Cancer Research Targets Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 4715-4719, September 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Badcock, G.
Right arrow Articles by Andrews, P. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Badcock, G.
Right arrow Articles by Andrews, P. W.
[Cancer Research 59, 4715-4719, September 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

The Human Embryonal Carcinoma Marker Antigen TRA-1-60 Is a Sialylated Keratan Sulfate Proteoglycan

Graeme Badcock, Christine Pigott, John Goepel and Peter W. Andrews1

Departments of Biomedical Science [G. B., C. P., P. W. A.] and Pathology [J. G.], University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and they are key components of germ cell tumors (GCTs). They express several high molecular weight glycoprotein antigens that are down-regulated upon differentiation. One of these antigens, defined by monoclonal antibody TRA-1-60, can be detected in the serum of GCT patients and provides a useful complement to the established serum markers human chorionic gonadotropin and {alpha}-fetoprotein, especially in those patients without elevated serum human chorionic gonadotropin or {alpha}-fetoprotein. To examine the relationship of the TRA-1-60-defined antigen to similar antigens defined by other monoclonal antibodies, we have carried out comparative Western blot and immunoprecipitation analyses of human GCT-derived cell lines with monoclonal antibodies TRA-1-60, TRA-1-81, GCTM2, and K21. The TRA-1-60 antigen was detected by Western blot analysis in extracts of all human EC cell lines and in clinical specimens of GCT tested as a diffuse band with a molecular weight of >200,000. A similar but noticeably fainter band was detected in GCT composed of seminoma only. The antigen was not expressed by GCT-derived lines without an EC phenotype. Affinity bead-purified TRA-1-60, TRA-1-81, GCTM2 and K21 antigens reacted in Western blot analysis with each of the other antibodies tested, indicating that the epitopes recognized by each antibody are carried by the same molecular species. This molecule could be metabolically labeled with inorganic [35S]sulfate and was degraded by keratanase. Glycopeptides produced from affinity-purified TRA-1-60 antigen by extensive digestion with Pronase exhibited a molecular weight in excess of 10,000 and were degraded by keratanase. The TRA-1-60 epitope was destroyed by digestion with neuraminidase, but the epitopes defined by TRA-1-81, GCTM2, and K21 were not. Our results indicate that human EC cells generally express a cell surface sialylated keratan sulfate proteoglycan that is subject to modification to yield a variety of epitopes, one of which is recognized by the monoclonal antibody TRA-1-60. Sensitivity to milk alkaline digestion suggests that the oligosaccharides of this proteoglycan are O-linked to a core polypeptide.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular germ cell tumors, which include EC,2 teratocarcinoma, and seminoma, are the most common solid tumors of young men (1 , 2) . Their incidence has increased 2–3-fold in most Western countries over the past 50 years (3) . The reasons for this increasing incidence are unclear, although a role for environmental estrogen analogues affecting germ cell development in utero has been proposed. Fortunately, these tumors are highly susceptible to chemotherapy, and current treatment regimens, including monitoring of serum tumor marker levels, are very successful (4) . Nevertheless, treatment failures occur, and even successful treatment is often accompanied by significant morbidity.

The serum tumor markers HCG and AFP are especially valuable tools for monitoring the treatment of patients (5 , 6) . They are widely used in clinical practice to detect tumor recurrence after primary treatment and to minimize the need for chemotherapy. However, a significant proportion of patients with GCT are negative for either HCG or AFP or for both markers. Furthermore, the expression patterns of these markers may change over time as the tumors evolve, with initially positive tumors sometimes becoming "marker negative" during treatment and follow-up. This probably reflects the fact that, in these histologically complex tumors, HCG and AFP are most likely produced by trophoblastic and yolk sac cells, respectively, rather than by the EC stem cells themselves. Whether HCG and/or AFP are detectable in the serum will depend upon the proportion of the trophoblastic and yolk sac elements present in the tumor.

EC cells are probably the single most important malignant component of nonseminomatous germ cell tumors and are thought to give rise to the other cell types of teratomas by differentiation. Therefore, a serum marker more directly linked to the presence of these stem cells would be a valuable complement to the existing markers, such as HCG and AFP. Several cell surface antigens characteristically expressed by human EC cells have been defined by a variety of monoclonal antibodies (e.g., Refs. 7, 8, 9, 10, 11 ). One of these antigens, TRA-1-60 (8) , is detectable in the serum of GCT patients whose tumors contain an EC component (12 , 13) . Moreover, as anticipated, serum TRA-1-60 antigen is often detectable even when HCG or AFP are absent, and it has, therefore, been suggested as an additional marker to complement serum HCG and AFP levels.

The TRA-1-60 antigen, like several other similar antigens reportedly expressed by human EC cells, is associated with a polydisperse glycoprotein with a molecular weight in excess of 200,000 (8 , 14) . Although the EC cell marker antigens SSEA3 and SSEA4 are glycolipids (9) , TRA-1-81, GCTM2, K4, and K21, for example, are monoclonal antibodies that identify proteins that are of similar size to that recognized by TRA-1-60, with similar, though not identical, patterns of expression on panels of tumor-derived cell lines (8 , 15) . However, the relationship of the TRA-1-60 antigen to these other similar antigens remains obscure. Sequential immunoprecipitation experiments have suggested that the TRA-1-60 and TRA-1-81 epitopes are associated with distinct molecular species (8) . Similarly, the K4- and K21-defined epitopes, which are reported to be carried by polylactosamine oligosaccharides, have also been detected associated with distinct polypeptides (11) . The antibody GCTM2 has been reported to recognize an epitope associated with a cell surface keratan sulfate, but whether the other antigens are also related to keratan sulfate has not been ascertained (16) .

We have now examined the relationship of the TRA-1-60-defined antigen from human EC cells to the antigens defined by monoclonal antibodies TRA-1-81, GCTM2, and K21. Our results suggest that these antibodies all recognize distinct epitopes formed by modifications of a common keratan sulfate core molecule that is characteristically expressed by human EC cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C under a humidified atmosphere of 10% CO2 in air. They were subcultured and reseeded at 5 x 106 cells per 75-cm2 culture flask after harvesting with 0.25% trypsin in Dulbecco’s PBS containing 1 mM EDTA. An exception was the NTERA2 pluripotent EC cell line, which was harvested for passage by scraping as described previously (17) . NTERA2 EC cells were induced to differentiate by culture in medium containing 10-5 M all-trans-retinoic acid (Eastman Kodak, Rochester, NY; Ref. 18 ). The germ cell tumor cell lines with an EC phenotype used were: 2102Ep cl.4D3 (7) , 833KE (19 , 20) , 1218E (20) , 1156QE (20) , and NTERA2 cl.D1 (17) . In addition, the following cell lines were used: 1411H, a yolk sac carcinoma line (21) ; 577 MF, a malignant teratoma cell line without an EC phenotype (20) ; BeWo, a gestational choriocarcinoma line (22) ; and HT29, a colorectal carcinoma line (23) .

Antibodies.
The antibodies TRA-1-60 and TRA-1-81 were obtained as culture supernatants from the respective hybridomas, as described previously (8) . GCTM2 (10) was provided by Dr. Martin Pera (Monash University, Clayton, Victoria, Australia), and K21 (11) was provided by Dr. Wolfgang Rettig (Memorial Sloan Kettering Institute, New York, NY). HRP-conjugated goat antimouse IgM (Sigma Chemical Co., Poole, Dorset, United Kingdom) was used as a detecting antibody in Western blots.

Affinity Purification Using Dynabeads.
M-450 Dynabeads, precoated with rat antimouse IgM (µ chain; 4.5-µm-diameter magnetic polystyrene beads; Dynal, Wirral, United Kingdom), were incubated for 1 h with primary antibody. After washing with PBS containing 0.1% BSA, these antibody-coated beads were incubated for 1 h with a lysate of 2102Ep EC cells prepared in 0.1% octylglucoside (Calbiochem), containing 0.1% BSA to prevent nonspecific binding. After extensive washing, the antigen-antibody complex was dissociated from the beads by boiling in electrophoresis loading buffer; after removal of the beads using a magnet, the resulting extract was subjected to SDS-PAGE.

Electrophoresis and Western Blot Analysis.
Cells were lysed in 0.1% octylglucoside (Calbiochem) in 0.05 M Tris-HCl (pH 6.8) with 0.1 M PMSF and subjected to SDS-PAGE according to the procedure of Laemmli (24) . For Western blot analysis, proteins were transferred electrophoretically from an SDS-polyacrylamide gel to a nitrocellulose membrane (0.45-µm pore size), which was then blocked with a 5% (w/v) solution of domestic fat-free milk powder containing 0.05% polysorbitan (Tween 20) in PBS for 2 h and successively incubated with primary monoclonal antibody in PBS (2 h at room temperature) and goat antimouse immunoglobulin conjugated with HRP. The blots were then developed using the Bio-Rad HRP Immuno-detection kit containing 4-chloro-1-napthol as the substrate for the HRP enzyme. For 35S-labeled samples, the gel was fixed and treated with the fluorographic reagent Amplify (Amersham, Little Chalfont, United Kingdom) for 20 min prior to exposure to Fuji RX X-ray film.

Metabolic Labeling with [35S]Sodium Sulfate.
2102Ep cells were cultured until just confluent in a 75-cm2 flask, washed once with sulfate-depleted medium (sulfate-depleted DMEM/Ham’s F-12; Life Technologies, Inc.) and cultured for a further 24 h in 10 ml of sulfate depleted medium including 10% dialyzed FCS and 125 µCi of [35S]sodium sulfate (DuPont-NEN, Stevenage, United Kingdom). Cells were then washed with PBS containing 0.1 mM PMSF and 10 mM magnesium sulfate and lysed at a proportion of 2 x 107 cells/ml in 0.05 M Tris-HCl containing 0.1% octylglucoside, 0.1 mM PMSF, and 1% BSA as a carrier protein.

Enzymatic Treatment.
Sialidase (Vibrio cholerae), {alpha}-fucosidase (chicken liver), endo-ß-galactosidase (Bacteroides fragilis), peptide-N-glycosidase F (Flavobacterium meningosepticum), endo-{alpha}-N-acetylgalactosaminidase (Streptococcus pneumoniae), chondroitinase ABC (Proteus vulgaris), and heparinase I (Flavobacterium heparinum) were all obtained from Oxford Glycosystems Ltd. (Abingdon, United Kingdom). Keratanase (Pseudomonas sp.) was obtained from ICN (Thame, United Kingdom). Cell lysates were prepared by osmotic shock in water for 5 min on ice; after centrifugation at 10,000 x g for 15 min to remove cell debris, the lysate was adjusted to the ionic strength and pH appropriate for each enzyme [heparinase I, 50 mM sodium acetate, 2.5 mM calcium acetate, and 50 mM NaCl (pH 7.0); chondroitinase ABC, 50 mM Tris-HCl (pH 7.3); keratanase, 50 M Tris-HCl (pH 7.4); and endo-ß-galactosidase, 50 mM sodium citrate/phosphate (pH 5.7)].

Glycopeptide Analysis.
Antigen labeled with 35S was purified using Dynabeads and digested with Pronase (Calbiochem; 1 mg/ml) in 0.05 M Tris-HCl (pH 8.0) at 37°C for 24 h. A further equal volume of enzyme solution was then added, and the incubation was repeated for another 24 h (25) . After heating to 100°C for 5 min to destroy the Pronase activity, the digest was either chromatographed directly on G-50 Sephadex in 0.1 M NaCl and 0.05 M Tris-HCl (pH 7.2 or adjusted to pH 7.4, the optimum for keratanase) and incubated for a further 24 h with 2 units of keratanase at 37°C before chromatography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To confirm that the TRA-1-60 antigen is generally expressed in a similar form by human EC cells, we carried out Western blot analysis of lysates from a series of GCT-derived cell lines (Fig. 1A)Citation and clinical biopsies of testicular GCT (Fig. 1B)Citation . As anticipated, all of the EC cell lines tested (2102Ep, NTERA2, TERA1, 833KE, 1218E, and 1156QE) expressed detectable TRA-1-60 antigen and, in each case, the antigen appeared as a similar diffuse band with a molecular weight of >200,000. A similar band was also detected in lysates of three of five teratocarcinomas, three of four seminomas, and one of one mixed tumor with EC and seminoma components. There were some minor differences in the pattern of the antigen within the diffuse band between samples, which might indicate some heterogeneity between different cells. In contrast to the EC cells, no antigen was detected in extracts of the cell lines that do not exhibit an EC phenotype, including: differentiated derivatives of NTERA2 EC cells; a yolk sac carcinoma line, 1411H; a malignant teratoma line, 577 MF; a choriocarcinoma line, BeWo; and a colorectal carcinoma cell line, HT29. These results correlate well with previous studies by immunobinding assay of cell lines and immunohistochemistry of tumor samples (8 , 15) ; it seems likely that the TRA-1-60 antigen detectable in the teratocarcinoma samples was due to the presence of EC elements in these tumors. It is notable that the antigen band was relatively weak in each of the seminoma samples, which may indicate a lower level of expression in these cells and explain why TRA-1-60 antigen is readily detectable in the serum of patients with histologically recognizable EC elements but not in the serum of seminoma patients (12) .



View larger version (64K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Western blot analysis of TRA-1-60 reactivity of detergent lysates of GCT-derived cell lines (A) and clinical biopsies of testicular GCT (B). The lysates prepared in octylglucoside were electrophoresed SDS on a 7% polyacrylamide gel. A, Lane 1, 2102Ep (EC); Lane 2, NTERA-2 (EC); Lane 3, TERA-1 (EC); Lane 4, 833KE (EC); Lane 5, 1218E (EC); Lanes 6–8, NTERA-2 cells induced to differentiate by retinoic acid for 7, 14, and 21 days, respectively; Lane 9, 1156QE (EC); Lane 10, 1411H (yolk sac carcinoma); Lane 11, 577 MF (malignant teratoma); Lane 12, BeWo (choriocarcinoma); and Lane 13, HT29 (colon carcinoma). Note that all of the EC cell lines tested (2102Ep, NTERA2, TERA1, 833KE, 1218E, and 1156QE) expressed a diffuse TRA-1-60 reactive band with molecular weight in excess of Mr 200,000. B, Lane 1, lymphoma; Lanes 2–6, teratocarcinomas; Lanes 7–10, seminomas; and Lane 11, mixed pathology tumor including seminoma and teratocarcinoma. Note that a similar pattern of reactivity is seen in three of five teratocarcinomas, three of four seminomas, and one of one mixed pathology samples of testicular GCT.

 
Other monoclonal antibodies that recognize antigens expressed by human EC cells and are associated with a similar diffuse high molecular weight protein upon SDS-PAGE, including TRA-1-81 (8) , GCTM2 (10) , and K21 (11) . The similar size and expression pattern of these antigens suggest that they may be related. To investigate this, we isolated the antigens detected by TRA-1-60, TRA-1-81, GCTM2, and K21 by affinity bead purification and carried out Western blots with each antibody in turn (Fig. 2)Citation . In each case, it was evident that the antigen recognized by one of the antibodies also carries epitopes that are detected by each of the others.



View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Coexpression of TRA-1-60, TRA-1-81, GCTM2, and K21 reactive epitopes on the same molecular species. Antigen was purified from a lysate of 2102Ep human EC cells using beads armed with the respective antibodies, as indicated. The resulting bound antigen was then solublized, electrophoresed, subjected to Western blot, and probed with each antibody. Lanes 1, TRA-1-60; Lanes 2, TRA-1-81; Lanes 3, GCTM2; and Lanes 4, K21. As can be seen, the antigen isolated with any one of the antibodies was reactive with each of the others on Western blot analysis.

 
These results and earlier results suggesting association with different molecular entities (8) would be compatible if a single core protein is subject to posttranslational modification. For example, glycosylation could yield the different epitopes, several of which may nevertheless be carried by the same core polypeptide; however, some molecular species might carry only a subset of possible epitopes. The K21 epitope has previously been reported to be associated with oligosaccharide structures, whereas the GCTM2 epitope has been reported to recognize a polypeptide epitope of a keratan sulfate proteoglycan. To test whether the TRA-1-60 epitope is also associated with an oligosaccharide, we investigated whether it is sensitive to a variety of enzymatic or chemical degradations.

Digestion of 2102Ep cell lysates with {alpha}-fucosidase, endo-ß-galactosidase, or endo-{alpha}-acetyl galactosaminidase had no detectable effect upon the TRA-1-60 antigen (data not shown). We have previously shown that TRA-1-60 reactivity of cells is destroyed by neuraminidase digestion (14) . When Western blots of 2102Ep EC cell lysates were incubated with neuraminidase, prior to probing with antibody, the TRA-1-60 epitope was also destroyed (Fig. 3)Citation . On the other hand, the reactivity of the TRA-1-81, GCTM2, and K21 epitopes with their respective antibodies was enhanced, suggesting that sialylation blocks or sterically hinders these epitopes. Lower molecular weight forms of GCTM2 and K21, in particular, were also revealed by neuraminidase treatment.



View larger version (53K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Neuraminidase sensitivity of the TRA-1-60 antigen in comparison to the TRA-1-81, K21, and GCTM2 antigens. Following electrophoresis of 2102Ep human EC cell lysates, Western blots were incubated with neuraminidase prior to probing with antibody. As shown, the reactivity with TRA-1-60 was eliminated, whereas reactivity with TRA-1-81, GCTM2, and K21 was enhanced.

 
Because the GCTM2 antigen was reported to be a keratan sulfate proteoglycan (16) , we determined that the TRA-1-60 and TRA-1-81 antigens can also be similarly metabolically labeled with inorganic [35S]sulfate. When these three antigens were isolated by affinity immunobead purification from 2102Ep EC cells that had been grown in the presence of inorganic [35S]sulfate and digested with neuraminidase prior to electrophoresis, all three showed similar upward shifts in apparent molecular weight (Fig. 4)Citation . Likewise, the K21 antigen has also been reported to be metabolically labeled by inorganic sulfate and to show an increase in apparent molecular weight when digested with neuraminidase (11) .



View larger version (81K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Neuraminidase sensitivity of 35S-labeled TRA-1-60, TRA-1-81, and GCTM2 antigens. Lysates were prepared from 2102Ep human EC cells metabolically labeled with inorganic 35S, and the antigens that were reactive with TRA-1-60, TRA-1-81, and GCTM2 were affinity-purified and analyzed by PAGE. An aliquot of purified antigen was digested with neuraminidase prior to SDS-PAGE. In each case, the antigen was strongly labeled with 35S. Upon digestion with neuraminidase before electrophoresis, the apparent size of each antigen was similarly shifted upward.

 
Taken together, these results suggest that the TRA-1-60 antigen is closely related to the other similar high molecular weight glycoprotein antigens expressed by human EC cells and that all represent modifications to a core keratan sulfate proteoglycan. To examine this hypothesis, we incubated affinity bead-purified TRA-1-60 antigen with several enzymes that are specific for degrading various proteoglycans, prior to electrophoresis and Western blot analysis. No effect was observed with hyaluronidase, heparinase, or chondroitinase (data not shown), but the antigenic activity was abolished by keratanase (Fig. 5A)Citation . When 35S-labeled TRA-1-60 antigen was similarly incubated with keratanase, it was also degraded (Fig. 5B)Citation . For comparison, in the latter experiment, a parallel aliquot of 35S-labeled antigen was incubated with neuraminidase because trace neuraminidase contamination of keratanase was reported by the manufacturer. As shown previously, neuraminidase increased the apparent molecular weight of the labeled TRA-1-60 antigen (Fig. 5BCitation , Lane 4) and did not degrade it in the same way as did keratanase (Fig. 5BCitation , Lane 5).



View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Sensitivity to keratanase. A, affinity-purified TRA-1-60 antigen was incubated in the absence (Lane 1) or presence (Lane 2) of keratanase, after which it was analyzed by electrophoresis and Western blot. Note that the antigen was almost completely destroyed by keratanase. B, 35S-labeled affinity purified TRA-1-60 antigen was incubated with no enzyme (Lane 3), neuraminidase (Lane 4), or keratanase (Lane 5). Note the marked upward shift in apparent molecular weight after neuraminidase treatment and substantial diminution of the labeled TRA-1-60 antigen after keratanase treatment.

 
To investigate further the nature of the oligosaccharide carbohydrate chains associated with the TRA-1-60 antigen, we digested affinity bead-purified 35S-labeled TRA-1-60 antigen with Pronase. This digestion did not yield any discrete peptides, and all of the label ran with the dye front upon SDS-PAGE (data not shown). When the Pronase-digested antigen was subjected to gel filtration on Sephadex G50, all of the label eluted in the void volume, indicating association with glycopeptides with molecular weights in excess of 10,000 (Fig. 6A)Citation . However, when the Pronase digest was further digested with keratanase, the 35S-labeled glycopeptides were substantially degraded, confirming the keratan sulfate character of the TRA-1-60 antigen (Fig. 6B)Citation .



View larger version (10K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. 35S-labeled TRA-1-60 antigen was affinity-purified and subject to extensive digestion with Pronase (two periods of 24 h with 1 mg/ml Pronase at 37°C). The resulting glycopeptides were chromatographed on G50 Sephadex (A). An aliquot of the Pronase-digested TRA-1-60 antigen was further incubated with 2 units of keratanase at 37°C for 24 h and chromatographed on the same column (B). Note that the Pronase-digested [35S]glycopeptides were almost exclusively found in the void column (Mr >10,000), whereas these were substantially degraded by digestion with keratanase.

 
To determine whether the oligosaccharides with which the TRA-1-60 epitope appears to be associated are O- or N-linked, we first tested whether the affinity-purified antigen was susceptible to digestion with either peptide-N-glycanase F or endo-{alpha}-N-acetylgalactosaminidase (O-glycanase). Neither enzyme affected its antigenicity or electrophoretic mobility (data not shown). However, the antigen was completely destroyed by mild alkaline digestion, which is consistent with the presence of O-glycolytic linkages (Fig. 7)Citation .



View larger version (91K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. TRA-1-60 antigenicity was destroyed by alkaline hydrolysis, suggesting O linkage of carbohydrate chains. Alkaline hydrolysis of the TRA-1-60 antigen 2102Ep cell lysates were incubated at 45°C for 24 h in PBS (pH 7.0; Lane 1) or under various conditions of alkaline hydrolysis: 0.05 M NaOH, 25°C, 24 h (Lane 2); 0.1 M NaOH, 25°C, 16 h (Lane 3); and 0.3 M NaOH, 45°C, 5 h (Lane 4).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The commonly prevailing view of the origin of GCT is that they arise from transformed PGCs during embryonic development. The evidence for this in humans is largely circumstantial (26) . However, in the laboratory mice of the 129/Sv strain, which develop spontaneous testicular teratomas, strong experimental evidence supports the hypothesis that these tumors originate from germ cells about the time that they enter the genital ridge (27) . Significant differences in the pathology of human and mouse GCT, notably, the absence of seminomas in the mouse, might suggest that the 129/Sv mouse does not provide a good model of the human disease. On the other hand, we have suggested that, in both cases, generation of a tumor could involve two steps (28) : first, a block to PGC differentiation and their transformation into permanently mitotic cells; and second, a switch in phenotype from that of PGC to that of ES cells, "short-circuiting" the changes that are normally the consequence of gametogenesis and fertilization. We would suggest that the second step is stochastic, with a high probability of occurring in the mouse but low probability in humans: in this model, the transformed PGCs would correspond to carcinomas in situ and seminomas, whereas the second step generates EC cells as caricatures (29) of normal ES cells.

Certainly, there is a close similarity in the gene expression pattern of PGCs and ES/EC cells. For example, both express high levels of alkaline phosphatase (30, 31, 32) and, in the mouse, the lactoseries carbohydrate antigen, SSEA1 (33) . They also characteristically express the stem cell transcription factor Oct-4 (34) . Unlike the mouse, human EC cells do not express SSEA1 (20) . By contrast, the TRA-1-60 antigen is strongly expressed by human but not by mouse EC cells (8) . Recently, TRA-1-60 has also been shown to be expressed by monkey (35) and by human ES cell lines (36 , 37) , supporting the view that human EC cells do resemble human ES cells. Furthermore, the TRA-1-60 antigen is expressed by human PGC as well as by carcinoma in situ cells (38) and seminoma cells (15) , albeit at a lower level than the expression by EC cells. Thus, in this regard, the pattern of TRA-1-60 expression in humans is reminiscent of SSEA1 expression in the mouse, although, whereas there is no evidence that the TRA-1-60 antigen is associated with glycolipids (39) , SSEA1 has been associated with both glycolipids and high molecular weight glycoproteins (40) .

Our results confirm previous data on the expression of TRA-1-60 by human EC cells and germ cell tumors and demonstrate that the epitope is associated with a polydisperse macromolecule of similar size range in all cases, including seminoma. Previous studies failed to detect TRA-1-60 antigen in the serum of seminoma patients but found high levels in patients with histologically identifiable EC (12) . The results presented here suggest that this difference is more likely a reflection of different levels of expression rather than the epitope being associated with distinct molecular forms of the antigen.

Previous studies of human EC cells have indicated that they characteristically express high molecular weight oligosaccharides associated with cell surface glycoproteins (41) . Our results suggest that many of the various related high molecular weight glycoprotein antigens that are typically expressed by human EC cells, including the TRA-1-60 antigen, are variants of a related macromolecule, namely, a keratan sulfate. Whereas the precise epitope structure recognized by TRA-1-60 remains to be established, it seems most likely that it represents a terminal modification to the core-sulfated, O-linked lactosaminoglycan that constitutes the carbohydrate of keratan sulfate. How this antigen is shed by EC cells and whether all forms are shed are unclear. Previously, we failed to detect by Western blot the shed form of the TRA-1-60 antigen in culture supernatants of EC cell lines, although it is associated with equally high molecular weight aggregates by gel filtration chromatography (14) . This suggests that the nature of the molecule is altered on shedding.

Previous data have clearly indicated that serum TRA-1-60 antigen is a useful marker to complement others, notably AFP and HCG, used to monitor treatment of GCT patients (12 , 13) . The GCTM2 antigen has also been reported in serum of GCT patients (42) . A knowledge of the structure of the core keratan sulfate and its associated epitopes may help in designing simpler assay systems. Furthermore, the common expression of this family of keratan sulfate proteoglycans on EC cells and probably on early ES cells suggests that it may play an important role in embryonic development and in development and progression of GCT. The parallel finding with SSEA1 expression in the mouse and the observation that SSEA-1 and TRA-1-60 antigens both involve high molecular weight lactosaminoglycan core carbohydrates suggest the hypothesis that TRA-1-60 and its related molecules may perform in humans whatever functions are performed by SSEA1-reactive glycoproteins in the mouse.


   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 To whom requests for reprints should be addressed. Phone: 44-(0)114-222-4173; Fax: 44-(0)114-276-5413; E-mail: P.W.Andrews{at}sheffield.ac.uk Back

2 The abbreviations used are: EC, embryonal carcinoma; HCG, human chorionic gonadotropin; AFP, {alpha}-fetoprotein; HRP, horseradish peroxidase; PMSF, phenylmethylsulfonyl fluoride; PGC, primordial germ cell; ES, embryonic stem; SSEA1, stage-specific embryonic antigen-1. Back

Received 3/11/99. Accepted 7/21/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Damjanov I. Pathogenesis of testicular germ cell tumours. Eur. Urol., 23: 2-7, 1993.
  2. Dixon F. J., Moore R. A. Atlas of Tumor Pathology: Tumors of the male sex organs (monograph) Armed Forces Institute of Pathology Washington, DC 1952.
  3. SkakkebæK E. E., Rajpert-de Meyts E., Jørgensen N., Carlsen E., Petersen P. M., Giwercman A., Andersen A-G., Jensen T. K., Andersson A-M., Müller J. Germ cell cancer and disorders of spermatogenesis: an environmental connection? Acta Pathol. Microbiol. Immunol. Scand., 106: 3-12, 1998.
  4. Horwich A. Testicular germ cell tumours: an introductory overview Horwich A. eds. . Testicular Cancer, : 1-17, Chapman and Hall London 1996.
  5. Bosl G. J., Geller N. L., Cirrincione C. Serum tumor markers in patients with metastatic germ cell tumors of the testis. A 10 year experience. Am. J. Med., 75: 29-35, 1983.[Medline]
  6. Mason M. D. Tumour markers Horwich A. eds. . Testicular Cancer, : 35-51, Chapman and Hall London 1996.
  7. Andrews P. W., Goodfellow P. N., Shevinsky L. H., Bronson D., Knowles B. B. Cell-surface antigens of a clonal human embryonal carcinoma cell line: morphological and antigenic differentiation in culture. Int. J. Cancer, 29: 523-531, 1982.[Medline]
  8. Andrews P. W., Banting G., Damjanov I., Arnaud D., Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma, 3: 347-361, 1984.[Medline]
  9. Kannagi R., Cochran N. A., Ishigami F., Hakomori S-i., Andrews P. W., Knowles B. B., Solter D. Stage-specific embryonic antigens SSEA-3 and -4 are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J., 2: 2355-2361, 1983.[Medline]
  10. Pera M. F., Blasco-Lafita M. J., Cooper S., Mason M. D., Mills J., Monaghan P. Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells. Differentiation, 39: 139-149, 1988.[Medline]
  11. Rettig W. J., Cordon-Cardo C., Ng J. S. C., Oettgen H. F., Old L. J, Lloyd K. O. High-molecular-weight glycoproteins of human teratocarcinoma defined by monoclonal antibodies to carbohydrate determinants. Cancer Res., 45: 815-821, 1985.[Abstract/Free Full Text]
  12. Marrink J., Andrews P. W., Van Brummen P. J., De Jong H. J., Sleijfer D. T., Schraffordt Koops H., Oosterhuis J. W. TRA-1-60: a new serum marker in patients with germ-cell tumours. Int. J. Cancer, 49: 368-372, 1991.[Medline]
  13. Gels M. E., Marrink J., Visser P., Sleijfer D. T., Droste J. H. J., Hoekstra H. J, Andrews P. W., Koops H. S. Importance of a new tumour marker TRA-1-60 in the follow-up of patients with clinical state I nonseminomatous testicular germ cell tumours. Ann. Surg. Oncol., 4: 321-327, 1997.[Medline]
  14. Andrews P. W., Marrink J., Hirka G., Von Keitz A., Sleijfer D. T., Gonczol E. The surface antigen phenotype of human embryonal carcinoma cells: modulation upon differentiation. Rec. Res. Cancer Res., 123: 63-83, 1991.[Medline]
  15. Andrews P. W., Casper J., Damjanov I., Duggan-Keene M., Giwercman A., Hata J., Von Keitz A., Looijenga L. H. J., Millan J. L., Oosterhuis J. W., Pera M. F., Sawada M., Schmoll H. J., Skakkebaek N. E., Van Putten W. Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours. Int. J. Cancer, 66: 806-816, 1996.[Medline]
  16. Cooper S., Pera M. F., Bennet W., Finch J. T. A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line. Biochem. J., 286: 959-966, 1992.
  17. Andrews P. W., Damjanov I., Simon D, Banting G., Carlin C., Dracopoli N. C., Fogh J. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2: differentiation in vivo and in vitro. Lab. Invest., 50: 147-162, 1984.[Medline]
  18. Andrews P. W. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev. Biol., 103: 285-293, 1984.[Medline]
  19. Bronson D., Andrews P. W., Solter D., Cervenka D., Lange J., Fraley E. E. A cell line derived from a metastasis of a human testicular germ-cell tumor. Cancer Res., 40: 2500-2506, 1980.[Abstract/Free Full Text]
  20. Andrews P. W., Bronson D., Benham F., Strickland S., Knowles B. B. A. Comparative study of eight cell lines derived from human testicular teratocarcinoma. Int. J. Cancer, 26: 269-280, 1980.[Medline]
  21. Vogelzang N. J., Bronson D. L., Savino D., Vessella R. L., Frayley E. A. Human embryonal-yolk sac carcinoma model system in athymic mice. Cancer (Phila.), 55: 2584-2593, 1985.[Medline]
  22. Pattillo R. A., Gey G. O. The establishment of a cell line of human hormone synthesizing trophoblastic cells in vitro. Cancer Res., 28: 1231-1236, 1968.[Abstract/Free Full Text]
  23. Fogh J., Trempe G. New human tumor cell lines Fogh J. eds. . Human Tumor Cells in Vitro, : 115-159, Plenum Press New York 1975.
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.[Medline]
  25. Cossu G., Andrews P. W., Warren L. Covalent binding of lactosaminoglycans and heparan sulphate to fibronectin synthesised by a human teratocarcinoma cell line. Biochem. Biophys. Res. Commun., 111: 952-957, 1983.[Medline]
  26. Rajpert-de Meyts E., Jørgensen N., Brøndum-Nielsen K., Müller J., Skakkebaek N. E. Developmental arrest of germ cells in the pathogenesis of germ cell neoplasia. Acta Pathol. Microbiol. Immunol. Scand., 106: 198-206, 1998.
  27. Stevens L. C. The biology of teratomas. Adv. Morphol., 6: 1-31, 1967.
  28. Andrews P. W. Teratocarcinomas and human embryology: pluripotent human EC cell lines. Acta Pathol. Microbiol. Immunol. Scand., 106: 158-168, 1998.
  29. Pierce G. B. Teratocarcinoma. Introduction and perspectives Sherman M. I. Solter D. eds. . Teratomas and Differentiation, : 3-12, Academic Press New York 1975.
  30. Bernstine E. G., Hooper M. L., Grandchamp S., Ephrussi B. Alkaline phosphatase activity in mouse teratoma. Proc. Natl. Acad. Sci. USA, 70: 3899-3902, 1973.[Abstract/Free Full Text]
  31. Chiquoine A. D. The identification, origin and migration of primordial germ cells in the mouse embryo. Anat. Res., 118: 135-149, 1956.[Medline]
  32. Benham F. J., Andrews P. W., Bronson D. L., Knowles B. B., Harris H. Alkaline phosphatase isozymes as possible markers of differentiation in human teratocarcinoma cell lines. Dev. Biol., 88: 279-287, 1981.[Medline]
  33. Solter D., Knowles B. B. Monoclonal antibody defining a stage specific mouse embryonic antigen SSEA-1. Proc. Natl. Acad. Sci. USA, 75: 5565-5569, 1978.[Abstract/Free Full Text]
  34. Brehm A., Ovitt C. E., Schler H. R. Oct 4: more than just a POUerful marker of the mammalian germ line. Acta Pathol. Microbiol. Immunol. Scand., 106: 114-126, 1998.
  35. Thomson J. A., Kalishman J., Golos T. G., Durning M., Harris C. P., Becker R. A., Hearn J. P. Isolation of a primate embryonic stem cell line. Proc. Natl. Acad. Sci. USA, 92: 7844-7848, 1995.[Abstract/Free Full Text]
  36. Thomson J. A., Itskovitz-Eldor J., Shapiro S. S., Waknitz M. A., Swiergiel J. J., Marshall V. S., Jones J. M. Embryonic stem cell lines derived from human blastocysts. Science (Washington DC), 282: 1145-1147, 1998.[Abstract/Free Full Text]
  37. Shamblott M. J., Axelman J., Wang S., Bugg E., Littlefield J. W., Donovan P. J., Blumenthal P. D., Huggins G. R., Gearhart J. D. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci., 95: 13726-13731, 1998.[Abstract/Free Full Text]
  38. Giwercman A., Andrews P. W., Jørgensen N., Mller J., Graem N., Skakkebaek N. E. Immunohistochemical expression of embryonal marker TRA-1-60 in carcinoma in situ and germ cell tumors of the testis. Cancer (Phila.), 72: 1308-1314, 1993.[Medline]
  39. Fenderson B. A., Radin N., Andrews P. W. Differentiation antigens of human germ cell tumours: distribution of carbohydrate epitopes on glycolipids and glycoproteins analyzed using PDMP, an inhibitor of glycolipid synthesis. Eur. Urol., 23: 30-37, 1993.[Medline]
  40. Childs R. A., Pennington J., Uemura K., Scudder P., Goodfellow P. N., Evans M. J., Feizi T. High molecular weight glycoproteins are the major carriers of the carbohydrate differentiation antigens I, i, and SSEA-1 of mouse teratocarcinoma cells. Biochem J., 215: 491-503, 1983.[Medline]
  41. Muramatsu H., Muramatsu T., Avner P. Biochemical properties of the high-molecular-weight glycopeptides released from the cell surface of human teratocarcinoma cells. Cancer Res., 42: 1749-1752, 1982.[Abstract/Free Full Text]
  42. Mason M. D., Pera M. F., Cooper S. Possible presence of an embryonal carcinoma-associated proteoglycan in the serum of patients with testicular germ cell tumours. Eur. J. Cancer, 27: 300-, 1991.



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
E. Rajpert-De Meyts
Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects
Hum. Reprod. Update, May 1, 2006; 12(3): 303 - 323.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Yamagishi, K. Suzuki, K. Imai, H. Mochizuki, K. Morikawa, M. Kyogashima, K. Kimata, and H. Watanabe
Purification, Characterization, and Molecular Cloning of a Novel Keratan Sulfate Hydrolase, Endo-{beta}-N-acetylglucosaminidase, from Bacillus circulans
J. Biol. Chem., July 3, 2003; 278(28): 25766 - 25772.
[Abstract] [Full Text] [PDF]

Home page
 ASH Education BookHome page
C. M. Verfaillie, M. F. Pera, and P. M. Lansdorp
Stem Cells: Hype and Reality
Hematology, January 1, 2002; 2002(1): 369 - 391.
[Abstract] [Full Text]

Home page
 Hum ReprodHome page
B.E. Reubinoff, M.F. Pera, G. Vajta, and A.O. Trounson
Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method
Hum. Reprod., October 1, 2001; 16(10): 2187 - 2194.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. Pera, B Reubinoff, and A Trounson
Human embryonic stem cells
J. Cell Sci., January 1, 2000; 113(1): 5 - 10.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Badcock, G.
Right arrow Articles by Andrews, P. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Badcock, G.
Right arrow Articles by Andrews, P. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online