Pedersen fetuin contains three adipogenic factors with distinct biochemical characteristics.

Crude Pedersen fetuin, derived from fetal bovine serum, contains adipogenic activity. Biochemical characterization was undertaken by following the differentiation of the 1246 adipogenic cell line. The present paper provides evidence that crude fetuin contains distinct proteins with adipogenic activity. By molecular sieve fractionation using Sephacryl S-300, the majority of adipogenic activity eluted in two distinct peaks, FI (molecular weight greater than 669 kDa) and FII (molecular weight ranging from 445 and 232 kDa). In addition a minor activity was found in a third peak, FIII (molecular weight around 69 kDa). Partial purification and biochemical characterization indicate that FI and FII are two distinct factors. FI has a PI higher than 9.4, is destroyed by alkaline treatment, and is stable when treated with acid. FII has a PI lower than 4.0, is alkali stable, but is destroyed completely by treatment with acid. Moreover, our data show that adipogenic factors are distinct from another protein alpha 2 macroglobulin known to be found in crude Pedersen fetuin. These results suggest that serum contains two large molecular weight proteins bearing adipogenic activity which could play an important role in the control of the adipose differentiation process.

Detection in non-erythroid cells of a factor with the binding characteristics of the erythroid cell transcription factor EF1.

The erythroid transcription factor erythroid factor-1 (EF1) plays a critical role in the transcription of erythroid-specific genes. Here we report the presence of a factor with the mobility and sequence-specific DNA-binding characteristics of EF1 at low abundance in a wide variety of non-erythroid cell types. This is the first report of an EF1-like activity in non-erythroid cells and indicates that this factor may play a role in the regulation of genes expressed in such cells.

Molecular analysis of early growth-associated events during the differentiation of F9 cells into embryoid bodies.

Mouse F9 teratocarcinoma cell lines can be induced to differentiate into either parietal endoderm or embryoid bodies which contain visceral endoderm-like cells. The nature of the early molecular events involved in these two differentiation pathways has not yet been fully elucidated. Moreover, since the process of differentiation is often accompanied by changes in cell growth, it is often difficult to determine which of the events that do occur during the early stages of differentiation are a direct result of the process of differentiation and which events are indirect results that occur as a consequence of altered cell growth. In the experiments reported here we have attempted to distinguish between these two possibilities by examining the patterns of expression of a representative group of growth-associated genes (i.e., c-myc, p53, and histone H3) when F9 cell aggregates are induced to differentiate into embryoid bodies containing visceral endoderm. By analysis of the patterns of growth-associated gene expression in both retinoic acid treated and nontreated F9 cell aggregates, we were able to classify early events as differentiation-specific events (events which occurred only following retinoic acid treatment of aggregates) or nondifferentiation-specific events caused by reduction in cell growth (events which occurred even when aggregates were not treated with retinoic acid). Our results show that F9 cells differentiated into embryoid bodies containing visceral endoderm-like cell exhibit an early reduction in both growth and c-myc mRNAs which is neither retinoic acid-specific nor differentiation-specific. However, following this initial response to aggregation, constant levels of c-myc mRNA are maintained despite continued reduction in growth. Thus, it appears that alteration in c-myc expression is a differentiation-specific event along the pathway to formation of visceral endoderm. Interestingly, however, the nature and time course of this alteration in c-myc expression in F9 cells' differentiation into visceral endoderm is different from that observed in F9 cells differentiated into parietal endoderm.

Changes in glycolipid expression in human testicular tumor.

Glycolipids were extracted from testicular tumor tissues of 13 patients, and their pattern of expression compared with that of normal testicular tissue. The most conspicuous and consistent change in the tumor extracts was marked accumulation of CTH (ceramide trihexoside). Structural analysis by enzyme cleavage showed that CTH which accumulated in the tumor tissue was Gb3 (Gal alpha 1-4Gal beta 1-4Glc beta 1-Cer). Immunohistochemistry using anti-Gb3 monoclonal antibody (MAb) (1A4) also indicated massive accumulation of Gb3 in the tumor tissue. Gb3 may be a new marker of testicular tumors, especially seminomas, for which useful markers are so far lacking.

Glial fibrillary acidic protein immunoreactivity of chondrocytes in immature and mature teratomas.

The immunoreactivity of chondrocytes for glial fibrillary acidic protein (GFAP), other intermediate filament proteins and S-100 protein was studied in formalin-fixed paraffin-embedded sections. A total of 95 cartilage specimens were examined from five immature teratomas, 12 mature teratomas, and a teratocarcinoma. GFAP-immunoreactive chondrocytes were abundant in immature cartilages, and as the cartilages maturated, these chondrocytes decreased and became distributed peripherally. Elastic cartilage had more GFAP-immunoreactive chondrocytes than non-elastic cartilage. GFAP-immunoreactive cartilage was often located close to central nervous tissue. Immunostaining for vimentin and S-100 protein revealed extensive distribution of immunoreactive chondrocytes in immature and mature cartilages, but in mature cartilage, chondrocytes at the center had less vimentin immunoreactivity. GFAP-immunoreactive chondrocytes also showed apparent immunostaining for vimentin. There was no difference in immunohistochemical staining for the alpha and beta subunits of S-100 protein. The immunoreactivities of teratoma cartilage specimens were quite similar to those of respiratory tract cartilage.

Absence of uvomorulin in a slowly compacting variant of H6 embryonal carcinoma cells.

When allowed to aggregate in calcium-containing medium, the H6 embryonal carcinoma cell variant named 6B(NG)C25 compacted more slowly than wild-type cells, and aggregates of hybrids between it and wild-type cells also compacted slowly, as if the variation (mutation) acted in a dominant fashion. In agreement with this, we now have found that the cell adhesion molecule uvomorulin is markedly reduced or absent in 6B(NG)C25 cells, as well as in the hybrids. A small amount of a higher-molecular-weight protein reacting with the antibody is present, which might represent residual uvomorulin migrating at a slower rate, an altered uvomorulin, the known precursor to uvomorulin, or an unrelated cross-reacting protein.

Embryonal carcinoma of the ovary: a six-year survival.

We report a case of embryonal carcinoma, stage II, arising in the right ovary of an 18-year-old woman. The elevated serum levels of alpha-fetoprotein (AFP) and urinary human chorionic gonadotropin (hCG) rapidly normalized after conservative surgery followed by combination chemotherapy. The tumor was composed of large primitive cells and some multinucleated giant cells. AFP and hCG were demonstrated immunohistochemically in each type of cells. She has been disease-free for 6 years.

The abnormal occurrence and the differentiation-dependent distribution of N-acetyl and N-glycolyl species of the ganglioside GM2 in human germ cell tumors. A study with specific monoclonal antibodies.

Human primary germ cell tumors were analyzed for the presence of the ganglioside GM2 using three specific monoclonal antibodies which can distinguish the molecular species of the sialic acid moiety: the antibody MK1-16 is specific for N-acetyl GM2, MK2-34 is specific for N-glycolyl GM2, and MK1-17 detects both N-acetyl and N-glycolyl GM2. When the occurrence of the GM2 antigen was tested in 107 cases of human germ cell tumors by the immunohistochemical technique using these antibodies, seminoma was characterized as having the highest frequency of N-acetyl GM2 (89.4%, 42 of 47 cases) among germ cell tumors, followed by embryonal carcinoma (40.0%), and teratocarcinoma (26.6%). Compared with this, yolk sac tumors and choriocarcinoma had a much lower positive incidence of the N-acetyl GM2 antigen. On the other hand, the N-glycolyl GM2 antigen was not found at all in 47 cases of seminoma (0%), and the positive incidence was very low in embryonal carcinoma (6.6%), although considerably higher incidences were obtained with choriocarcinoma (25.0%), yolk sac tumor (22.2%), and teratocarcinoma (13.3%). The presence and molecular species of the GM2 antigens in these human germ cell tumors were also ascertained chemically by the thin-layer chromatography (TLC) immunostaining of the ganglioside fractions prepared from primary germ cell tumors. These results indicate that seminoma specifically contains N-acetyl GM2 and no N-glycolyl GM2, suggesting that N-acetyl GM2 could be a good marker for seminoma. On the other hand, non-seminomatous germ cell tumors were characterized by the presence of N-glycolyl GM2, one of the Hanganutziu-Deicher antigens (H-D antigens). Moreover, the positive occurrence of N-glycolyl GM2 correlated very well with the degree of differentiation of non-seminomatous germ cell tumors, i.e., the differentiated tumors such as yolk sac tumors, choriocarcinoma, and teratocarcinoma had a higher positive incidence of N-glycolyl GM2 type H-D antigen but a lower positive incidence of N-acetyl GM2 when compared with embryonal carcinoma, the most undifferentiated tumors among non-seminomatous germ cell tumors.

Lineage-specific patterns of p21ras proteins in immortalized cell lines derived from mouse teratocarcinoma.

The expression of proteins coded by the ras oncogene family was examined in mouse embryonal carcinoma (EC) cells and in immortalized cell lines derived from EC. These cell lines, which correspond to early stages of differentiation, express the simian virus 40 (SV40) T antigen and still proliferate. By 2-D gel electrophoresis of the immune complexes formed with monoclonal anti-ras antibodies, it was possible to distinguish the products of the Ha-, N- and Ki-ras genes and to correlate the observed patterns with the differentiation state of the cells. We show in this report (1) that the 2-D gel pattern of ras protein is identical for the various EC tested and is not influenced by SV40 transformation, (2) that p21Ki-ras is not detected in EC cells, although some EC cell lines are known to express a Ki-ras transcript, and (3) that the complex patterns of N- and Ha-ras observed in EC cells becomes simpler as differentiation proceeds, with a different, characteristic pattern for neuroectodermal, mesodermal and endodermal derivatives. Such patterns could prove useful as differentiation markers.

An immunohistochemical study of neuropeptides and neuronal cytoskeletal proteins in the neuroepithelial component of a spontaneous murine ovarian teratoma. Primitive neuroepithelium displays immunoreactivity for neuropeptides and neuron-associated beta-tubulin isotype.

Approximately one third of the female mice of the LTXBO strain develop spontaneous ovarian teratomas. These tumors contain a large neuroepithelial component, which includes primitive neural structures resembling embryonic neural tubes (medulloepithelial rosettes), ependymoblastic and ependymal rosettes, neuroblasts, mature ganglionic neurons, myelinated neurites, and astrocytes. The purpose of this study was to characterize these tumors according to the immunohistochemical location of some well-characterized trophic and regulatory neuropeptides and neurotransmitters, several neuronal-associated cytoskeletal proteins, and other proteins indicative of neuronal and glial differentiation. Medulloepithelial rosettes showed focal serotonin-like, opioid peptide-like and gamma-amino butyric acid-like immunoreactivity, and displayed immunostaining for the neuron-associated class III beta-tubulin isotype. The mature ganglion cells were also immunoreactive for these markers, and, in addition, for somatostatin, cholecystokinin, bombesin, glucagon, vasoactive intestinal peptide, and neuropeptide Y. Mature ganglion cells were also immunoreactive for proteins associated with the neuronal cytoskeleton (including microtubule-associated proteins, MAP2 and tau, and higher molecular weight phosphorylated and non-phosphorylated neurofilament subunits), neuron-specific enolase, and synaptophysin. Undifferentiated stem cells, ependymoblastic and ependymal rosettes, and astroglia all stained with a monoclonal antibody that recognizes all mammalian beta-tubulin isotypes, but did not react with antibodies to neuronal-associated cytoskeletal proteins or neuropeptides. Neuropeptide-like immunoreactivity and demonstration of the class III beta-tubulin isotype indicate early neuronal commitment in neoplastic primitive neuroepithelium. These patterns of immunoreactivity closely follow those encountered in the normal neurocytogenesis of the mammalian and avian forebrain, and increase the precision with which the early stages of progressive neuroepithelial differentiation can be analyzed in human embryonal tumors of the CNS.

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.

Adenomatoid tumor of the epididymis with special reference to immunohistochemical study of 3 cases.

Three cases of epididymal adenomatoid tumor are presented. The adenoid compositions of the tumors lined by epithelial cells showed a canalicular pattern with large vascular spaces, tubular pattern with glandlike regions or plexiform pattern with connective tissue strands. Immunohistochemistry demonstrated positive cytoplasmic staining for keratin, but negative for carcinoembryonic antigen and factor VIII-related antigen in each neoplastic tissue. These findings support the mesothelial origin of the epididymal adenomatoid tumors.

[Mixed ovarian germinal neoplasia: adult embryonal carcinoma and dysgerminoma with trophoblasts]. / Neoplasia germinale ovarica complessa: carcinoma embrionale adulto e disgerminoma con trofoblasto.

Malignant mixed germ cell tumor of the ovary: embryonal carcinoma and dysgerminoma with trophoblastic cells. A case of ovarian neoplasia in a 19-years-old woman is described showing mixed germ cell tumor patterns. The main component is a solid embryonal carcinoma with mainly syncytial-like, highly anaplastic cells, displaying diffuse CK-immunoreactivities and scattered PLAP-positive cells. Many CK- and beta-HCG-positive syncytiotrophoblastic and intermediate trophoblastic cells are present. A second component is a dysgerminoma with lymphoid stroma and diffuse PLAP-cytomembrane immunoreactivities: rare cells, to be identified as intermediate trophoblast cells, are CK- and strongly beta-HCG-positive. Many syncytiotrophoblastic cells with a brisk CK- and beta-HCG-positivities are also noted. The embryonal carcinoma component is metastasized to the controlateral ovary, uterus and omentum. A complete immunohistochemical analysis is recommended to properly diagnose germ cell neoplasias of the ovary both for descriptive and prognostic-therapeutic purposes. The very rare presence in the same ovarian tumor of mixed patterns as adult embryonal carcinoma and dysgerminoma with trophoblastic cells, is stressed.

[Primary teratocarcinoma of the pineal region]. / Teratocarcinoma primitivo della regione pineale.

Primary Teratocarcinoma of Pineal Region. A case of a 12 year old boy admitted for intracranial hypertension of sudden onset has been reported. CT scanning and MR showed a triventricular hydrocephalus due to a space-occupying lesion of the pineal region. Tonic-clonic fits of the upper limbs and Parinaud syndrome were followed by loss of consciousness. Intervention I: ventriculo-peritoneal shunt with sampling of CSF and assay for beta-HCG, alpha FP and CEA, which proved negative. Cytology for neoplastic cells in cerebrospinal fluid was negative. Intervention II: grossly total removal of the tumor. This was followed by partial remission of Parinaud syndrome, total remission of the hypertensive symptoms and discharge on day 12. The 3 cm. whitish-pink tumor of rubbery consistency proved on histological examination to be a teratocarcinoma. The patient was further submitted to chemioterapy and irradiation but died 7 months after the second intervention. This is a rare tumor, much more than teratoma of the pineal gland, which is relatively frequent. It is interesting histologically because of the presence not only of chondroid and mesenchymal portions but also of adamantinomatous rudiments and of epithelial zones resembling embryonal carcinoma of the testis.

Retroperitoneal immature teratoma producing alpha-fetoprotein.

An alpha-fetoprotein producing retroperitoneal immature teratoma is demonstrated and the aetiology of AFP arising probably from small island of hepatoid cells or various tubular epithelial cells in the immature teratoma is discussed.

[A transforming growth factor-beta like growth factor in mouse embryonal carcinoma cells].

Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.

Solid teratomas and mixed müllerian tumors of the ovary: a clinical, histological, and immunocytochemical comparative study.

The differential diagnosis between solid teratomas and malignant mixed Müllerian tumors (MMTs) can be difficult, because both tumours have an epithelial as well as a mesenchymal component. Sometimes an MMT is incorrectly diagnosed as a solid teratoma. This has prognostic and therapeutic consequences, because MMTs have a worse prognosis. In this study 20 solid ovarian teratomas and 15 MMTs of the ovary have been compared according to their clinical, histomorphological, and immunocytochemical characteristics. Teratomas occur in the younger age group and are characterized by the presence of argyrophil cells which are immunoreactive to a wide range of neurohormonal peptides and are in addition characterized by the presence of GFAP immunoreactive tissues (glial fibrillar acidic protein). MMTs occur in the older age group and are characterized by the presence of sarcomatous elements and by the absence of an organoid arrangement, neuroectoderm, skin, argyrophilia, and immunoreactivity to GFAP and neurofilament.

[Testicular germinal tumors. Current therapeutic principles]. / Tumeurs germinales testiculaires. Principes thérapeutiques actuels.

The cure rate for germinal tumors of the testes are now very high, greater than 80%, all stages and histological types taken as a whole. This calls for a revision of therapeutic indications, in light of these results and modern staging methods. In localized stages, or tumors associated with a good prognosis, therapeutic de-escalation is in progress, in order to limit the side effects. In invasive forms or forms associated with a poor prognosis, the use of more aggressive methods, such as high dose chemotherapy, should further improve prognosis.

A study of the sedimentation properties of the U-snRNPs of mouse embryoid bodies (OTT6050) by sucrose gradient centrifugation under physiological ionic conditions and electrophoretic analysis of the RNA in various fractions.

Fractionation of nuclear extracts of mouse embryoid bodies (EBs; OTT6050) on sucrose gradients (5-20%) under conditions of physiological ionic strength (150 mM NaCl, 1mM MgCl2) and an analysis of the RNA in various fractions by gel electrophoresis revealed that the major U small nuclear ribonucleoproteins (U-snRNPs; U1a, U1b, U2, U4, U5 and U6-snRNPs) sediment over a wide region of the gradient, although they sediment preponderantly in the light region of the gradient. This result suggests that, under these experimental conditions, some of the populations of snRNPs exist as free particles separated from large nuclear RNP particles, while some of the populations associate with them in EBs. Furthermore, all species of these major U-snRNPs appear to associate with the larger nuclear RNP particles of EBs, since all these species sediment in the heavier fractions (approximate greater than or equal to 60S) of the 15-40%/50% gradients. The relative abundance of the various species of major U-snRNPs can also be observed to vary among the fractions of the gradients. A similar analysis of the post-mitochondrial cytoplasmic fraction showed that some leakage of the major U-snRNPs, but not the selective leakage of any particular species of U-snRNP, from the nuclear fraction, occurred during aqueous fractionation of the cells. Some species of RNA, larger than the snRNAs U1a/b and U2 respectively were also detected in the cytoplasmic fraction.

Immunohistochemistry of a spontaneous murine ovarian teratoma with neuroepithelial differentiation. Neuron-associated beta-tubulin as a marker for primitive neuroepithelium.

Spontaneous ovarian teratomas develop in a large proportion of female LT strain mice. These tumors display a large neuroectodermal component with morphologic differentiation ranging from primitive neuroepithelium (medulloepithelial and ependymoblastic rosettes) to mature neurons, and provide a useful system for the study of various asynchronous stages of neuroepithelial differentiation. The aim of this study was to assess the expression of various cytoskeletal proteins in conjunction with other differentiation-related antigens in these tumors. We found that the medulloepithelial rosettes reacted with only two anti-beta-tubulin monoclonal antibodies. One of these (TU27) recognizes an epitope common to all of the mammalian beta-tubulin isotypes. The other monoclonal antibody (TUJ1) recognizes an epitope unique to class III beta-tubulin isotypes (neuronal-associated). Whereas immunoreactivity in the ependymoblastic rosettes was limited to TU27, differentiating polar neuroblasts reacted with both TU27 and TUJ1 and expressed neuron-specific enolase, synaptophysin, and the 68 kilodalton subunit of neurofilament protein. In well-differentiated foci, mature neurons were positive for all three neurofilament protein subunits (68, 168 and 200 kilodaltons), microtubule-associated-protein 2, synaptophysin, and neuron-specific-enolase, and reacted with both TU27 and TUJ1. By contrast, glial elements expressed glial fibrillary acidic and S-100 proteins, Leu-7 and TU27 but not TUJ1. Myelin basic protein and myelin-associated glycoprotein reactivity was found in the neuropile of these mature areas. The neuroepithelial components were negative for retinal S-antigen and cytokeratin. The expression of the class III beta-tubulin isotype by medulloepithelial rosettes suggests that this isotype may be one of the earliest markers to signal neuronal commitment in primitive neuroepithelium.