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Trichosanthin is the active protein component in the Chinese herb Trichosanthes kirilowi, which has distinct pharmacological properties. The cytotoxicity of trichosanthin was demonstrated by its selective inhibition of various choriocarcinoma cells. When Jar cells were treated with trichosanthin, the influx of calcium into the cells was observed by confocal laser scanning microscopy. When the distribution of trichosanthin-binding proteins on Jar cells was studied, two classes of binding sites for trichosanthin were shown by radioligand binding assay. Furthermore, the cytoplasmic membrane of Jar cells was biotinylated and the trichosanthin-binding proteins were isolated with trichosanthin-coupled Sepharose beads. Two protein bands with molecular masses of about 50 kDa and 60 kDa were revealed, further characterization of which should shed light on the mechanism of the selective cytotoxicity of trichosanthin to Jar cells.

Trichosanthin (TCS) is a ribosome inactivating protein (RIP). It is generally believed that its many biological activities act through inhibition of ribosomes resulting in a decrease in protein synthesis. It has been hypothesized that the rate of entry of TCS into cells to reach ribosomes is an important factor in determining its biological activity. To prove this hypothesis, we have mapped out and compared the intracellular routing of TCS in two cell lines, namely the choriocarcinoma JAR cell line, which is known to be highly sensitive to the toxic effects of TCS, and the hepatoma H35 cell line, to which TCS shows minimal toxicity. Results from laser scanning confocal microscopy indicated that fluorescein isothiocyanate labeled TCS quickly accumulated inside JAR cells within 4 h of incubation while only a low level of fluorescent signals was detected in H35 cells during the same period of time. When TCS was conjugated with gold particles (Au) and its intracellular locations were traced with a transmission electron microscope, it was found that most of TCS were bound to coated pits on the JAR cell surface and were rapidly internalized within an hour. By 4 h, TCS reached almost every cytoplasmic region including ribosomes, and the JAR cell began to degenerate. In H35 cells, however, the binding of TCS to coated pits was not observed, but instead, a small amount of TCS was found to penetrate the cell non-specifically by direct diffusion across the cell membrane. Our observations suggest that most of TCS enter JAR cells via a specific receptor mediated pathway, which allows a swift transport of TCS across the membrane and a rapid accumulation of intracellular TCS, while in H35 cells, TCS takes a slow and non-specific route. The receptor-mediated uptake together with the specific intracellular routing of TCS may partly account for the differential vulnerability of the choriocarcinoma cell line towards the toxicity of TCS.

To investigate the role of ras genes in trophoblastic cell lineage, we transfected the viral H- or K-ras oncogene into a human choriocarcinoma cell line, CCI, and analysed the biological properties of CCI cells expressing an activated ras oncogene. All v-H-ras-expressing clones distinctively formed the hemispherical domes, which represents an in vitro morphological expression of vectorial transport function and are characteristic of the polarized epithelial cells, but none of v-K-ras-expressing clones and control clones did. Microscopic observation demonstrated that those domes were cavities filled with fluid which accumulated between the cell layer and the surface of culture dish. Scanning electron microscopy revealed that the domes were aggregates of round cells with long numerous microvilli and were morphologically similar to a blastocyst. Furthermore, Na(+)-K(+)-ATPase activity, which is associated with the vectorial fluid transport in transporting epithelial cells, was significantly higher in the v-H-ras-expressing clones than that in the v-K-ras-expressing clones and the parental cells. Those domes flattened within 24 h after treatment with a specific inhibitor of Na(+)-K(+)-ATPase, ouabain, and the number of domes decreased in dose-dependent manner, indicating that Na(+)-K(+)-ATPase activity was required for maintainance of domes. These results suggest that up-regulated activity of H-ras but not of K-ras facilitates the vectorial fluid transport through a chorionic cell layer and leads to the dome formation. The function of II-ras in trophoblasts, may therefore, be essential for embryogenesis, especially for supplying the nutrients.

Transmission electron microscopy was used to clarify the detailed morphology of the "intermediate type" trophoblast cell in normal and tumor issue. 67 normal placental villi specimen, 10 placental bed specimens and 10 malignant mole, 10 hydatidiform mole, 5 choriocarcinoma specimen (the last three types taken before chemotherapy) were examined. Results showed that the transitional type trophoblasts of the placenta were developed from cytotrophoblasts through differentiation and fusion to syncytiotrophoblasts which showed features of maturation and aging, having features of cytotrophoblast nuclei and syncytiotrophoblast cytoplasm. The transitional trophoblast of placental bed showed similar morphology as that of transitional type cells of villi. The morphology of transitional type cells of villi. The morphology of transitional type cells of trophoblastic tumors had both normal morphology and cellular hyperplasia, atypia and features of tumor ultrastructure. The prominent feature was the high electron density of the granules and polymorphic cysts crowded in villi, demonstrating that the morphology of "intermediate type" trophoblasts in placental and tumor tissue are similar, whereas heterotype cellular morphology is present in varying degrees in tumor tissue.

Immunocytochemistry with a monoclonal antiepidermal growth factor (anti-EGF) receptor antibody directed against the extracellular domain which can inhibit ligand binding to the receptors showed that nuclei of choriocarcinoma JEG-3 cells and normal placental trophoblasts were distinctly immunostained for EGF receptors. This finding led us to investigate the structure and function of nuclear EGF receptors. Western immunoblotting revealed that cell membranes, isolated intact pure nuclei, and nuclear membranes contain a 170-kilodalton EGF receptor protein. Covalent receptor cross-linking demonstrated that the 170-kilodalton receptor protein in nuclei and nuclear membranes can bind [125I]EGF just as in cell membranes, and that this binding is inhibited by excess unlabeled EGF. As in cell membranes, the addition of EGF resulted in an increased receptor autophosphorylation in the nuclei and nuclear membranes. In addition, the activated receptor kinase stimulated, and in some cases inhibited, tyrosine phosphorylation of a number of lower molecular size proteins, especially in nuclei and nuclear membranes. Although the identity of these proteins is not known, none of them could bind [125I]EGF. The addition of EGF to isolated nuclei resulted in a time-dependent specific transcriptional inhibition of hCG/LH receptor gene. In summary, our data demonstrating the presence of functional nuclear EGF receptors are novel, potentially important, and go against the traditional concepts of growth factors action. The nuclear receptors have the capacity to transduce signals from EGF and may mediate intracrine and paracrine actions of EGF in the regulation of trophoblast functions.

Previous experiments demonstrated that human cytotrophoblasts and cells of the choriocarcinoma cell line JAr interact in vitro. As a result of this interaction there is an increased synthesis of CG and hPL, probably as a result of the increased CG and hPL synthesis by the cytotrophoblasts. In the present investigation we studied this interaction in greater detail and found that both cytotrophoblasts and JAr cells undergo changes in their biological properties as a result of this interaction. JAr cells and cytotrophoblasts cocultured for 72 hr were fractionated according to their size by centrifugal elutriation. The number of cells in the fraction which contain the largest cells was very significantly increased as a result of the coculture. This increase was due to an increase in the number of cells of both cell types. This fraction was the most active one in the synthesis of CG and hPL. The synthesis of DNA by the JAr nuclei in this fraction of the cocultured cells was almost completely inhibited but in the parallel fraction of the JAr cells cultivated alone the level of DNA synthesis was equal to that of all other JAr cell fractions. Heterokaryons are formed in the coculture. In these heterokaryons a factor which inhibits DNA synthesis in the cytotrophoblasts may inhibit DNA synthesis in JAr nuclei and at least be partly responsible for the inhibition of DNA synthesis observed.

Maternal glucose is a major substrate for placental and fetal metabolism. The kinetics of its uptake into placental trophoblast cells has not been characterised yet and was therefore investigated in the present study. In addition to trophoblast cells isolated from human term placentae, JEG-3 and JAR choriocarcinoma cells were used. Measurements were carried out in 5 s intervals until 30 s with the non-metabolisable glucose analogue 3-O-[14C]methyl-D-glucose using confluent cells adhering to glass coverslips. L-[1-14C]glucose was used to correct for extracellular trapped tracer and diffusion. The uptake was rapid and saturable. It reached equilibrium after 30 s at 20 degrees C and could be inhibited by 0.4 mmol/l cytochalasin B up to 98%. The choriocarcinoma cells took up twice as much glucose as trophoblast cells. Fitting the experimental data to the Michaelis-Menten equation by non-linear regression failed to adequately describe the data, even when a contribution of diffusion to total uptake was considered. Introducing the Hill coefficient n into the Michaelis-Menten equation significantly improved the quality of the fits as was assessed by three statistical criteria. Using this equation modified for allosteric kinetics (v = k[To] [S]n)/(Km + [S]n)), parameters were calculated as Km = 12 mmol/l, Vmax = 17 fmol/l s-1 per cell, n = 1.1 for trophoblast cells; Km = 13 mmol/l, Vmax = 27 fmol/l s-1 per cell, n = 1.2 for JEG-3 cells and Km = 29 mmol/l, Vmax = fmol/l s-1 per cell, n = 1.4 for JAR cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Five cases of non-molar trophoblastic disease including one placental site trophoblastic tumor (PSTT), two exaggerated placental sites and two choriocarcinomas were compared with each other and with normal chorionic villi and placental site. This involved light microscopic, immunohistochemical and ultrastructural studies. Comparison of PSTT with choriocarcinoma suggested that the former represented a neoplastic transformation of placental site intermediate trophoblast. The PSTT showed a characteristic immunohistochemical distribution of human placental lactogen and human chorionic gonadotropin, resembling that of the placental site intermediate trophoblast. Placental site trophoblastic tumor cells were also characterized ultrastructurally by prominent perinuclear filaments, abundant rough endoplasmic reticulum, or both. Infiltrating intermediate trophoblasts in exaggerated placental sites were similar to PSTT cells rather than normal placental site intermediate trophoblasts. However cells with vacuolated cytoplasm or spindle-shaped intermediate trophoblastic cells were observed more frequently in the PSTT than the exaggerated placental sites. The intermediate trophoblastic cells in the choriocarcinomas showed a morphologically transitional form from cytotrophoblastic cell to syncytiotrophoblastic cell, but did not share unique ultrastructural similarities with placental site intermediate trophoblasts.

A total of 120 paraffin-embedded gestational trophoblastic tumor tissue blocks was selected and divided into 5 groups: (1) 20 cases of normal chorionic villi. (2) 40 cases of hydatidiform mole with no malignant change during a following-up period of at least two years. (3) 40 cases of hydatidiform mole which developed into invasive mole or choriocarcinoma. (4) 10 cases of invasive mole. (5) 10 cases of choriocarcinoma. Nucleolar organizer regions (NORs) was Ag-stained and AgNOR dots were counted using the Plotion's method. The result showed that there was significant difference between group 1 and group 2 (P < 0.005), group 2 and group 3 (P < 0.001), group 3 and group 4 (P < 0.05). Taking the AgNOR count 4.00 as a standard, 75% of the cases in group 2 (mean = 2.730) was below this standard. The study suggested that with the increase of malignancy of trophoblastic tumor, the AgNOR count increased correspondingly. A quantitative study of AgNOR might be a useful measure to detect the early malignant change of hydatidiform mole.

Some tumors frequently encountered in other organs and usually with a high grade of malignancy and a poor prognosis have been recently described in the urinary bladder, very often in close relationship with a pre-existing transitional cell carcinoma. Of these, primary choriocarcinoma of the urinary bladder is one of the most uncommon and its histogenesis much discussed. It is important to identify this tumor type, since a change in the oncologic treatment may be warranted. We report an additional case of this rare bladder tumor with clinicopathologic study and discuss the histogenetic and therapeutic aspects.

Background: The hematopoietic growth factor colony-stimulating factor-1 (CSF-1) and its receptor (encoded by the proto-oncogene c-fms) are implicated in the regulation of human placental development., Experimental Design: In this study, we have performed a detailed immunohistochemical localization of the CSF-1 receptor (CSF-1R) on cells of the uteroplacental interface in human first trimester pregnancy, supplemented by Northern blot, in situ hybridization, and flow cytometric analysis. CSF-1R expression by JEG-3 and JAR choriocarcinoma cells was also investigated., Results: c-fms mRNA was detected in primary cultures of first trimester trophoblast and was localized to the extravillous cytotrophoblast columns streaming off the anchoring villi. CSF-1R was expressed by fetal Hofbauer cells in the mesenchyme of the chorionic villi, and this expression increased considerably from first trimester to term. No expression was seen on first trimester and term villous cytotrophoblast. CSF-1R expression on villous syncytiotrophoblast was absent at 6 weeks, strongest at 8-10 weeks, and faded by 12 weeks. First trimester extravillous cytotrophoblast columns were strongly and consistently positive for CSF-1R expression, as was the superficial shell of extravillous trophoblast over the maternal decidua. However, with deeper invasion and terminal differentiation into placental bed giant cells, this expression became weak or absent. Endovascular trophoblast was also only weakly positive for CSF-1R. At the implantation site itself, large numbers of decidual macrophages and CD3-, CD56bright large granular lymphocytes were seen. The macrophages expressed CSF-1R strongly, but large granular lymphocytes, decidual stromal cells, glandular epithelium, and endothelial cells were found to be negative for CSF-1R expression. No CSF-1R expression was detected in JAR choriocarcinoma cells, but CSF-1R was present in first trimester cultured trophoblast and JEG-3 choriocarcinoma cells, although this was shown to be intracellular., Conclusions: These results suggest that CSF-1 may regulate invasion and differentiation of human placental trophoblast, depending upon the spatial and temporal distribution of its receptor. CSF-1 may also influence placental development and function by acting via decidual and fetal macrophages, which are the other cell populations expressing the receptor.

Background: There have been 12 documented cases of choriocarcinoma arising in the urinary bladder, either alone or in combination with other epithelial tumors. It has been shown that some high-grade transitional cell carcinomas (TCC), without obvious syncytiotrophoblastic elements, can produce human chorionic gonadotrophins (HCG)., Methods: A case of choriocarcinoma, in association with high-grade TCC of the renal pelvis, was encountered in an 80-year-old man. For additional evaluation of HCG production by TCC, 25 consecutive cases of invasive high-grade TCC of the bladder were stained with an anti-HCG antibody. Immunogold staining also was performed in two of the cases studied., Results: Immunoperoxidase staining of the renal pelvis tumor showed focal positivity for HCG within the TCC and a more intense reaction as the tumor cells differentiated into choriocarcinoma elements. Seven of the 25 cases (28%) displayed varying degrees of reactivity within individual cells or groups of cells. In an additional case, typical syncytiotrophoblastic giant cells without cytotrophoblasts were seen in a high-grade TCC. Immunogold studies demonstrated positive labeling in the cytoplasm of carcinoma cells in a case of TCC without syncytiotrophoblasts and in the syncytiotrophoblastic giant cells in the one case in which these were present., Conclusions: The findings support a metaplastic origin of cases of choriocarcinoma arising primarily in the urothelial tract.

The relationship of adenotin, a low-affinity adenosine-binding protein, to adenosine receptors was examined in two human tissues and two mammalian cultured cell lines. An adenosine A2 receptor exists in the membranes from platelets, PC-12 cells, and JAR cells as shown by a stimulation of adenylate cyclase related to 5'-N-ethylcarboxamidoadenosine (NECA) or a NECA-related increase in intracellular cAMP levels. In contrast, binding studies with tritiated NECA revealed typical adenotin-like low-affinity binding sites on the membranes from the sources studied with agonist potencies as follows: NECA greater than 2-chloroadenosine greater than R-PIA. No evidence was found of coupling to a guanine nucleotide regulatory protein. Solubilization of platelet and placental membranes and precipitation with polyethylene glycol separated adenotin or the adenotin-like protein from a second adenosine binding site in each tissue. The pharmacologic properties of the precipitated binding sites were compatible with an adenosine A2 receptor in platelets and an adenosine A1 receptor in placenta. Our observations indicate that adenotin-like proteins exist outside the placenta. In addition, adenotin and adenotin-like proteins coexist with the adenosine A1 or A2 receptor in a number of cells and tissues and do not couple to a guanine nucleotide regulatory protein and stimulate adenylate cyclase. Therefore, adenotin is pharmacologically distinct from adenosine receptors, and its function remains to be discovered.

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.

A continuous in vitro cell line of rat choriocarcinoma has been established. It is composed of pure trophoblast cells which multiply and differentiate. The morphology of the cells is very similar to normal rat cytotrophoblasts and giant cells. The cultured cells contain cytokeratin, alkaline phosphatase and express the receptors for Bandeira simplificifolia Agglutinin-I (BSA-I). They are hormonally active as demonstrated by the presence of lactogen and progesterone in the supernatant of the culture. The injected cells develop into choriocarcinoma in syngeneic as well as allogeneic rats. The morphological, biological and immunohistochemical features of these tumors are identical to those described in the transplantable neoplasm from which the in vitro line was established. The presence of Y chromosome in cultured cells proves the paternal origin of the primary tumor developed from extra-embryonic membranes in fetectomized rat and makes this neoplasm similar to human post-gestation choriocarcinoma.

Treatment of human choriocarcinoma cells with cytochalasin B at the doses of inhibiting cell division but not inhibiting nuclear division led to in vitro formation of the multinucleated syncytiotrophoblast-like ( STL ) cells. A concomitant increase in human chorionic gonadotropin (hCG) secretion per cell was noted. Immunocytochemical staining demonstrated the predominant localization of hCG in the STL cells. These results indicate that multinucleation stimulates hCG synthesis and secretion in the choriocarcinoma cells.

Treatment of choriocarcinoma should be eradicated of trophoblastic cells which undergo destruction in both of primary and metastatic sites and Methotrexate (MTX) has a most effective value. While MTX is being administration to the patients, there is no determination of clear relationship between proliferation of trophoblastic cells and production of human chorionic gonadotropin (hCG). In this report, we attempted to clarify the changes of the dividing compartments in cell cycle of human choriocarcinoma in vitro in the administration of MTX (concentration in 5 x 10(-6) M and 5 x 10(-7) M) to the experimental cells. Cell samples were stained by propidium iodide (PI) and fluorescein isothiocyanate (FITC), then were measured nucleic acid and protein content by means of flow microfluorometry (FMF). MTX was remarkably increased the intensity of FITC associated with protein production, in dose that is not only killing effect to the cells but inhibition of DNA synthesis. Moreover, the most difference in both subjects were as follows; Ever when MTX in low concentration was removed after 48 hours, 50% of experimental cells recovered to the level of controls in contrast to the persistence of remarkable inhibition of DNA synthesis in high concentration.

Experimental choriocarcinomas of the rat were studied with electron microscopy and compared with the ultrastructure of rat trophoblasts at mid-and late-gestational stage. The choriocarcinoma numbered m-803 contained abundant cytoplasmic organellae and was quite similar to the trophospongial cell at late-gestational stage. The M-801 closely resembled the trophospongial cell at mid-gestational stage. The m-673, which has 3-beta-ol dehydrogenase enzyme system, could not be studied sufficiently inspite of the examination of more than 30 blocks of epon embedded specimen, because of the degeneration and necrosis occurring in the specimens.

Examples of focal clearing of endometrial epithelial nuclei associated with the presence of trophoblastic tissue have been observed recently in our surgical pathology laboratories. These nuclear alterations were initially observed in endometria from first- and second-trimester spontaneous abortions, term pregnancies, and a uterus harboring choriocarcinoma. Subsequently, an identical change was seen in small foci of endometriosis removed during postpartum tubal ligation. The prominent vacuolated appearance to the nuclei resembled, in some cases, the inclusions seen in herpesvirus infection, but electron-microscopic study showed that the nuclear clearing was due to replacement of normal chromatin by a network of fine filamentous material rather than herpesvirus DNA. A subsequent review of 200 consecutive first-trimester abortions found less-striking degrees of this change in 7% of cases. The endometrial nuclear clearing was focal; often the endometrium also contained cells which demonstrated the Arias-Stella reaction. The etiology of this type of nuclear clearing is not known, but the fibrils may represent a thread-like substructure of normal chromatin which forms as a result of hormonal stimulation of endometrial epithelial cells.

The secretory potential of human chorionic gonadotropin (hCG) in eight strains of human choriocarcinoma cell lines was examined in vitro and the correlation between the secretory potential and certain cell biological characteristics was studied. Multinucleated giant cells (syncytiotrophoblast-like cells) were occasionally observed only in high hCG-secreting cell lines in accordance with inhibition of growth. Appearance of abundant dilated rough endoplasmic reticulum and aggregated glycogen particles in the cytoplasm seemed to represent ultrastructural characteristics of high hCG-secreting cell lines. A shift of nuclear DNA to hyperploidy, its wide distribution, and S phase accumulation of cells by flow cytometry were also characteristic of them. However, no correlation was noted between the hCG secretory potential and population doubling time of the cell lines in vitro.

The development of metastases represents the lethal event in the clinical course of most neoplastic diseases. The complex mechanism responsible for the spread of cancer are governed by interactions between tumour cells and the host tissues. Choriocarcinoma cells and cultured blood vessels were used to study the interaction between tumour and endothelial cells. Seeding of tumour cells onto blood vessel wall caused retraction of endothelial cells leading to the exposure of underlying tissue. Subsequently, the tumour cells penetrated the sub-endothelial connective tissue and the internal elastic lamina.

Indirect immunofluorescence microscopy using anti-actin serum has been used to investigate the distribution of actin-containing polymers in BeWo cells. This cell line, derived from a human choriocarcinoma, contains tissue that, like its tissue of origin, is partly syncytial. The syncytial nature has been inferred from study of Nomarski optical sections and from transmission electron microscopy. The multinucleated plaques of tissue possess a syncytioskeleton with a number of actin-containing features characteristic of cultured cells. These include stress fibres, cortical layers and ruffled membranes. Other actin-containing structures are more typical of the related non-pathological syncytiotrophoblast. These include a dense population of microvilli. The overall organization of the actin syncytioskeletons bears no obvious relationship to the number or position of nuclei in the syncytium. Indirect immunofluorescence microscopy has also been employed to localize the protein tubulin in BeWo cells. The microtubules do not appear to be spatially organized by a particular nucleus. Rather, there are numerous microtubule-organizing centres (MTOCs) that exist in the cytoplasm and do not have the expected numerical and positional relationship to nuclei. From these data it appears that polymeric cytoskeletal elements in these syncytia are organized in a manner not immediately subordinate to syncytial nuclei.

Ten trophoblastic tumors, including seven classical choriocarcinomas, two choriocarcinomas with atypical histology, and one placental-site trophoblastic tumor (PSTT), were studied to compare their fine structural features. Ultrastructurally, the classical choriocarcinomas showed well-defined cytotrophoblasts and syncytiotrophoblasts. The cytotrophoblasts were primitive epithelial cells, while the syncytiotrophoblasts were complex cells with multiple nuclei and dense cytoplasm containing dilated endoplasmic reticulum, lysosomes, vesicles, and tonofilaments. The syncytiotrophoblast cell membranes often contained numerous microvilli. In the choriocarcinomas, scattered intermediate trophoblasts showed features transitional between the cytotrophoblasts and the syncytiotrophoblasts, with moderately complex cytoplasm containing some of the organelles found in the syncytiotrophoblasts. Histologically, the atypical choriocarcinomas showed a predominance of mononucleate and binucleate cells and indistinct syncytiotrophoblasts. Ultrastructurally, these atypical tumors were composed largely of intermediate trophoblasts, yet contained scattered syncytiotrophoblasts with microvilli in compressed aggregates. The PSTT was composed primarily of intermediate trophoblasts that contained prominent paranuclear filaments not seen in the intermediate trophoblasts of the choriocarcinomas. Rare cells resembling syncytiotrophoblasts were found in the PSTT, but no cytotrophoblasts were observed. Immunoreactivity for human chorionic gonadotropin and human placental lactogen was found in the intermediate trophoblasts and syncytiotrophoblasts of both the choriocarcinomas and the PSTT, demonstrating functional homology between these tumors despite some ultrastructural differences. These results demonstrate ultrastructural features of trophoblastic cells that correlate with the morphologic diversity seen in these tumors by light microscopy. Furthermore, the comparisons suggest that the PSTT is composed of a distinct form of intermediate trophoblast that appears to reflect its origin from the extravillous trophoblast.

An inguinal sweat gland carcinoma is described including the unusual occurrence of alpha hCG. The alpha hCG was demonstrated in the tumor presenting choriocarcinomatous differentiation and undifferentiated polygonal cells in ordinary tumor nests. beta hCG and SP-1 were weakly and sporadically demonstrated in a small area of the tumor presenting choriocarcinomatous differentiation. On the other hand, hPL and histochemical reaction of placental type alkaline phosphatase were not observed in the choriocarcinomatous and undifferentiated tumor cells. CEA was observed in both ordinary sweat gland carcinoma nests and choriocarcinomatous differentiated regions. In the ordinary tumor nests, enzyme histochemical reactions of phosphorylase and SDH were positive. And S 100 protein was sporadically demonstrated. In spite of chemotherapy and radiotherapy, the patient died after a short duration of the disease.

A 45-year-old woman suffered from hypertension and congestive heart failure for 5 months. She was found to have a choriocarcinoma of the left mesosalpinx. The final event was, in all probability, a massive pulmonary tumor embolus which occurred shortly after removal of the uterus and adnexae. The tumor was studied by immunohistochemical methods as well as ultrastructurally, and the findings are illustrated and described.

Human chorionic gonadotrophin(hCG)-producing cells in the human placenta and in testicular germ cell tumors have been investigated by ultrastructural immunohistochemistry using labeled Fab'fragments of anti-hCG-alpha and anti-hCG-beta. Most reaction products were demonstrated in the rough endoplasmic reticulum (rER) and perinuclear spaces of syncytiotrophoblastic giant cells ( STGC ) occurring in embryonal carcinomas and in syncytial cells observed in choriocarcinomas and in normal placenta. The rER of placental and choriocarcinomatous syncytial cells was highly developed and invariably showed a positive reaction. However, the rER of STGCs varied both with regard to its degree of development and its immunoreactivity with hCG. Localization of hCG in the Golgi apparatus and in cytoplasmic granules was indefinite. Mononuclear cells reacting positively with anti-hCG-alpha or -beta were also demonstrated. Cytotrophoblast-like cells which failed to react with anti-hCG-alpha or -beta were noted adjacent to sTGCs and the significance of these cells is discussed.

Hydatidiform moles can be divided into two distinct syndromes: partial, or transitional, and classic. Both classic and partial moles appear to result from abnormal fertilization but differ in the type of abnormal fertilization, karyotype, histology, epidemiology and malignant potential. Using chromosomal banding polymorphism, classic hydatidiform moles have been shown to be androgenetic in origin, developing from a sperm with the egg nucleus either absent or inactivated. No maternal chromosomes are transmitted to the classic mole. Studies using HLA and enzyme heterozygosity have suggested that fertilization occurs by a haploid sperm with duplication of its chromosomes and without cell division, giving the 46XX karyotype found in classic moles. About 4% of classic moles are 46XY and are also androgenetic but result from dispermic fertilization. The partial mole consists of hydropic villi, but some normal villi also are present. An embryo, cord and fetal membranes generally can also be found, and the karyotype frequently is aneuploid (usually triploid) and not the 46XX or 46XY of the classic mole. In contrast to the classic mole, the partial mole has a maternal chromosomal contribution. Preliminary data suggest that many partial moles arise from dispermic fertilization, with participation of the maternal genome giving a triploid karyotype. The malignant potential of the partial mole is still controversial, but preliminary data indicate that 2.1% of partial moles require treatment as compared to 10% of classic moles.

The range of ultrastructural morphology was studied in 107 male germ cell neoplasm to assess relationships among tumor subtypes and to consolidate diagnostic criteria. Eighty-three pure-pattern neoplasms including 47 seminomas, 26 embryonal carcinomas, 10 endodermal sinus tumors, and 24 mixed germ cell tumors were analyzed. In the seminoma category, 4 cases showing cell surface specialization in keeping with early carcinomatous transformation were noted. The finding suggested a closer link between seminoma and nonseminomatous germ cell tumors than had been traditionally recognized and was in keeping with other clinical, histologic, biochemical, and xenograft observations. Subtypes of nonseminomatous germ cell tumors also exhibited a continuum of ultrastructural morphology with some types such as embryonal carcinoma and endodermal sinus tumor often blending together. At a practical level, electron microscopy has been of value in selected differential diagnoses such as seminoma versus lymphoma.

We report a case of choriocarcinoma in situ arising from a term placenta in an otherwise normal pregnancy that resulted in fetal hydrops and intrauterine fetal death from chronic fetal-maternal hemorrhage (FMH). The clinical and pathologic features are described and compared with the few similar cases reported and with an additional placental choriocarcinoma found in our files. We also describe the clinical and pathologic observations of two chorangiomas that caused massive FMH and led to fetal death.

Fluorescence microscopy of living first trimester human placental cells and choriocarcinoma cells in cultures was carried out using the vital fluorescent dye Rhodamine 123. The length and distribution of mitochondria and, in the normal cells, their reaction to colchicine treatment is described. It appears that in the presence of colchicine the distribution of mitochondria in normal placental cells becomes more restricted to the perinuclear cytoplasm and that the mean length of mitochondria is reduced. However, the total length of all the mitochondria in treated cells is not significantly different from that in paired cells which were not exposed to the drug. It is inferred from this result that mitochondrial shortening in the presence of colchicine is not an elastic shortening consequent on the removal of cytoskeletal elements which promote extension of the organelle. Rather it is brought about by fragmentation of the organelle into tandem segments.

The human gastric cancer cell lines, MKN1 and SCH, were biochemically and biologically characterized according to the monophenotypic expression of placental alkaline phosphatase (PLAP)-like enzymes. The MKN1 cell line, derived from adenosquamous carcinoma, showed the same enzyme properties as the Regan isoenzyme, whereas the SCH cell line, derived from primary gastric choriocarcinoma, had properties identical with the Nagao isoenzyme. Regan isoenzyme activity expressed by MKN1 cells was stimulated by glucocorticoid and suppressed by retinoic acid. Both agents had no significant effect on SCH cells. On the other hand, Nagao isoenzyme activity expressed by SCH cells was stimulated by sodium butyrate, which had no stimulatory effect on MKN1 cells. Moreover, the PLAP-like activity of MKN1 cells showed no observable relationship to human chorionic gonadotropin (hCG)-producibility. Whereas expression of the Nagao isoenzyme by the SCH cell line is presumably a result of functional differentiation in the trophoblastic direction, that of the Regan isoenzyme by the MKN1 cell line is probably not. Perhaps the Regan isoenzyme is related to carcinogenesis.

In this study, the ultrastructure of the human placental 'nematosome' or 'glomerular body' is described. It is usually found as electron-dense aggregates forming an inclusion 0.3 to 0.5 micron in diameter and of variable length, and occurs within cytotrophoblast cells in different configurations which include spirals, concentric rings, parallel arrays, random coils and paracrystalline structures. Analysis of random electron micrographs of placentae from normal and complicated pregnancies showed that the nematosome is present in low frequencies in the first and second trimesters but is more commonly found at full term; their incidence did not differ markedly from normal in any of the pathological conditions studied, ranging from 10.4 to 21.9 per cent, and their frequency suggests that there may be several nematosomes per cell. The relationship of the nematosome to the centriole is discussed, and it is suggested that morphometric analysis may elucidate the three-dimensional structure of nematosomes, their distribution within cells and their relationship to the plasma and other membranes.

The authors report a case of pure nongestational choriocarcinoma of the ovary (NGCO) in a prepubertal female patient and emphasize the electron microscopic and immunohistochemical findings. Pure NGCO accounts for 0.6% or less of all ovarian neoplasms. Distinction from gestational choriocarcinoma of the ovary (GCO) is important because of the worse prognosis of NGCO. No distinctive ultrastructural or immunohistochemical differences were found between NGCO and GCO. Cytogenetic studies may be indicated in future cases to investigate potential reasons for the difference in prognosis.

Choriocarcinoma is a common cancer in the tropical and subtropical areas including Taiwan, Hong Kong and Japan. An experimental model consisting of monolayer of decidual or smooth muscle cells growing on collagen gels was established to study the invasiveness of choriocarcinoma cells in vitro. It was found that tumour cells induced retraction of stromal cells in the process of invasion.

An autopsy case of primary gastric choriocarcinoma in a 55-year-old male is presented. The tumor was diagnosed as choriocarcinoma of the stomach from a histological examination of biopsy specimens. The level of human chorionic gonadotropin (HCG) was significantly increased in the serum and urine. A histological examination of autopsy specimens showed the tumor of the stomach to be a pure choriocarcinoma composed of syncytiotrophoblasts and cytotrophoblasts with a mixture of eosinophilic necrotic tissues, but with no elements of adenocarcinoma. The tumor showed metastases and/or invasions to the liver, lungs, pancreas, omentum, pleura, peritoneum and lymph nodes. Positive immuno-histochemical staining for the beta-subunit of HCG (HCG-beta) in the gastric tumor was demonstrated in the form of granular diffuse deposits in the cytoplasm of trophoblasts and predominated in syncytiotrophoblasts over transitional cells. Under electron microscopic observation, positive immunostaining for HCG-beta was observed in the perinuclear space, cisternae of the rough endoplasmic reticulum (RER) and secretory vesicles of syncytiotrophoblasts and transitional cells.

Tumor cells obtained from the metastatic focus of the lung of a 51-year-old woman with choriocarcinoma were transplanted in the subcutaneous tissue of the nude mouse and studied with an electron microscope. At the junctional zone between the tumor and the invaded cells a desmosome-like structure, between the transplanted tumor cells and endothelial cells, was observed in the nude mouse. Although this finding seems quite unusual, it may be related to the unique character of the early trophoblast.

The ultrastructure of eight 7,12-dimethylbenz (a) anthracene (DMBA) or 4-nitroquinoline-N-oxide (4-NQO) induced malignant tumors of placental and uterine origin in the rat were studied. Three of eight tumors had the ultrastructural characteristics of choriocarcinoma. The other five tumors had different fine structures. Two were classified as squamous cell carcinoma, one was as a leiomyosarcoma and the other two were undifferentiated sarcoma.

A case is reported of a 19-year-old man who presented with a mixed germ cell tumor metastatic from the testicle. The predominant component of the lymph node metastases was choriocarcinoma. Chorionic villous-like structures were identified in several areas of choriocarcinoma. To my knowledge, there is no previous report of chorionic villous-like structures in choriocarcinoma in a male patient.

Changes in the substratum anchorage of cells and nuclei were examined during methotrexate (MTX)-induced cytodifferentiation of BeWo human choriocarcinoma cells. During this process cytotrophoblast-like cells (CTLs) transform into giant mono- and multinucleated syncytiotrophoblast-like cells (STLs). Cells treated with MTX for 24 h exhibited significantly faster rates of substratum detachment by EDTA, trypsin-EDTA, EDTA-glycine, and DMSO than did uninduced controls. The decrease in cell-substratum adhesiveness occurred prior to the onset of morphological transformation. By 48 h, when morphological transformation was first observed, there had occurred a marked change in nuclear-cytoskeletal anchorage to the substratum, as evidenced by a difference in sensitivity of Triton-extracted STL and CTL monolayers to detachment by KI. STL monolayers were completely detached within 5 min of exposure to 0.3 M KI, while CTL monolayers remained firmly attached to the substratum for at least 3 h. KI-extracted residues were examined by electron microscopy and found to consist of nuclear shells attached to intermediate filaments. When cytoskeletal residues and KI-extracted proteins of STL and CTL cells were compared by two-dimensional polyacrylamide gel electrophoresis (PAGE), qualitative and quantitative differences were seen in a number of minor components. Thus the sensitivity of STL nuclear-cytoskeletal monolayers to removal by KI, an effective actin depolymerizing agent, may involve changes in the organization, stability, or interactions of actin with other components of the cytoskeletal framework.

Observation of 24 gestational trophoblastic tumors, i.e. 20 hydatidiform moles, 1 invasive hydatidiform mole, 1 non-metastatic and 2 metastatic choriocarcinomas, is reported. The diagnoses were based on case record data, clinical findings, echography and radioimmunoassay of human gonadotropin (HCG). The therapy was oriented towards maintaining fertility in reproductive women. For treatment with cytostatic drugs the patients were allocated to a low risk and a high risk group according to history, diagnosis and former therapy, and an appropriate mono- or combined therapy was started. 17 of 24 patients were cured by surgery in an initial therapy phase. 7 tumors were progressive, 6 of which responded to secondary surgical or cytostatic treatment. One patient died from metastases of choriocarcinoma. The total cure rate of 96% was rendered possible by regular follow-up including bimanual palpation, echography, HCG-assay and chest X-rays.

A human gestational uterine choriocarcinoma cell line (NJG) was established in vitro. The NJG cell line had been subcultured more than 73 times over 7 years. The cells consisted mainly of Langhans-like cells, while multinucleated cells were occasionally seen. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 80 hrs. Electron-microscopically, desmosomes were characteristically observed, which suggested that the cultured cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a hypertriploid mode and 4 marker chromosomes. Immunocyto- and-histochemical staining for tumor markers of the cultured cells and the transplanted tumors to nude mice showed close similarity. SP1 was occasionally demonstrated and PP5 was faintly stained only in the heterotransplanted tumors, although PP10 and PP12 were not demonstrable. HCG was localized not only in syncytial cells, but also in Langhans cells, and syncytial cells without hCG were also observed in the tumors. A single cell clone was also established from the original NJG cells by a limiting dilution method. The heterotransplanted tumor consisted of Langhans and syncytial cells, indicating that both cells originated from the same cell.

A carcinoma of the urinary bladder with a distinct choriocarcinomatous component that developed in a postmenopausal woman was associated with high titers of circulating chorionic gonadotropin. The diagnosis was supported by histochemical demonstration of human chorionic gonadotropin in syncytial tumor giant cells and electron microscopic features consistent with syncytiotrophoblastic cell differentiation.

A primary pancreatic choriocarcinoma was discovered at surgery in a patient with a clinical diagnosis of pancreatic inflammatory pseudocyst. To our knowledge, this is the first example of an extragonadal choriocarcinoma reported as a primary at this site. We consider this neoplasm to be another example of a visceral choriocarcinoma arising from a primary carcinoma through a process of metaplasia. This case illustrates the inherent difficulties in the preoperative evaluation of cystic lesions of the pancreas, and the importance of recognizing endocrinologic manifestations of malignant disease.

JAr choriocarcinoma cells grew as multicellular spheroids, and exhibited a logarithmic pattern of growth, reaching geometric mean diameters of at least 1200 microns after 21 days in culture. HCG, E2 and P4 were secreted into the culture medium throughout the entire culture period, in proportion to spheroid size. This, along with the presence of lipid droplets, non-staining glycogen, Golgi apparatus and microvilli on the spheroid surface and in intercellular spaces indicated that the JAr cells within spheroids were functionally active. While spheroids of less than 800 microns GMD attached to culture dishes and produced cellular outgrowth, larger spheroids showed impaired attachment, and a delay in the subsequent production of outgrowth, which was not correlated with the development of a necrotic core. This outgrowth contained more multinucleated cells and cellular projections than that of smaller spheroids, suggesting that the cells in larger spheroids had undergone early differentiative changes. The spheroid culture system will be applied to the study of processes, such as implantation, which may require a three-dimensional arrangement of placental cells.