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PubMed2004

OCT4 staining in testicular tumors: a sensitive and specific marker for seminoma and embryonal carcinoma.

OCT4 (POU5F1) is a transcription factor expressed in embryonic stem and germ cells and is involved in the regulation and maintenance of pluripotency. It has been detected in primary testicular germ cell tumors with pluripotent potential, seminoma, and embryonal carcinoma. We undertook immunohistochemical staining of OCT4 in a wide variety of primary testicular neoplasms (germ cell tumors and other tumors) to assess the specificity and usefulness of this marker as a diagnostic tool. We examined histologic sections from 91 primary testicular neoplasms, including 64 cases of mixed germ cell tumors containing embryonal carcinoma (54), seminoma (51), yolk sac tumor (38), mature teratoma (31), immature teratoma (20), and choriocarcinoma (15). In addition, we examined sections from spermatocytic seminomas (5), Leydig cell tumors (8), Sertoli cell tumors (6), unclassified sex-cord stromal tumors (4), adenomatoid tumors (2), testicular tumor of adrenogenital syndrome (1), and granulosa cell tumor (1). Each tumor was examined with hematoxylin and eosin staining and with antibodies to OCT4. In all cases of mixed germ cell tumor with components of embryonal carcinoma (54) and seminoma (51), there was greater than 90% nuclear staining of the embryonal carcinoma and seminoma tumor cells with little to no background staining. In all but 1 of these cases (embryonal carcinoma), there was strong (3+) staining intensity. The other germ cell tumor components (yolk sac tumor, mature teratoma, immature teratoma, and choriocarcinoma) showed no staining. Syncytiotrophoblast cells, which were present in 15 of the cases, were also completely negative, as were all 5 of the spermatocytic seminomas. The 22 cases of non-germ cell tumors were all immunohistochemically negative for OCT4. Fifteen of the 54 germ cell tumors containing embryonal carcinoma were also examined with antibodies to CD30. These embryonal carcinoma components were all positive for CD30 with staining of greater than 90% of the tumor cells but with variable staining intensity. We conclude that immunostaining with antibodies to OCT4 is a useful diagnostic tool in the identification of primary testicular embryonal carcinomas and "usual," but not spermatocytic, seminomas. OCT4 immunostaining has comparable sensitivity but greater consistency compared with CD30 in the diagnosis of embryonal carcinoma.

Adenomatoid Tumor↗

PubMed1990

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells.

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.

Actins↗

PubMed1991

Analysis of the binding proteins and activity of the long terminal repeat of Moloney murine leukemia virus during differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cell lines were established which carry the stably integrated chloramphenicol acetyltransferase (CAT) gene under the control of the transcriptional elements of the long terminal repeat (LTR) of Moloney murine leukemia virus. The activity of three elements of the stably integrated LTR was analyzed in undifferentiated EC cells (stable CAT assay). Results of the study are summarized as follows. (i) In the stable assay, the promoter region of the LTR was inactive in undifferentiated ECA2 and F9 cells, and the level of the activity was 10(-4) of that in NIH 3T3 cells. (ii) In contrast to the results of the transient assay, the enhancer was active in undifferentiated ECA2 cells and in F9 cells. It activated CAT activity more than 60-fold and about 8-fold in ECA2 cells and F9 cells, respectively. (iii) Suppression by ELP, the embryonal LTR-binding protein, was more pronounced in the stable assay than in the transient assay. These data suggest that, when compared with NIH 3T3 cells, a major factor for the inactivity of the LTR in EC cells is the inefficiency of the promoter in this assay. Transcriptional activity of the LTR was analyzed during the differentiation of EC cells. In the case of ECA2 cells, the magnitude of activation by the enhancer did not change during differentiation. The activity of the promoter increased about 10-fold, and the suppression by ELP became negligible 4 days after the induction of differentiation. Upon differentiation of F9 cells, the activity of the enhancer increased more than 300-fold, but the promoter remained inactive. The pattern of LTR-binding proteins also varied during the differentiation of EC cells. Our present data suggest that the activity of LTR elements as assayed by the stable assay differs from the activity as assayed by the transient assay. It also indicates that the activity of these elements exhibits cell-type-specific changes during the differentiation of EC cells.

Animals↗

PubMed1985

Functional analysis of the mouse H-2Kb gene promoter in embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells do not express the major H-2 class I transplantation antigens. The latter, however, become detectable upon in vitro differentiation of EC cells. Neither class I H-2 genes nor the gene coding for beta-2 microglobulin (beta 2m) is transcribed in EC cells. We have constructed two hybrid plasmids containing the 5' flanking region of an H-2Kb gene followed by the coding regions of either the herpes simplex virus thymidine kinase (H-2 tk) or the chloramphenicol acetyl transferase (H-2 CAT) genes. Upon transfer into EC cells, the H-2 tk hybrid gene is expressed in F9 tk- cell lines which thus acquire a stable tk+ phenotype. When such transformed clones are induced to differentiate in vitro, tk activity shows a moderate increase, which reflects an increase in transcription of the hybrid gene. In transient transformation experiments, EC cells were found to express the H-2 CAT hybrid gene as well. We conclude that the 2 kilobase pair region of the H-2Kb gene which we used contains an active promoter region, but does not include all the elements required for the correct regulation of the H-2Kb gene.

Acetyltransferases↗

PubMed1987

Pluripotent differentiation of single F9 embryonal carcinoma cells.

F9 embryonal carcinoma cells were induced to form a variety of differentiated cell types in monolayer culture. Cells with the morphological, histochemical and immunocytochemical properties of parietal and visceral endoderm, neurones and adipocytes were identified. Cells expressing Thy-1 antigen and large, multinucleated cells expressing cytoplasmic fibronectin were also observed. Various cell types were found together in colonies derived from individual F9 cells, allowing us to conclude that F9 cells are pluripotent in vitro.

Animals↗

PubMed1990

Analysis of a nontumorigenic embryonal carcinoma cell line.

Embryonal carcinoma (EC) cells have proven to be of particular value in studies of both oncogenesis and mammalian development as well as in evaluating the relationship between these two phenomena. We have infected EC cells with a retrovirus in an effort to obtain by insertional mutagenesis cell lines defective in either differentiative or oncogenic potentials. One such cell line, identified originally by its unique morphological phenotype, is abnormal with respect to both parameters. These cells do not differentiate along typical EC cell lineages, possibly having lost their ability to elaborate endodermal derivatives. They do, however, retain certain cell surface markers characteristic of EC cells and lose these markers after exposure to retinoic acid. Most significantly, they also fail to form tumors in vivo in syngeneic mice, although they grow as well as the parental cells in vitro. Southern blot analysis indicates that this variant cell line has a single viral insert and the original cell was probably hemizygous for the insertion site, suggesting that a single gene may regulate both the tumorigenic and differentiative capacities of the cell.

Animals↗

PubMed1996

Head-activator and the neuroectodermal differentiation of P19 mouse embryonal carcinoma cells.

P19 embryonal carcinoma cells differentiate into neuroectodermal derivates upon aggregation and treatment with retinoic acid (RA). Such RA-induced P19 cells, concomitant with neuronal differentiation, produce the neuropeptide head activator (HA) and release HA into the culture medium. The amount of HA produced by such differentiating P19 cells is more than 100-fold higher than that of uninduced P19 cells. RA-induced P19 cells respond to HA treatment with a two-fold increase in total number of neuronally differentiating P19 progeny. As early response to HA, an increase in intracellular calcium concentration is observed.

Animals↗

PubMed1986

Negative regulation of the major histocompatibility class I gene in undifferentiated embryonal carcinoma cells.

Murine embryonal carcinoma F9 cells, which do not express appreciable levels of major histocompatibility complex (MHC) class I mRNA, start to express the mRNA and proteins upon differentiation induced by retinoic acid (RA). To investigate the molecular mechanism of this regulation, we examined in F9 cells transient expression of the chloramphenicol acetyltransferase (CAT) gene directed by the 5' flanking region of a MHC class I gene, H-2Ld. The native 1.4-kilobase H-2Ld 5' upstream region gave very low CAT activity in undifferentiated F9 cells. Deletion between positions -210 and -135 relative to the cap site resulted in a 4- to 5-fold increase in CAT activity as compared with constructs containing the region. However, all of these constructs, regardless of the deletion, expressed comparable CAT activity in differentiated F9 cells. These data suggest the presence of a negative cis-acting element that is under developmental control. Further analysis revealed that the sequence conferring the negative regulation resides between positions -195 and -161. This region, highly conserved among the MHC class I genes, is found to be capable of increasing CAT activity in NIH 3T3 cells that express the class I genes constitutively. Further, this regulatory sequence, when connected to the simian virus 40 promoter, produced repressive and enhancing effects in F9 and NIH 3T3 cells, respectively. Based on these results, we suggest that the expression of MHC class I genes during development involves switching from negative to positive regulation dictated by the class I regulatory element located between positions -195 and -161.

Acetyltransferases↗

PubMed1987

A cellular enhancer of retrovirus gene expression in embryonal carcinoma cells.

Murine embryonal carcinoma (EC) cells are refractory to infection by retroviruses because retroviral long terminal repeat (LTR) enhancers have little activity in EC cells. A previous report described the isolation of clonal EC cell lines that express the integrated neomycin-resistance gene (neo) linked to the Moloney murine leukemia virus LTR. The expression of the neo gene was explained by a cis-acting mechanism [Taketo, M., Gilboa, E. & Sherman, M. I. (1985) Proc. Natl. Acad. Sci. USA 82, 2422-2426]. From one such EC cell line, we isolated the flanking cellular sequence 5' to the proviral genome, ligated it to various test constructs, and transfected into the parental EC cells. The cellular sequence increased expression of the LTR-linked neo gene significantly, in a manner independent of its orientation and position. The neo mRNA was initiated at the bona fide promoter of the LTR. By deletion analyses, we defined a region of DNA essential for the enhancer activity and determined its sequence. This region contains distinctly characteristic stretches as well as some similarity to various viral and cellular enhancers. Thus the LTR-linked neo gene is expressed because the provirus is integrated in the vicinity of this enhancer that is active in undifferentiated EC cells.

Animals↗

PubMed1983

Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells.

F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.

Animals↗

PubMed1984

Inhibitory effect of mouse interferon on the growth of an embryonal carcinoma in mice.

Embryonal carcinoma cells (F9) were resistant to the inhibitory effects of interferon (IFN) on viral multiplication and cell division in vitro. Nevertheless, daily administration of IFN resulted in a marked inhibition of the growth of this carcinoma injected intraperitoneally (ip) and increased mouse survival time. An incidental finding was that daily IFN treatment increased the serum level of alpha-fetoprotein in normal 129 mice.

Animals↗

PubMed1990

A dominant negative mutation of the alpha retinoic acid receptor gene in a retinoic acid-nonresponsive embryonal carcinoma cell.

Pluripotential embryonal carcinoma cells such as those of the P19 line differentiate when exposed to retinoic acid (RA). The RAC65 cell line is a mutant clone of P19 cells selected to be RA nonresponsive. RAC65 cells carry a rearrangement affecting one of the genes encoding a nuclear retinoic acid receptor (RAR alpha). The mutant gene encodes a protein, RAR alpha', that has lost its 70 C-terminal amino acids, thus truncating the RA-binding domain. The RAR alpha' was found to be a dominant repressor of transcription from an RA-responsive target gene; however, expression of RAR alpha' was insufficient to confer RA nonresponsiveness, suggesting that RAC65 cells carry an additional mutation(s) affecting RA-induced genes.

Amino Acid Sequence↗

PubMed1985

X chromosome reactivation in mouse embryonal carcinoma cells.

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

Animals↗

PubMed1988

Regulated expression of a transfected human cardiac actin gene during differentiation of multipotential murine embryonal carcinoma cells.

P19 embryonal carcinoma (EC) cells are multipotential stem cells which can be induced to differentiate in vitro into a variety of cell types, including cardiac muscle cells. A cloned human cardiac actin (CH-actin) gene was transfected into P19 cells, and stable transformants were isolated. Low levels of CH-actin mRNA were present in transformed EC cells, but a marked increase in the level of CH-actin mRNA was found as these cells differentiated into cardiac muscle. The accumulation of CH-actin mRNA paralleled that of the endogenous mouse cardiac actin mRNA. A chimeric gene, which consisted of the CH-actin promoter linked to the herpes simplex virus thymidine kinase coding region, was constructed and transfected into P19 cells. In these transformants, the thymidine kinase protein was located almost exclusively in cardiac muscle cells and was generally not detectable in EC or other nonmuscle cells. These results suggest that the transfected CH-actin promoter functions in the appropriate developmental and tissue-specific manner during the differentiation of multipotential EC cells in culture.

Actins↗

PubMed1995

Retinoic acid induces activin receptor IIB mRNA in F9 embryonal carcinoma cells.

Mouse embryonal carcinoma F9 cells are pluripotent stem cells and differentiate into primitive endodermal cells upon treatment with retinoic acid (RA). We have recently shown that in F9 cells RA regulates gene expression of activin receptor type II (ActR-II), whose ligand is a potent differentiation agent. The present study examined the regulation of the newly cloned activin receptor type IIB (ActR-IIB) gene by RA. F9 cells expressed equal amounts of three ActR-IIB transcripts of 8.0, 7.5 and 4.0 kb. Both 9-cis-RA (c-RA) and all-trans-RA (t-RA) induced ActR-IIB gene expression in a dose-dependent manner. At 10(-9) M c-RA exerted no effect, while 10(-5) M c-RA increased the 8.0 kb ActR-IIB transcript about sevenfold. In contrast, t-RA induced the 8.0 kb ActR-IIB transcript fivefold at 10(-9) M and up to eightfold at 10(-5) M. The inductive effect on the 8.0 kb transcript was greater than that on the 7.5 kb transcript, and was least effective on the 4.0 kb transcript, suggesting that these three mRNA isoforms may originate from different promoters. Both cycloheximide and actinomycin D inhibited the inductive effect of t-RA on ActR-IIB gene expression, in contrast to ActR-II whose gene expression was not suppressed by cycloheximide but abolished by actinomycin D. Thus, endodermal differentiation of F9 cells is associated with activation of ActR-IIB gene and the mechanisms involved in the regulation of ActR-II and IIB gene expression are different.

Activin Receptors↗

PubMed1988

Two transient increases in c-myc gene expression during neuroectodermal differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells of the P19 line can be induced to differentiate into neurons, astrocytes and fibroblasts by exposure to retinoic acid (RA), whereas treatment of the EC cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal tissues, including cardiac and skeletal muscle. In RA- but not DMSO-treated cultures, the level of c-myc mRNA underwent two transient increases. The concentration of c-myc mRNA in RA-treated cultures increased 3-fold after 3 h in the presence of the drug, returned to normal by 9 h, increased again 5-fold from 48 to 96 h, and finally decreased below pre-treatment values by 144 h. Increased levels of c-myc protein were observed at the times of elevated c-myc mRNA. Nuclear run-on assays of the c-myc gene and measurements of c-myc mRNA stability indicated that both transcriptional and post-transcriptional mechanisms contribute to the modulated expression of the c-myc gene. The two transient increases in c-myc expression suggest a role for the c-myc protein in the differentiation of cells along neuroectodermal lineages.

Animals↗

PubMed1985

Metabolism of retinoids by embryonal carcinoma cells.

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.

Animals↗

PubMed1990

Effects of NGF and dibutyryl cAMP on neuronal differentiation of embryonal carcinoma cells.

The P19S18O1A1 embryonal carcinoma cell line is capable of neuronal differentiation and is therefore useful in studying neuronal development and the influence of growth modulators on neuronal differentiation. We report here on the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (db cAMP), individually and combined, on differentiation of P19S18O1A1 cells. NGF alone did not induce any significant neuron-like changes in cultures exposed to NGF for as long as 12 days. Treatment with db cAMP resulted in changes in a significant population of the cells, including development of a neuron-like morphology, seen at both the light and electron microscopic level, loss of stage-specific embryonic antigen expression and the appearance of two neuronal markers, neurofilament protein and neuron-specific enolase. These changes were similar to changes seen when embryonal carcinoma (EC) cells are treated with retinoic acid. NGF in combination with dibutyryl cyclic adenosine monophosphate brought about similar changes as dibutyryl cyclic adenosine monophosphate alone, and was therefore not synergistic for induction of neuronal properties. In retinoic acid-treated cultures, the neuron-like cells had ultrastructural features very similar to neurons in non-tumorous, normal tissue, with typical organelles, such as one nucleolus, neurotubules and neurofilaments, while db cAMP-treated EC cells showed similar findings at the electronmicroscopic level. The results suggest that db cAMP can induce the neuronal phenotype in EC cells alone without pre-treatment with retinoic acid.

Bucladesine↗