Mol. Hum. Reprod. Advance Access originally published online on August 16, 2008
Molecular Human Reproduction 2008 14(9):547-554; doi:10.1093/molehr/gan046
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Specific tumour-associated methylation in normal human term placenta and first-trimester cytotrophoblasts
1Developmental Epigenetics Research, Murdoch Children's Research Institute, Royal Children's Hospital, VIC 3052, Australia 2Department of Paediatrics, University of Melbourne Parkville, VIC 3052, Australia 3 Institute of Cell and Molecular Science, Barts and the London, 4 Newark Street, London E1 2AT, UK 4 Monash Institute of Medical Research, Monash University, Clayton, VIC 3168, Australia 5 Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK 6 UCL, Cancer Institute, University College London, London WC1E 6DD, UK
7 Correspondence address. Tel: +61-03-8341 6341; E-mail: richard.saffery{at}mcri.edu.au
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Abstract |
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Human placentation displays many similarities with tumourigenesis, including rapid cell division, migration and invasion, overlapping gene expression profiles and escape from immune detection. Recent data have identified promoter methylation in the Ras association factor and adenomatous polyposis coli tumour suppressor genes as part of this process. However, the extent of tumour-associated methylation in the placenta remains unclear. Using whole genome methylation data as a starting point, we have examined this phenomenon in placental tissue. We found no evidence for methylation of the majority of common tumour suppressor genes in term placentas, but identified methylation in several genes previously described in some human tumours. Notably, promoter methylation of four independent negative regulators of Wnt signalling has now been identified in human placental tissue and purified trophoblasts. Methylation is present in baboon, but not in mouse placentas. This supports a role for elevated Wnt signalling in primate trophoblast invasiveness and placentation. Examination of invasive choriocarcinoma cell lines revealed altered methylation patterns consistent with a role of methylation change in gestational trophoblastic disease. This distinct pattern of tumour-associated methylation implicates a coordinated series of epigenetic silencing events, similar to those associated with some tumours, in the distinct features of normal human placental invasion and function.
Key words: placenta/cytotrophoblasts/DNA methylation/Wnt signalling/choriocarcinoma
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Introduction |
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One of the hallmarks of cancer is the silencing of tumour suppressor genes by promoter methylation. Many human tumours are characterized by a specific profile of gene promoter methylation. This CpG island methylator phenotype (CIMP) varies both between tumour types, but also within defined classes of tumours (Esteller et al., 2001). Genes silenced in tumours have a diverse range of cellular functions including regulation of cell division and cycling, cell migration, apoptosis and cell–cell adhesion (Esteller et al., 2001). Interestingly, although many of these genes have large, potentially methylatable, CpG islands, most have yet to be shown to undergo regulation by methylation during the course of normal human development. Such methylation can therefore generally be considered disease-specific.
The similarities between tumourigenesis and the process of placentation have been documented over many years. These include high cell proliferation rate, lack of cell-contact inhibition, cell migration and invasion, activation of telomerase, de novo vascularization and a capacity to escape effectors of the immune system. Specific similarities include the expression of common growth factors, cell adhesion molecules, matrix-digesting enzymes and proto-oncogene products (Soundararajan and Rao, 2004; Ferretti et al., 2007). Interestingly, some tumours also express pregnancy or trophoblast-specific antigens (Salahshor et al., 2005; Koslowski et al., 2007). In addition, normal human cytotrophoblasts express functional tumour-associated genes, several of which are essential for the development of certain malignancies (Ferretti et al., 2007; Koslowski et al., 2007).
As with many cancers, it is becoming increasingly clear that DNA methylation also plays a pivotal role in the invasive behaviour of trophoblast cells and that this involves distinct methylation profiles at genes such as E-cadherin (Rahnama et al., 2006). This is best illustrated by the demonstration that inhibition of DNA methylation disrupts trophoblast invasive and migratory potential (Rahnama et al., 2006; Serman et al., 2007).
Alterations in the expression of tumour suppressor gene expression profile have been identified in placentas from pre-eclamptic pregnancies (Heikkila et al., 2005) whereas aberrant tumour suppressor gene methylation status has been associated with gestational trophoblastic diseases, specifically hydatidiform mole and choriocarcinoma (Xue et al., 2004).
In combination, these data suggest that trophoblasts and cancer cells may use common epigenetic modification to facilitate their proliferative, migratory and invasive properties (Ferretti et al., 2007). However, the major difference between placentation and tumour development is that whereas tumours tend to grow in an uncontrolled fashion, trophoblast cells possess several mechanisms to precisely regulate the extent of growth and invasion (Soundararajan and Rao, 2004).
In light of the recent observations of epigenetic regulation of some tumour suppressor genes in human placentas (Dokras et al., 2006; Chiu et al., 2007; Wong et al., 2008), we set out to identify the extent of other tumour-associated promoter methylation in human placenta tissue and to assess corresponding methylation levels in gestational disease-derived choriocarcinoma cell lines.
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Placental samples and cell lines
We had access to freshly delivered full term human placenta and first-trimester termination tissue, baboon and mouse placentas obtained from unrelated research projects, and several different human choriocarcinoma cell lines. Tissues were harvested for research purposes with appropriate human ethics clearances from the Royal Women's Hospital (03/51), Mercy Hospital for Women (R07/15) and Monash Medical Centre (07084C).
For full term human placentas, a 5 mm3 tissue piece was isolated from 
5 mm below the outermost layer of the placenta. Whole fresh mouse or baboon chorionic villous samples (CVS) and full term placental tissues were macerated with a scalpel blade prior to DNA isolation.
First-trimester placental tissue
Normal first-trimester placental tissue was obtained from healthy women of reproductive age who underwent elective termination of pregnancy for psycho-social reasons (amenorrhoea: 8–12 weeks). Chorionic villi were washed four times in sterile saline and transferred to DMEM/F12 medium containing antibiotic–antimycotic mixture (Invitrogen, Carlsbad, CA, USA). Tissue was processed within 2 h of collection.
Cytotrophoblasts
Trophoblast cells were isolated as previously described (Tapia et al. 2008) and purity of preparations determined using antibodies to the trophoblast-specific cytokeratin-7 (CK-7, 1:100 dilution, clone OV-TL 12/30, DakoCytomation, Glostrup, Denmark) using immunocytochemistry. Cells were fixed in ethanol, rehydrated and treated with 0.3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity. Cells were then washed with tris-buffered saline (TBS) and incubated with non-immune block for 30 min. Primary antibody was applied and incubated at 4°C for 16 h followed by biotinylated horse anti-mouse IgG (1:200) for 30 min, then streptavidin–biotin–peroxidase complex ABC (DakoCytomation) according to the manufacturer's instructions. Peroxidase activity was visualized by application of diaminobenzidine substrate (DakoCyomation) for 3–5 min. Cells were counterstained with Harris haematoxylin (Sigma Aldrich, St Louis, MO, USA), air dried and mounted. Only cell preparations in which 95% of the cells were positive for CK7 were used for subsequent experiments.
CVS tissue
For CVS samples, contaminating maternal blood and decidua were removed and 5–10 mg of cleaned villi was separated from the diagnostic sample, washed in isotonic saline, pelleted by centrifugation and stored at –20°C until DNA extraction and bisulphite modification.
A cell culture from each villi sample was established by finely macerating the villi and resuspending in 1.0 ml of 0.25% trypsin with incubation of the suspension at 37°C for 45 min. Cells were then resuspended in 7 ml of RPMI 1640 medium (SAFC Biosciences, Lenexa, KS, USA) with 20% FCS, pelleted at 560g and resuspended in a small volume of AmnioMAX medium (GIBCO Invitrogen) supplemented with 0.5 mg/ml amphotericin B. The cell suspension was then inoculated onto glass cover slips inside 35 mm petri dishes that were incubated at 37°C in 5% CO2 in humidified air and flooded with 2 ml of AmnioMAX medium the following day. Cells were grown to 95% confluency, trypsinized, pelleted and stored at –20°C until DNA extraction and bisulphite modification. Cultured cells were isolated following 14–18 days in culture with a maximum of three passages.
Choriocarcinoma cell lines
JEG-3 (HTB-36; Kohler and Bridson, 1971) and JAR cells (HTB-144; Pattillo et al., 1971) were maintained in RPMI: 10% FCS and BeWO (CCL-98; Pattillo and Gey, 1968) in Hams F12: 10% FCS at 37°C in 5% CO2. Cells were grown for only a few passages and were split (1:2–1:5) prior to confluency.
Genomic DNA isolation
Tissue samples were incubated at 50°C overnight with shaking in DNA extraction buffer [100 mM NaCl, 10 mM Tris–HCl pH8, 25 mM EDTA, 0.5% SDS] with 200 µg/ml Proteinase K. DNA was isolated by two rounds of phenol:chloroform extraction, followed by RNAse A treatment, precipitation in absolute ethanol containing 10% sodium acetate (3 M, pH 5.2), and resuspended in 100 µl nuclease-free water (Ambion, USA). DNA was stored at –20°C until needed.
DNA methylation analysis
Bisulphite DNA sequencing was carried out as previously described (Clark et al., 1994). DNA samples were processed using the Methyl EasyTM bisulphite modification kit (Human Genetic Signatures, Sydney, Australia) according to the manufacturer's instructions. This results in selective conversion of unmethylated cytosine nucleotides to uracil, which following amplification by PCR, are converted to thymidine. Methylated cytosines in the original DNA are protected from conversion and remain unchanged following amplification. Resultant amplicons were cloned using either the pGEMT EasyTM (Promega, Madison, WI, USA) or TOPO TA (Invitrogen, Carlsbad, California, USA) Cloning Kits (Invitrogen) for automated fluorescent DNA sequencing as described in Wong et al. (2006). Data were analysed using BiQ Analyser software (Bock et al., 2005) and individual cloned PCR products showing <80% conversion (following bisulphite treatment) were not included in downstream analysis. Details of PCR primers directed to bisulphite-modified DNA are detailed in Supplementary Table 1. Between 5 and 12 individual DNA clones were sequenced for each starting genomic DNA with results displayed using standard ‘lollipop’ diagrams. Each line represents an individual cloned promoter sequence with circles denoting sites of potential CpG methylation. Closed circles denote methylated CpG sites. Positions of circles in all figures approximate the locations of CpG sites within the assayed regions.
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Identification of candidate tumour-associated methylation in human placental tissue
Recent advances in epigenomics, primarily resulting from the combination of traditional methylation and chromatin analytical techniques with microarray analysis, have resulted in the generation of large amounts of genome-wide epigenetic data (Weber et al., 2005; Wilson et al., 2006; Birney et al., 2007; McCann et al., 2007; Thurman et al., 2007; Zhang et al., 2008). As part of a recent genomic methylation mapping study, Rakyan et al. identified a list of putative tissue-specific differentially methylated regions (tDMRs) in several human tissues, including full term human placenta (Down et al., 2008; Rakyan et al., 2008).
In order to identify genes highly enriched for methylation in human placenta, we ranked tDMRs, according to a placental methylation ratio (PMR). This was calculated by dividing the total methylation level observed in whole human placenta, by the average methylation level seen in all other tissues examined (Supplementary Table 1). Genes ranked in the top 100, previously reported to show tumour-associated methylation, were then identified based on ontology and/or database searches. Those genes that showed a >2.5-fold increase in methylation level in the placenta compared with other human tissues, and had a tDMR associated with the 5' end of the gene, were then subjected to further analysis. Using these criteria, we identified eight potential candidates for further investigation (Table I).
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Importantly, the adenomatous polyposis coli (APC) and Ras association factor (RASSF1A) genes, previously identified as showing specific placental promoter methylation (Chiu et al., 2007; Wong et al., 2008), were identified as part of this analysis (numbers 42 and 77 in the PMR rankings; Supplementary Table 1). However, promoter methylation of common tumour suppressor genes p53, p16(INK4A), p14(ARF), CDKN2B, MGMT, Rb, TIMP-3, CDH-1, HIC-1, DAPK-1 and PTEN was not detected using this approach, consistent with previous data demonstrating expression of several of these proteins in different trophoblast cell types (Quenby et al., 1998) and a lack of methylation of some in placental tissue (Xue et al., 2004; Chiu et al., 2007).
We designed specific bisulphite DNA sequencing assays for methylation of gene regulatory regions spanning (or overlapping) tDMRs of interest (Supplementary Table 1). Of six genes analysed in this manner, all were confirmed as showing some methylation in human term placental tissue (Table I). Interestingly, several distinct patterns of methylation were observed. Whereas CD44, KIAA0101 and EGR4 showed a methylation pattern consistent with monoallelic gene silencing (as previously described for the APC gene; Fig. 1), SIM1 and SFRP showed a consistently methylated profile (Figs 1 and 2) and EN-1 was more highly variable between different tissue and cell samples (see below).
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We also examined the methylation status of the tumour suppressor gene Deleted in Colorectal Carcinoma (DCC-1; PMR rank 316) and the p53-associated NADPH: quinone oxidoreductase gene (NQO1; PMR ranking 696), but did not detect any placental methylation at these loci in any placental tissues or cells tested (data not shown).
Methylation of multiple Wnt pathway inhibitory genes in human term placentas and first-trimester cells
Most strikingly, we identified evidence of promoter methylation of two negative regulators of Wnt signalling (soluble frizzled receptor protein-2, SFRP2; Rattner et al., 1997) and engrailed-1 (En-1; Bachar-Dahan et al., 2006) in both full term human placental tissue and purified first-trimester cytotrophoblast cells (Figs 2
–4). We have previously demonstrated a similar methylation profile for the APC gene promoter in human placental tissue (Wong et al., 2008) and have now confirmed that this methylation is present in first-trimester cytotrophoblasts (Supplementary Figure 1). However, whereas APC promoter methylation is distributed in a pattern consistent with monoallelic gene silencing (Wong et al., 2008), SFRP2 showed increased methylation in all cells tested (Fig. 3). Examination of methylation status of SFRP-1, -4 and -5 gene promoters revealed a complete lack of methylation in these regions in placental cells suggesting some specificity in Wnt pathway activation in the placenta (data not shown).
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Another potential Wnt negative-regulatory gene, EN-1, showed generally lower methylation levels, with significant variability in both full term placental tissue and first-trimester cytotrophoblast cells (Figs 3 and 4). Interestingly, this methylation was almost completely absent in CVS samples cultured for several passages (Fig. 3), indicating that this methylation was most likely present in cells that do not adapt and divide in the culturing conditions used (such as trophoblasts or lymphocytes).
Comparative methylation patterns in mouse and baboon placental tissue
We also examined methylation at the corresponding gene promoter regions in E15 mouse placentas and in CVS and full term baboon placental tissue. No methylation was detected for any of the genes examined in the mouse (Fig. 4 and Supplementary Figure 2) whereas methylation of both SFRP2 and APC genes was detected in the baboon placental samples (Supplementary Figure 2). This profound difference in epigenetic profile reflects a wider disparity between mouse and human placentation that includes numerous functional (including endocrine profile and degree of invasiveness) and structural differences (Carter, 2007).
Methylation status in choriocarcinoma cell lines
We also examined methylation of each of these genes in choriocarcinoma (CCA)-derived cell lines JAR-1, BeWo and/or JEG-3. Although studies have investigated the methylation status of some tumour suppressor genes in complete hydatidiform mole or CCA, none of the genes examined in this study has been previously investigated. Interestingly, whereas SFRP2, APC, EN-1, SIM-1, CD44 and EGR4 showed hypermethylation in CCA cell lines tested, KIAA0101 showed a decrease in methylation relative to primary placental tissue (Table I; Supplementary Figure 3). We also observed complete methylation of the DCC-1 gene in all CCA cell lines tested (data not shown).
Discussion
Considerable evidence for a role of epigenetic modification (including DNA methylation) in placental function has been collected over decades, primarily in relation to imprinted genes expressed in a parent-of-origin manner (Wagschal and Feil, 2006). These are postulated to play a role in balancing the paternal drive to maximize growth of related offspring with the maternal drive to balance resource allocation to progeny (Abu-Amero et al., 2006). DNA methylation is also an important regulator of placental function generally: single dose 5-deoxy azacytidine (DNA methylation inhibitor) given to pregnant rats, disrupts trophoblast proliferation (Serman et al., 2007) and disrupts trophoblast migration in choriocarcinoma-derived cell lines (Rahnama et al., 2006).
The pseudo-malignant nature of the placenta has been commented on for many years. Trophoblasts of the human placenta proliferate, migrate and invade the uterine wall and its vasculature in a manner that is recapitulated in many malignant tumours. The key difference is the spatial and temporal limitations of placental trophoblast growth under normal conditions (Armant, 2005). This prevents placental overgrowth, hyper-invasiveness and potential malignancy. It is this aspect of placental development that is conspicuously absent in many cancers, representing a fundamental difference between the two processes (Currie and Bagshawe, 1967; Soundararajan and Rao, 2004; Salahshor et al., 2005; Ferretti et al., 2007).
Data presented in this study provide compelling evidence for a small subset of tumour-associated methylation in a small subset of genes as part of normal human placentation. However, unlike the situation in many cancers, there is little evidence of a general silencing of many different classes of tumour suppressors (Rakyan et al., 2008). Nevertheless, all of the methylation described herein has been previously reported in at least one tumour type (Table I) and, in the case of SFRP2, APC and RASSF1, has been described in a wide variety of different human cancers. Such methylation has also been linked to silencing of gene expression (Harada et al., 2002; Yan et al., 2003; Deng et al., 2004; Hagihara et al., 2004; Miyamoto et al., 2005; Cheng et al., 2007). Generally speaking, these genes have not been shown to undergo methylation-induced silencing as part of a normal development.
We have identified DNA methylation in complex cell populations (term placental tissue and CVS), partially purified trophoblasts (first-trimester CK-7 positive cytotrophoblasts) and transformed invasive EVT-like cell lines (JEG-3 and others). Methylation was detected in each of these samples albeit with slightly different patterns for different genes. We did not see similar methylation in placenta-derived leucocytes or fibroblasts isolated from different density bands following percoll gradient centrifugation (data not shown). On the basis of these data, we can conclude that the methylation we are seeing is localized to the cytotrophoblast compartment. However, given that in some cases a pattern consistent with monoallelic methylation is observed, we cannot exclude the possibility that a specific population of trophoblasts within the purified cytotrophoblasts is the primary source of the observed DNA methylation.
It is clear that invasive differentiation of trophoblasts is essential for normal fetal growth and that Wnt/β-catenin signalling is implicated in this process (see above). The canonical Wnt/β-catenin signalling pathway has a pivotal role in the regulation of different aspects of cellular functioning and specialization via nuclear accumulation of β-catenin and regulation of TCF transcription factors (Willert and Jones, 2006). In cancer, APC and other negative regulators become targets of mutations or epigenetic inactivation leading to constitutive Wnt pathway activation (Aguilera et al., 2007). In addition, active Wnt signalling has been implicated in the survival, differentiation and invasion of human trophoblasts with nuclear β-catenin accumulation apparent in extravillous trophoblast cells (Pollheimer et al., 2006).
Wnt signalling is largely mediated through the membrane-bound frizzled receptors. The related secreted frizzled receptor proteins (SFRP-1, -2, -4 and -5) lack signalling capacity and inhibit Wnt receptor binding to down-regulate Wnt signalling (Rattner et al., 1997; Suzuki et al., 2004). Epigenetic down-regulation of SFRP genes is instrumental in the early stages of Wnt pathway activation in tumourigenesis (Suzuki et al., 2004). In addition, recent data have implicated decreasing SFRP4 expression with Wnt pathway activation in trophoblast cells of the basal zone in the rat (Hewitt et al., 2006). This is interesting in light of our demonstration of a lack of corresponding methylation in SFRP-4 in human placental tissue.
Engrailed proteins (En-1 and -2) are highly conserved homeodomain-containing DNA-binding proteins that also play important roles in many different developmental processes (Joyner and Martin, 1987; Hidalgo, 1996; Joyner, 1996). Data have emerged implicating En-1 in the negative regulation β-catenin transcriptional activity via a novel proteasomal degradation mechanism. Thus, En-1 acts as a negative regulator of Wnt pathway signalling (Bachar-Dahan et al., 2006), albeit via a distinct mechanism to SFRP proteins or the multi-protein destruction complex containing APC. Similarly, loss of RASSF1A results in the nuclear accumulation of β-catenin due to the disruption of β-TrCP-mediated β-catenin degradation (Estrabaud et al., 2007). This has been postulated to occur in the presence of other cooperative Wnt signalling activation processes (van der Weyden et al., 2008) in a similar manner to that demonstrated by co-inactivation of SFRP and APC genes in human colon cancer (Taketo, 2004).
The demonstration of a coordinated methylation-induced reduction in expression of APC, SFRP-2, RASSF1A or En-1 genes in combination will increase Wnt signalling with associated downstream consequences on gene expression profile in human trophoblast and/or other placental cells. These cumulative data highlight the importance of Wnt signalling in human placentation. However, the different patterns of methylation seen for each of the Wnt pathway inhibitory genes raises the possibility of cell-type-specific up-regulation of Wnt signalling via different mechanisms in different cell types. For example, whereas the APC gene shows a pattern of methylation consistent with monoallelic methylation in full term placental tissue, CVS samples and purified first-trimester cytotrophoblasts, the SFRP2 gene promoter shows near complete methylation in each of these cell populations. In addition, whereas APC methylation is lost with CVS culturing, near complete SFRP2 methylation is found in cultured CVS samples. The En-1 gene shows a lower level of methylation than APC or SFRP2 in term placenta, CVS, and first-trimester EVTs, but all three genes are almost completely methylated in CCA cell lines. We have now begun to further define the subcellular localization of this methylation in a wider variety of purified placental subtypes.
It is particularly interesting that we did not detect methylation in any of the genes studied in mouse placentas which show a lower degree of invasiveness in comparison with primate counterparts. However, the previous demonstration of methylation of the SFRP-4 gene in the rat would also suggest a role for increased Wnt signalling in rodent placentation, possibly via an alternate epigenetic regulatory mechanism. The role that such specific methylation plays in trophoblast function or invasion will require further investigation but will undoubtedly provide valuable insights into the necessity for Wnt signalling in specific trophoblast functions.
Trophoblast hyperplasia is a common feature of complete hydatidiform moles with the potential for malignant transformation to choriocarcinoma (Li et al., 2002). Such tumours are often associated with abnormal expression of cell cycle regulatory proteins including cyclins, and tumour suppressors such as p53 (Ichikawa et al., 1998). Trophoblasts that comprise the choriocarcinoma resemble the primitive trophoblast of the pre-villous stage during placental development, arrested in specific stages of differentiation (Shih Ie and Kurman, 2002). The increasing levels of methylation of the SFRP2, APC, EN-1, SIM-1, CD44, EGR4 and DCC-1 in all CCA cell lines provides compelling evidence for a link between the further down-regulation of these genes and the tumourigenic processes leading to choriocarcinoma. The significance of the differential methylation profile exhibited by KIAA0101 remains unclear but likely reflects a differing cellular localization of methylation within the placenta, or differing role in trophoblast tumourigenesis.
The placenta is a complex tissue that displays many cell subtypes likely to display a range of methylation profiles. In the case of the Wnt signalling pathway, a series of coordinated methylation events appears to contribute to the activation of this signalling cascade in a similar manner to that often seen in cancer. Further definition of the mechanisms regulating this methylation in the placenta may therefore provide valuable insights into cancer progression in humans.
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Supplementary Data |
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Supplementary data are available at http://molehr.oxfordjournals.org/.
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R.S. and J.M.C. are supported by National Health and Medical Research Council (Australia) RD Wright Fellowships.
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We would like to thank Dr Nicole Brooks and Ms Tina Vaino (Mercy Hospital for Women, Australia) and Ms Sarah Healy (Royal Women's Hospital) for help with collection of placental tissue. Professor Lois Salamonson (Prince Henry's Institute of Medical Research, Australia) and Professor Samuel Breit (University of NSW, Australia) for CCA cancer cell lines, Drs Patrick Western and Craig Smith (Murdoch Childrens Research Institute) for mouse placental tissue, Dr Mark Pertile for human CVS and Prof Anne-Maree Hennessey and Dr Neroli Sunderland (Royal Prince Alfred Hospital) for baboon placenta and CVS tissue. Special thanks to Prof Bob Williamson for comments relating to the manuscript.
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References |
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Submitted on June 19, 2008; resubmitted on July 31, 2008; accepted on August 6, 2008.
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