Alterations in the developmental potential of embryonal carcinoma cells in mixed aggregates of nullipotent and pluripotent cells.

Pluripotent embryonal carcinoma cells can be triggered to differentiate in vitro by allowing them to form multicellular aggregates. Nullipotent embryonal carcinoma cells form aggregates, but further development is blocked. Pluripotent and nullipotent embryonal carcinoma cell lines were co-cultured to form mixed aggregates in order to determine whether a developmental signal produced by the pluripotent cell could induce the nullipotent cells to differentiate. Unlike pure pluripotent cell aggregates, aggregates from cultures initiated with a 1:1 mixture of pluripotent (PSA-1) and nullipotent (F9) cells formed endoderm but failed to differentiate further. The nullipotent cells did not produce a detectable soluble inhibitor of differentiation. A hypoxanthine phosphoribosyltransferase-deficient subclone of the nullipotent cell line was used so that the fate of both nullipotent and pluripotent cells could be followed in autoradiographs of histological sections of aggregates labeled with ³H-hypoxanthine. Seven day old aggregates of pure pluripotent cell cultures contained endoderm, ectoderm and embryonal carcinoma cells. On the other hand, in 7 day old mixed cell aggregates, almost all the pluripotent cells became endoderm located on the outer surface of the aggregate. The nullipotent cells in the mixed aggregates assumed an internal position and remained embryonal carcinoma cells. Following the efficiency of plating of pluripotential cells in pure and mixed aggregates as a function of time showed that viable pluripotent embryonal carcinoma cells were lost at a 10 fold greater rate in mixed cell aggregates than in pure pluripotent cell aggregates. We conclude that nullipotent embryonal carcinoma cells in mixed aggregates with pluripotent cells exert a limitation on the ability of these pluripotent cells to differentiate.     

Vitronectin production by human yolk sac carcinoma cells resembling parietal endoderm

Normal mesenchymal cells, normal epithelial cells and many transformed epithelial cells require serum attachment factors and extracellular matrix proteins for growth and differentiation in vitro, and recent evidence strongly supports a role for extracellular matrix molecules in the regulation of cell movement in vivo during early embryogenesis. We previously described the isolation and characterization of cell lines representative of three types of stem cells most commonly found in human adult testicular teratomas, namely embryonal carcinoma cells, yolk sac carcinoma cells resembling visceral endoderm and yolk sac carcinoma cells resembling parietal endoderm (endodermal sinus tumour cells). Of these three cell types, only endodermal sinus tumour cells, which show particularly malignant behaviour in vivo, have no serum requirement for attachment and growth in vitro. Supernatants from endodermal sinus tumour cells support the attachment of embryonal carcinoma cells in serum-free medium. We demonstrate here that endodermal sinus tumour cells, but not other cell types isolated from testicular teratomas, secrete the serum attachment protein, vitronectin (also known as serum-spreading factor, S-protein or epibolin), as well as fibronectin, laminin and type IV collagen, into serum-free medium. Purified vitronectin from medium conditioned by endodermal sinus tumour cells supported both attachment and spreading of embryonal carcinoma cells in vitro, whereas cells attached but did not spread properly on surfaces coated with fibronectin or laminin. Peptides containing the RGD cell recognition sequence common to many attachment proteins blocked attachment of endodermal sinus tumour cells to untreated tissue-culture plastic in serum-free medium. The results suggest a possible role for vitronectin in regulating cell motility and growth in early development, and in the invasion and spread of teratomas in vivo.     

A new differentiated cell line (Dif 5) derived by retinoic acid treatment of F9 teratocarcinoma cells capable of extracellular matrix production and growth in the absence of serum

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 μM retinoic acid are described.Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize α-fetoprotein but does secrete plasminogen activator.An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF.The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.     

High molecular weight extracellular proteins synthesized by endoderm cells derived from mouse teratocarcinoma cells and normal extraembryonic membranes

Rabbit antiserum raised against teratocarcinoma embryoid bodies reacts with two extracellular, collagenase-resistant glycoproteins, PYS A and B, with molecular weights of approximately 350,000 and 220,000 daltons. The 220,000-dalton protein is distinguishable from fibronectin. The two proteins are synthesized and secreted into the medium in large amounts by the teratocarcinoma-derived parietal endoderm line PYS-1, and by normal parietal endoderm cells from the 10.5-day embryo. There was no detectable synthesis of PYS A and B by normal visceral endoderm cells isolated from the 10.5-day embryo, and only trace amounts of PYS A were synthesized by the teratocarcinoma-derived visceral endoderm line PSA5E and by mesodermal cells isolated from the visceral yolk sac. The two proteins therefore seem to be good biochemical markers for distinguishing parietal from visceral endoderm cells. Synthesis and secretion of PYS A and B could not be detected in undifferentiated embryonal carcinoma cells or in endoderm cells derived from them in the presence of retinoic acid.     

Basement membrane components secreted by mouse yolk sac carcinoma cell lines

Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac carcinoma respectively. Cell lines NE and ME were composed of a monomorphous cell population; however, the morphology of ME was growth-medium-dependent. LRD was composed of a heterogeneous cell population and formed embryoid bodies. NE secreted soluble laminin, osteonectin, entactin and fibronectin but did not form visible pericellular matrix. ME formed pericellular matrix which was composed of laminin and entactin, but did not contain fibronectin. The LRD cells formed pericellular matrix which was composed of laminin, entactin and fibronectin. Whereas laminin from ME and LRD reacted with polyclonal antibodies and a monoclonal antibody to parietal yolk sac laminin, the laminin from NE cells was unreactive with the monoclonal antibody. Osteonectin was found in the supernatant of LRD and ME, but could not be demonstrated immunohistochemically in the extracellular matrix. We conclude that some extracellular matrix components, such as laminin and fibronectin, are produced not only by yolk sac carcinoma cells but by nullipotent EC as well, although the latter do not assemble them into extracellular matrix. Laminin produced by EC is immunochemically different from laminin secreted by yolk sac carcinoma. The extracellular matrix produced by mixed parieto-visceral yolk sac carcinoma is different from the matrix laid down by the pure PYS in that the latter does not contain fibronectin. The lack of osteonectin in the extracellular matrix of yolk sac carcinoma cells indicates that not all polypeptides secreted by these cell lines are incorporated into the extracellular matrix. The new cell lines described in this paper differ with regard to their capacity to form extracellular matrix and secrete its various components. Hence they could be used for further studies of basement membrane assembly in vitro.     

Covalent binding of lactosaminoglycans and heparan sulphate to fibronectin synthesized by a human teratocarcinoma cell line

Fibronectin synthesized by the human teratocarcinoma cell line 2102Ep carries covalently bound lactosaminoglycan and heparan sulphate, whereas human fibroblast fibronectin does not. Murine embryonal carcinoma cells synthesize similarly modified fibronectin suggesting that this type of glycosylation may be generally important in early embryogenesis.     

Two multipotent embryonal carcinoma cell lines irreversibly differentiate in defined media

Two unrelated multipotent embryonal carcinoma cell lines, OC-15S1 and 1003, have been cultured in hormone-supplemented defined media in order to identify the signals that influence their differentiation. Previous studies have shown that F9 embryonal carcinoma cells can be grown for many generations in the defined medium, EM-3, which contains fibronectin, insulin, and transferrin in place of serum. F9 cells, which only differentiate into a few cell types, undergo little or no differentiation in EM-3 unless an inducer is present (A. Rizzino and C. Crowley, 1980, Proc. Natl. Acad. Sci. USA77, 457–461). This report demonstrates that, in contrast to F9, OC-15S1 and 1003 embryonal carcinoma cells do not proliferate in EM-3. Instead, the cells differentiate. However, the differentiated cells do not survive in EM-3 unless it is supplemented with factors such as purified serum lipoproteins. In EM-3 containing high-density lipoprotein, a population of differentiated cells, devoid of embryonal carcinoma cells, is formed. The differentiated cells that appear exhibit an epithelioid morphology throughout the culture. These cells also secrete plasminogen activator and two different criteria argue that it is the type released by parietal endoderm. This suggests that, under the influence of the defined medium, both multipotent embryonal carcinoma cell lines differentiate at high frequency into parietal endoderm. It was also determined that fibronectin promotes the differentiation of OC-15S1 and 1003 in serum-containing media, and this suggests that fibronectin is at least partly responsible for the differentiation observed in EM-3 plus high-density lipoprotein. In light of these findings, it is suggested that fibronectin may directly influence cellular differentiation during early mammalian development.     

Identification of murine extra-embryonic endodermal cells by reaction with teratocarcinoma basement membrane antiserum

Embryonal carcinoma cells from the PSA1 cell line will differentiate in vitro to form structures called embryoid bodies composed of an inner core of embryonal carcinoma cells surrounded by a basement membrane matrix and an outer layer of extra-embryonic endodermal cells. Immunization of rabbits with basement membranes isolated from embryoid bodies resulted in an antiserum, which binds to fixed extra-embryonic endodermal cells of either embryonic or teratocarcinoma origin but does not bind substantially to mouse embryonal carcinoma cells, fibroblasts, myoblasts or erythroleukemic cells. The F9-22 embryonal carcinoma cell line normally differentiates only to a very limited extent in vitro or in vivo. However, incubation of these cells in medium containing retinoic acid results in the appearance of cells resembling extra-embryonic endoderm. The embryoid body basement membrane antibodies were used to measure, by flow microfluorometry, the appearance of reactive cells in F9-22 cultures treated with retinoic acid. The kinetics of appearance of cells reactive with the basement membrane antibodies are similar to the kinetics of appearance of cells secreting plasminogen activator, a known marker of extraembryonic endoderm.     

Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast

NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found. Our results further establish that the ability of a cell to organize fibronectin into an extracellular matrix is dependent on certain gangliosides, but they also indicate that cell adhesion to fibronectin is independent of these gangliosides. We suggest that matrix organization and cell attachment and spreading are based on separate mechanisms and that these functions are associated with different cell surface "receptors."     

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.     

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells

In early postimplantation mouse development, transferrin synthesis appears to be a marker of visceral endoderm cell types. Transferrin was identified using immunoperoxidase staining, in the proximal (visceral) endoderm of the sixth-day egg cylinder, in some tissues at later stages, and in the visceral yolk sac (VYS) at all stages examined. Since the location of a plasma protein does not necessarily indicate its site of synthesis, the incorporation of labeled amino acids into transferrin was studied. Synthesis could be detected in egg cylinders on the seventh day of gestation onwards and in the VYS at all stages. However, although endoderm was the likely tissue source, its ability to synthesize transferrin after its isolation from the embryo was either much reduced or absent. The data are suggestive of a modulating influence by mesoderm and other cell types on transferrin synthesis in visceral endoderm cells. Three types of endoderm-like cells which are produced by teratocarcinoma embryonal carcinoma (EC) cells were analyzed for transferrin synthesis to assess possible parallels with the embryo. Embryoid bodies from PSA1 EC cells contained some outer endoderm cells which stained for transferrin and others which did not. The endoderm line PSA5E but not PYS-2 synthesized transferrin. The third type of endoderm-like cell (END cells) synthesized very little (OC15S1) or no (PC13 clone 5) transferrin. The conclusion that PSA5E, OC15 END, and some differentiated PSA1 cells have visceral endoderm-like character while PYS-2 reflects parietal endoderm phenotype is in agreement with published data.     

Expression and structure of human SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some of these cells show an unusual expression pattern

A mouse SPARC cDNA clone was used to elucidate the expression of SPARC mRNA in normal diploid human cells as well as in tumor cells. Among 40 cell lines examined, 19 showed expression. The mRNA transcribed by the majority of the expressors are 2.1 kb with a trace amount of 3 kb. However, three cell types, undifferentiated basal keratinocytes, their differentiated derivatives, and breast adenocarcinoma cells, showed an expression pattern distinct from the typical one, having abundant 3-kb mRNA but no detectable 2.1-kb mRNA. The mRNA was translated and the product secreted. This expression pattern was not observed before in human cells and was not found in tumor cells of keratinocytes, squamous carcinoma cells, or many other adenocarcinoma cells. We showed by Northern hybridization that the SPARC-expressing melanocytic melanoma cell lines produced laminin, a component of extracellular matrix. Other cell types expressing the SPARC mRNA were also reported to synthesize extracellular matrix components. Thus, our results indicate an association between SPARC gene expression and production of extracellular matrix. However, the opposite is not true since non-SPARC-producers may or may not produce extracellular matrix. For example, A431 cell line, which does not express SPARC mRNA, is known to produce extracellular matrix components while the normal diploid melanocytes and undifferentiated embryonal carcinoma cells, which do not express SPARC mRNA, do not produce extracellular matrix component.     

Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early stages of development

The patterns of protein synthesis in teratocarcinoma stem cells (embryonal carcinoma cells) and in mouse embryos at various stages of preimplantation development were studied using SDS-polyacrylamide slab gel electrophoresis with autoradiography. Significant differences were observed in comparisons of embryonal carcinoma cells with isolated inner cell masses (ICMs) or with embryonic cells at earlier stages of development. However, no such differences in the overall pattern of protein synthesis were found when the embryonal carcinoma cells were compared with the embryonic ectoderm (that portion of the ICM which remains after endoderm differentiation). Both synthesize at least one prominent 55,000-dalton protein that is not detected in embryonic cells at earlier stages of development. This protein can thus be used as a biochemical marker of ectoderm formation during embryonic development. The pattern of protein synthesis common to embryonal carcinoma cells and embryonic ectoderm is not shared by other cultured cell types.     

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

Summary The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.     

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.     

Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells

Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.     

The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid

Treatment of embryonal carcinoma cells F9 with retinoic acid results in the appearance of epithelioid cells resembling endoderm which synthesize basement membrane protein and plasminogen activator. Concomitant with the appearance of these properties of differentiated cells, the epithelial cells cease to express SSEA-1, an antigenic determinant characteristic of teratocarcinoma stem cells and early mouse embryos. Our evidence indicates that the phenotypic changes that accompany retinoic acid treatment of embryonal carcinoma cells are irreversible and a consequence of the differentiation of the cells into endoderm.