-derived cell lines (Tomasseto et al., 1995). It can be postulated that the high level of HER-2/neu copy number is related to the chromosome 17 structural rearrangement observed in FbEM-1 cells. Concerning the primary lesion, none of the cells demonstrated a parallel increase in both chromosome 17 centromere and Her-2/neu copies, which suggests that the increase of copy in this sample was not due to aneuploidy alone. At present, the molecular genetic mechanisms of HER-2/neu amplification in endometriotic lesions are not known. In this respect, consistent but variable expression of HER-2/neu gene at the protein level was previously demonstrated in a series of advanced stage endometriotic lesions from various locations (Bergkvist et al., 1991).
In conclusion, this study reveals that the endometriosis-derived FbEM-1 cell line exhibits a highly complex molecular cytogenetic pattern including the unbalanced translocation der(5)t(5;6)(q34;p11) and rearrangements of chromosomes 1, 5, and 6. The molecular cytogenetic analysis of FbEM-1 cells and of the original tissues, yielded data of partial genetic resemblance involving the 6p and 17q chromosomal arms. In addition, a high level of HER-2/neu gene amplification was observed in both FbEM-1 cells and the ovarian endometrioma lesion. These findings and data from molecular cytogenetic analysis of larger series of primary endometriotic lesions should highlight chromosomal sites of putative oncogenes and/or tumour suppressor genes involved in the development of endometriosis.
Notes
5 To whom correspondence should be addressed at: INSERM U 507, Hôpital Necker, 161, Rue de Sevres, 75015-Paris, France. E-mail: gogusev{at}necker.fr 
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Submitted on January 10, 2000; accepted on June 19, 2000.
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