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Brachyury Is Expressed by Human Teratocarcinoma Cells in the Absence of Mesodermal Differentiation¹

Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

	Abstract

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Reverse transcription-PCR and Northern and Western blot analyses indicate that mRNA and protein encoded by the Brachyury gene are expressed by the pluripotent human embryonal carcinoma cell line NTERA2 and are only modestly down-regulated during retinoic acid-induced differentiation. This differentiation occurs along a neural lineage, with no obvious evidence of the formation of mesodermal derivatives. Several other human embryonal carcinoma cell lines that do not differentiate, a yolk sac carcinoma cell line and two choriocarcinoma cell lines, also express readily detectable levels of Brachyury mRNA and protein. Thus, in human teratocarcinomas, Brachyury expression is not necessarily an indicator of commitment to mesodermal differentiation.

	Introduction

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Teratocarcinomas are a subset of GCTs,³ the most common type of cancer occurring in young men. They contain a disorganized array of embryonic and extraembryonic tissues and provide a paradigm for the notion that the development and progression of cancer involve aberrations in the same mechanisms that regulate cell differentiation during embryogenesis (1) . Typically, teratocarcinomas contain highly malignant stem cells, EC cells, that differentiate to form multiple cell lineages leading to a range of terminally differentiated cells, often with the loss of malignancy, in a caricature of embryogenesis (2) . In the laboratory mouse, it has long been known that EC cells closely resemble ES cells of the early embryo, and they have been widely used as tools for investigating aspects of embryonic cell differentiation (3 , 4) . Likewise, recent studies have confirmed the similarity of human tumor-derived EC cells to human embryo-derived ES cells (5 , 6) . However, it is also evident that human EC and ES cells differ from their murine counterparts. For example, human EC and ES cells characteristically express the globoseries glycolipid antigens SSEA3 and SSEA4 but not the lactoseries glycolipid antigen SSEA1 (6 , 7) , whereas murine ES and EC cells are typically SSEA3(-) and SSEA4(-), but SSEA1(+) (8, 9, 10, 11) . Furthermore, human EC and ES cells commonly seem able to differentiate into trophectoderm, a pathway that is, apparently, usually closed to murine EC and ES cells (6 , 7) .

Many human EC cell lines have apparently lost the capacity for differentiation, perhaps because genetic variants that cause the loss of pluripotency have a selective advantage for tumor growth, because differentiation often appears to result in loss of a malignant phenotype. However, NTERA2 is one human EC cell line that does differentiate extensively and irreversibly in response to retinoic acid to yield a variety of terminally differentiated cells including neurons (12) . This differentiation is marked by loss of characteristic EC cell surface antigens and acquisition of new differentiation-specific antigens (13) and by the induction of various developmentally regulated genes, such as those of the HOX cluster, which are activated in a retinoic acid dosage-dependent manner (14 , 15) . Another developmental gene activated during NTERA2 differentiation is Wnt13 (but not Wnt1), in contrast to the differentiation of some murine EC cells (16) .

Although the most noticeable cells in differentiated NTERA2 cultures are postmitotic neurons that express a variety of distinctive neural features (12 , 17, 18, 19) , only a small fraction (typically <10%) of the differentiated cells adopt this phenotype. Indeed, many of the differentiated cells derived from NTERA2 EC cells appear to be nonneural, and questions remain as to their identity. One possibility is that some of these nonneural, differentiated NTERA2 cells represent a mesodermal lineage, although no evidence of skeletal muscle differentiation has been forthcoming (12) . Accordingly, we sought to test this hypothesis by examining the expression of Brachyury during NTERA2 differentiation.

Brachyury (or T) was first identified in the laboratory mouse as a dominant short tail mutant that is also a recessive lethal; homozygous T/T embryos die in mid-gestation due to a failure of posterior mesoderm (20 , 21) . Following cloning of the murine Brachyury gene (22) and its homologues in other species (23, 24, 25, 26, 27, 28, 29) , Brachyury has generally proved a valuable marker for recognition of mesodermal differentiation (30) . For example, apart from expression in embryos themselves, Brachyury has been reported to be activated during the differentiation of certain murine EC and ES cell lines differentiating along mesodermal lineages in vitro (31, 32, 33) .

The human homologue of the mouse Brachyury has been cloned, and its expression was detected by RT-PCR in the notochord remnant, the nucleus pulposus, of human abortuses at 14–15 weeks gestation, but not in the fetal intestine or muscle, or in 14-week spinal cord (29) . Both the gene and its predicted protein show strong homology to the mouse Brachyury. It was therefore with some surprise that we discovered that Brachyury is expressed generally by undifferentiated human EC cells as well as by the differentiating derivatives of NTERA2 EC cells and by yolk sac carcinoma and choriocarcinoma cells.

	Results

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Brachyury Is Detectable in the Pluripotent Human EC Cell Line NTERA2.
To determine whether Brachyury is expressed and developmentally regulated in differentiating cultures of NTERA2 EC cells, RNA from the undifferentiated EC cells and from the cells induced to differentiate with 10^-5 M retinoic acid was initially analyzed by RT-PCR. A product of the expected size of 252 bp was obtained in each case (Fig. 1A) . The RT-PCR product was confirmed to correspond to Brachyury by Southern blotting using a specific probe generated from a plasmid containing the first exon of human Brachyury, provided by Dr. D. Stott (University of Warwick, Coventry, United Kingdom). Finally, the PCR product was cloned and shown to have a sequence identical to that of corresponding segment of human Brachyury exon 1.

View larger version (46K): [in this window] [in a new window]   Fig. 1. PCR analysis of Brachyury expression in differentiating NTERA2 cells induced with retinoic acid. A, RT-PCR from duplicate reactions with RNA isolated from NTERA2 EC cells (Lane 1) and cells differentiating in response to retinoic acid for 4 and 14 days (Lanes 2 and 3). B, Brachyury representation in cDNA libraries (34) prepared from NTERA2 EC cells (Lanes 1), ME311(+) retinoic acid-induced NTERA2 cells (nonneural; Lane 2), and purified NTERA2 derived neurons (Lane 3). The PCR products were detected by Southern blotting.

To confirm the PCR results in a more quantitative manner, Northern blot analysis was carried out on mRNA isolated from differentiated NTERA2 cells. Brachyury mRNA was clearly detected in the untreated EC cells and, with little change in levels, on retinoic acid induction (Fig. 2) . The Brachyury message also appeared to persist in the purified neuronal preparation (Fig. 2 , Lane 1), although at a very low level compared with the other stages of NTERA2 differentiation.

View larger version (77K): [in this window] [in a new window]   Fig. 2. A, Northern blot analysis of Brachyury mRNA expression in NTERA2-derived cells. Lane 1, purified neurons; Lane 2, cells treated for 12 days with retinoic acid; Lane 3, cells treated for 8 days with retinoic acid; Lane 4, cells treated with a 4-day retinoic acid treatment; Lane 5, EC cells. B, ß-actin control probe.

RT-PCR indicated general expression of Brachyury in all of the other human EC and other GCT-related cell lines tested (data not shown), and Northern blot analysis revealed a mRNA transcript of approximately 2 kb corresponding to human Brachyury in each case (Fig. 3) . Thus, Brachyury expression seems to be a typical feature of a wide range of these GCT-related cell lines, including several EC cells that do not differentiate significantly.

View larger version (81K): [in this window] [in a new window]   Fig. 3. mRNA (5 µg/lane) was electrophoresed from a variety of cell lines of germ cell origin and subjected to Northern analysis. Lane 1, BeWo; Lane 2, TERA1; Lane 3, 577 MF; Lane 4, 2102Ep; Lane 5, 1156QE; Lane 6, 833KE; Lane 7, JAR.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Western blot analysis of BRACHYURY protein expression in various human GCT- and choriocarcinoma-derived cell lines: NTERA2 EC cells (Lane 1); NTERA2 cells differentiated in retinoic acid for 8 days (Lane 2) and 13 days (Lane 3); 2102Ep EC cell clones cl.4D3 (Lane 4) and cl.2A6 (Lane 5); 833KE (Lane 6); TERA1 (Lane 7); 1156QE (Lane 8); 1777N RpMet (Lane 9); 1411H (Lane 10); BeWo (Lane 11); JAR (Lane 12); and 577 MF (Lane 13).

	Discussion

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Several additional genes containing a consensus sequence, the T-box, in common with Brachyury have been reported, notably the T-box genes, Tbx1–5 (37) . However, it is very unlikely that any of these were detected in the present experiments. The RT-PCR reactions and the Northern analyses were tested by hybridization with a probe derived from the first exon of human Brachyury. Analysis using a BLAST search algorithm⁴ indicated that the probe would only detect the published human Brachyury sequence and not sequences derived from other related human T-box genes. Furthermore, sequencing of RT-PCR-derived clones indicated that the amplified DNA did indeed correspond to human Brachyury.

With the exception of NTERA2, the EC cell lines studied show little or no evidence of an ability to differentiate (7 , 8 , 36 , 38) . Previously, we had failed to find expression of desmin in retinoic acid-induced NTERA2 cells (12) , whereas we have also failed to find evidence of MyoD or cardiac actin,⁵ again implying the absence of muscle differentiation Thus, there is no evidence for a propensity for mesodermal differentiation by the human EC cells studied, despite their expression of Brachyury.

The expression of Brachyury by undifferentiated human EC cells may appear somewhat surprising. During the embryogenesis of several species, Brachyury expression has been specifically associated with the appearance of mesodermal precursor cells (30) . For example, in the mouse, Brachyury is expressed in the cells adjacent to the primitive streak from the onset of gastrulation and in the primitive streak cells induced by the endoderm to form the mesoderm, and it continues to be expressed as these cells differentiate (39) . In Xenopus embryos, Brachyury is evidently sufficient to induce mesoderm (40) . On the other hand, studies of chimeric T/T +/+ mouse embryos suggest that Brachyury is not required for the formation of mesoderm in mammals but rather plays a role in migration of these cells from the primitive streak (41) . Consistent with this view is the observation that although Brachyury is substantially up-regulated when P19 murine EC cells are induced to differentiate into mesodermal derivatives, its overexpression alone is not sufficient to cause the formation of mesoderm cells (31) . Indeed, although Brachyury marks embryonic cells that become mesoderm, its expression occurs at such an early stage of their commitment that they are still competent to form other cell types (42) . Thus, Brachyury expression may not necessarily be a marker for commitment, but rather one of competence for mesodermal differentiation.

Recent studies of mouse ES cells in which E-cadherin expression was disrupted suggest a corresponding induction of Brachyury expression (43) . Indeed, we have found that NTERA2 EC cells express particularly low levels of E-cadherin (44) , and we wondered whether this might contribute to the expression of Brachyury in these cells. However, the other human EC cell lines that we find express Brachyury also express substantially higher levels of E-cadherin (44) . Therefore, it seems unlikely that low E-cadherin expression can explain the general expression of Brachyury by human EC cells.

The significance of readily detectable levels of Brachyury in the range of human embryonic tumors studied here is unclear, but this study serves to indicate further differences between these human tumors and their murine counterparts. One possibility is that the results reflect the tumor derivation of EC cells and are not necessarily indicative of the phenotype of human embryo-derived ES cells. On the other hand, at least with respect to expression of characteristic surface antigens, the phenotypes of human EC and ES cells are reported to be similar (5) . Low levels of Brachyury have been reported in some murine EC cell lines (31) , but we are not aware of evidence of expression in the extraembryonic cells to which choriocarcinoma and yolk sac carcinomas correspond. Given that Brachyury expression in murine embryonic cells is associated with competence for mesoderm differentiation, its presence in nullipotent human EC cells might indicate a similar competence, although the corollary of their nullipotency would then be an active repression of differentiation in such cells. Such a repression would clearly have a selective advantage for faster, more aggressive tumor growth.

	Materials and Methods

TOP Abstract Introduction Results Discussion Materials and Methods References

 
Cell Culture.
NTERA2 clone D1 (NTERA2) human EC cells were maintained as undifferentiated stock cultures in DMEM supplemented with 10% FCS as described previously (45) . To induce differentiation, these cells were harvested using trypsin:EDTA (0.25% trypsin; 1 mM EDTA) and seeded at 10⁶ cells/75-cm² tissue culture flask in medium containing 10^-5 M all-trans retinoic acid (Eastman Kodak) (12) . Neurons were purified from retinoic acid-induced differentiated NTERA2 cultures after 3–4 weeks using the technique described by Pleasure et al. (19) .

Several other cell lines derived from human testicular GCTs were also studied. These included the EC cell lines 1156QE, 2102Ep, TERA1, and 833KE (8 , 38 , 46 , 47) ; the yolk sac carcinoma cell line 1411H (48) ; and the malignant teratoma cell line 577 MF (38) . In addition, we also analyzed the gestational choriocarcinoma cell lines JAR (49) and BeWo (50) .

PCR Analysis.
Polyadenylated RNA was reverse-transcribed using 300 units of Moloney murine leukemia virus reverse transcriptase (Promega) for 2 h at 37°C in the presence of poly(dT)₁₈/random primer, 200 µM deoxynucleotide triphosphate in 50 µl of reaction buffer [final concentration, 50 mM Tris-HCl (pH 8.5), 40 mM KCl, 10 mM DTT, 7 mM MgCl₂, and 0.1 mg ml^-1 BSA]. For PCR, RT cDNA (50 ng) derived in this way or pooled cDNA obtained from cDNA libraries was incubated with 20 pmol of primers (size, 20–30 mer), 200 µM each deoxynucleotide triphosphate, PCR reaction buffer, 2.5 mM MgCl₂, 1 unit of Taq DNA polymerase, and distilled H₂O to 30 µl.

PCR primers corresponding to exon 1 of human Brachyury (GenBank accession number NM003181; Ref. 29 ) were as follows: (a) Brachyury, forward primer 5'-TAAGGTGGATCTTCAGGTAGC-3' (bp 127–146 of GenBank accession number NM 003181); and (b) Brachyury, reverse primer 5'-CATCTCATTGGTGAGCTCCCT-3' (358–377 bp).

The following PCR cycle was used: (a) 93°C, 3 min (1 cycle); (b) 94°C, annealing temperature (58^oC), 1.5 min, 72°C, 1.5 min (35 cycles); and (c) 72°C, 5 min (1 cycle).

PCR products were analyzed by electrophoresis on 1% agarose gels containing 1x Tris-acetate-EDTA buffer. Selected DNA bands were excised, purified by ethanol precipitation, and cloned by ligating into precut T/A vectors (Promega) that were used to transform DH5- Escherichia coli. Cloned DNA fragments were sequenced using the Prism fluorescence-labeled chain terminator sequencing kit (Perkin-Elmer) and analyzed by the in-house sequencing service (Krebs Institute, University of Sheffield, Sheffield, United Kingdom).

Southern and Northern Blot Analyses.
For Southern blotting, DNA was separated on horizontal 1–1.5% agarose gels and transferred in 0.4 N NaOH to Hybond membranes (Amersham-Pharmacia, Ltd.). ³²P-labeled DNA probes were hybridized at 65°C in 5x saline-sodium phosphate-EDTA, 5x Denhardt’s solution, 0.5% (w/v) SDS, and 0.5 mg of denatured salmon sperm DNA.

For Northern analysis, 5 µg of mRNA were loaded per lane on a 1% MOPS acid gel (51 ; 3–5 mm thick) and electrophoresed in 1x MOPS buffer [0.04 M MOPS, 0.01 M sodium acetate, and 1 mM EDTA (pH 7.2)] at 60 V for 3–4 h. RNA was transferred in 10x SSC to a Gene Screen Plus nylon membrane (DuPont). Hybridization was performed at 42°C in 50% (w/v) formamide, 5x saline-sodium phosphate-EDTA, 5x Denhardt’s solution, 1% SDS, and 10% dextran sulfate sodium salt (M_r 500,000). Hybridization in Southern and Northern analyses was visualized by a Bio-Rad phosphorimager.

Western Blot Analysis.
Monolayers of cells were rinsed three times with ice-cold PBS and incubated with 1 ml of lysis buffer/75-cm² flask (1% v/v NP40, 1% w/v sodium deoxycholate, and 0.1 mM phenylmethylsulfonyl fluoride in PBS) for 15 min at 4°C. Cell lysates were passed through a 21-gauge needle to shear the DNA, followed by a freeze/thaw cycle and centrifugation (30 min, 4°C, 15,000 x g) to remove insoluble material. Protein concentrations were determined using the Bio-Rad assay. SDS-PAGE was carried out by the method of Laemmli (52) using 16 µg of protein per lane of a 10% polyacrylamide gel. Subsequently, the separated proteins were transferred electrophoretically to a nitrocellulose membrane (pore size, 0.45 µm), which was then washed with PBS and 0.05% Tween (PBS-T) and blocked using 5% milk powder dissolved in PBS-T (60 min, room temperature). The blots were incubated with a purified polyclonal antibody raised against a peptide composed of the first 129 amino acids of mouse BRACHYURY protein (24) . After washing, the blots were incubated with horseradish peroxidase-labeled secondary antibody; and antibody was visualized by using the enhanced chemiluminescence technique (Amersham-Pharmacia).

	Acknowledgments

 
We are grateful to Dr. B. G. Herrmann of the Max-Planck Institute, Freiburg, Germany for providing a polyclonal antibody to the BRACHYURY protein and to Dr. D. Stott of the University of Warwick, Coventry, United Kingdom for a DNA clone of exon 1 of human Brachyury. We thank Christine Pigott for excellent technical assistance.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by grants from the Wellcome Trust and the EU Human Capital Mobility program.

² To whom requests for reprints should be addressed, at Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. Phone: 44-0-114-222-4173; Fax: 44-0-114-222-2399; E-mail: P.W.Andrews{at}Sheffield.ac.uk

³ The abbreviations used are: GCT, germ cell tumor; RT-PCR, reverse transcription-PCR; EC, embryonal carcinoma; ES, embryonic stem; MOPS, 4-morpholinepropanesulfonic acid.

⁴ www.ncbi.nlm.nih.gov.

⁵ Unpublished results.

Received for publication 11/30/99. Revision received 1/13/00. Accepted for publication 1/14/00.

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