Search

Acrylonitrile (AN), which is widely utilized in the manufacture of plastics, acrylamide, acrylic fibers, and resins, is also one of main components of cigarette smoke (CS). In this study, we examined the effects of AN on the cell viability and apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cell lines. A cell viability assay confirmed that AN decreased the cell proliferation of JEG-3 and BeWo cells in a dose-dependent manner. Additionally, Western blot assay revealed that protein expression of cyclin D and cyclin E decreased, while protein expression of p21 and p27 increased in response to AN treatment for 48 hr. The changes in reactive oxygen species (ROS) levels in JEG-3 and BeWo cells exposed to AN were also measured by a dichlorofluorescein diacetate (DCFH-DA) assay, which revealed that ROS levels increased in response to AN treatment for 48 hr. Moreover, western blot assay confirmed that AN treatment of JEG-3 and BeWo cells for 4 hr promoted the expression of phosphorylated eukaryotic initiation factor 2 alpha protein (p-eIF2α), C/EBP homologous protein (CHOP) and caspase 12, which are known to be involved in ROS-mediated endoplasmic reticulum stress (ER-stress)-related apoptosis. Overall, the protein expression of p53 and Bax (a pro-apoptosis marker) increased, while the expression of Bcl-xl (an anti-apoptotic marker) decreased and the number of apoptotic cells increased in response to AN treatment for 48 hr. Taken together, these results suggest that AN has the potential to induce apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cells by activating ROS.

Cigarette smoke (CS) contains over 60 well established carcinogens. In this study, we examined the effects of benzo(a)pyrene (B(a)P), a main CS component, on the viability and apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cell lines. An MTT assay confirmed that B(a)P decreased the cell viability of JEG-3 and BeWo cells in a dose-dependent manner. Additionally, Western blot (WB) assay revealed that protein expression of cyclin D and cyclin E decreased, while protein expression of p21 and p27 was increased in response to B(a)P treatment for 48 h. The changes in reactive oxygen species (ROS) levels in JEG-3 and BeWo cells exposed to B(a)P were also measured by a dichlorofluorescein diacetate (DCF-DA) assay, which revealed that ROS levels increased in response to B(a)P treatment for 48 h. WB assay also confirmed that each B(a)P treatment of JEG-3 and BeWo cells for 4 h promoted the expression of phosphorylated eukaryotic initiation factor 2 alpha protein (p-eIF2α) and C/EBP homologous protein (CHOP), which are known to be involved in ROS-mediated endoplasmic reticulum stress (ER-stress) related apoptosis. Overall, the protein expression of Bax (a pro-apoptosis marker) increased, while the expression of Bcl-xl (an anti-apoptotic marker) decreased and the number of apoptotic cells increased in response to B(a)P treatment for 48 h. Taken together, these results suggest that B(a)P has the potential to induce apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cells by increasing the ROS level and simultaneously activating ER-stress., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The goal of the present study was to investigate the action of TBBPA on PPARγ protein expression in vitro in human choriocarcinoma-derived placental JEG-3 cells. We also analyzed TBBPA for its action on placental secretion of progesterone and β-hCG, cell viability, and apoptosis. Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level. This alteration in PPARγ protein expression was accompanied by a decreased β-hCG level. Interestingly, co-treatment with the PPARγ antagonist GW9662 reversed the TBBPA-mediated changes in PPARγ protein expression but, according to β-hCG secretion, potentiated an inhibitory effect of TBBPA. Additionally, in our study, we assessed the ability of TBBPA to increase progesterone levels in JEG-3 cells compared with those of controls. Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control. The apoptotic action of TBBPA was also confirmed by Hoechst 33342 staining. These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells. Although co-treatment with antagonist of PPARγ reversed the TBBPA-mediated increase in this protein expression and restored it to the control level, it did not reverse the effect on β-hCG secretion. This indicated that the mechanism of TBBPA-induced changes in β-hCG secretion is PPARγ-independent.

Aims and Objectives: Gestational choriocarcinoma is a malignant form of gestational trophoblastic disease with a highly aggressive biologic behavior and responds well to chemotherapy. The objective of this study is to analyse the various histological features of this neoplasm as seen in Ahmadu Bello University Teaching hospital, ( ABUTH ) Zaria, determine its incidence, and compare with other studies., Materials and Methods: The bench registers were used to retrieve the request forms, slides, and tissue blocks. The slides were all stained with standard haematoxylin and Eosin. The histological criteria published by Gehrig and van Lee was used to diagnose the tumours and grading of the cases from grade I to III., Results: Forty three cases were studied and these formed 4.9% of all products of conception and 37.7% of all gestational trophoblastic diseases. The peak age of incidence was in the third and fourth decades of life with vaginal bleeding as the leading mode of presentation. Extensive histopathological analysis and grading revealed haemorrhage, necrosis diamorphic appearance and pleomorphism as the most frequent features., Conclusion: Gestational choriocarcinoma is a common problem in Zaria, North- Western Nigeria with an incidence of 1 in 1039 deliveries. Haemorrhage, necrosis, diamorphic appearance and pleomorphism were the most frequent histological features. Health education and early detection are of paramount importance in reducing morbidity and mortality.

Invasiveness of BeWo cells has been assessed in a variety of assay systems including matrigel and mouse. At the same time BeWo cells are mostly used as model system for trophoblast fusion. Here we aimed to test the properties of BeWo cells in a combined approach. We forced BeWo cells to differentiate by culturing the cells in the presence of forskolin and then used these cells for invasion assays on the chorioallantoic membrane (CAM) of the turkey. The chorioallantoic membranes of turkey eggs were incubated with medium containing forskolin, BeWo cells cultured in medium alone, BeWo cells cultured in forskolin and washed, and BeWo cells cultured in forskolin and used directly for application. Suspensions were applied onto ten CAM per condition. For local tumor formation eggs were checked for tumor development every 24h macroscopically for up to 12 days and immunohistochemistry for cytokeratin 18 and Ki-67 were used for further analysis. Forskolin alone did not have any deleterious effect on the CAM. When the CAM was incubated with BeWo cells cultured in medium 40% of the eggs developed a macroscopically visible tumor. BeWo cells stimulated with forskolin and washed induced tumor growth in 50% of the eggs, while forskolin stimulated BeWo cells applied directly onto the CAM induced tumor growth in 70% of the eggs. Forced differentiation of BeWo cells by forskolin may lead to syncytial fusion in a plastic culture dish. Under the conditions used here, i.e. in direct contact to a living tissue, forskolin-induced differentiation of BeWo cells leads to an increase in tumor formation in the CAM. Thus BeWo cells may use signaling pathways to decide for both differentiation pathways similar to primary trophoblast depending on the environment., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The syncytiotrophoblast of the human placenta arises from fusion of stem cells called cytotrophoblasts. The molecular mechanisms associated with cell fusion and syncytiation of cytotrophoblastic cells remain largely unknown. In the present study, we investigated the morphological and electrical properties of BeWo cells, a human choriocarcinoma-derived trophoblast cell model, with several features of the human cytotrophoblast. Cultured cells tended to cluster, but only fused into small, multinucleated syncytia in the presence of cAMP (72 h). The morphological features of both the actin and microtubular cytoskeletons indicated that within 72 h of constant exposure to cAMP, intracellular cortical actin cytoskeleton disappeared, which was the most prominent inducing factor of multi-nucleation. The presence of the cation channel protein, polycystin-2 (PC2), a TRP-type cation channel, associated with placental ion transport in term human syncytiotrophoblast, co-localised with acetylated tubulin in midbodies, but was found non-functional under any conditions. Different electrical phenotypes were observed among control BeWo cells, where only 26% (8 of 31 cells) displayed a voltage-dependent outwardly rectifying conductance. Most quiescent BeWo cells had, however, a low, slightly outwardly rectifying basal whole cell conductance. Acute exposure to intracellular cAMP (<15 min) increased the whole cell conductance by 122%, from 0.72 nS/cell to 1.60 nS/cell, and eliminated the voltage-regulated conductance. The encompassed evidence indicates that the early events in BeWo cell fusion and syncytiation occur by cAMP-associated changes in ionic conductance but not morphological changes associated to chronic exposure to the second messenger. This suggests a tight regulation, and important contribution of cation conductances in cytotrophoblastic cells prior to syncytiation.

Placental hypoxia has been implicated in pregnancy pathologies, including fetal growth restriction and preeclampsia; however, the mechanism by which the trophoblast cell responds to hypoxia has not been adequately explored. Glucose transport, a process crucial to fetoplacental growth, is upregulated by hypoxia in a number of cell types. We investigated the effects of hypoxia on the regulation of trophoblast glucose transporter (GLUT) expression and activity in BeWo choriocarcinoma cells, a trophoblast cell model, and human placental villous tissue explants. GLUT1 expression in BeWo cells was upregulated by the hypoxia-inducing chemical agents desferroxamine and cobalt chloride. Reductions in oxygen tension resulted in dose-dependent increases in GLUT1 and GLUT3 expression. Exposure of cells to hypoxic conditions also resulted in an increase in transepithelial glucose transport. A role for hypoxia-inducible factor (HIF)-1 was suggested by the increase in HIF-1alpha as a result of hypoxia and by the increase in GLUT1 expression following treatment of BeWo with MG-132, a proteasomal inhibitor that increases HIF-1 levels. The function of HIF-1 was confirmed in experiments where the hypoxic upregulation of GLUT1 and GLUT3 was inhibited by antisense HIF-1alpha. In contrast to BeWo cells, hypoxia produced minimal increases in GLUT1 expression in explants; however, treatment with MG-132 did upregulate syncytial basal membrane GLUT1. Our results show that GLUTs are upregulated by hypoxia via a HIF-1-mediated pathway in trophoblast cells and suggest that the GLUT response to hypoxia in vivo will be determined not only by low oxygen tension but also by other factors that modulate HIF-1 levels.

p-Nonylphenol (p-NP) is a metabolite of alkylphenol ethoxylates used as surfactants in the manufacturing industry. Although it is reported to have estrogenic activity and to be transferred from the mother to the embryo, no data are available on its effects on the development of the human placenta. In the present study, we investigated estrogen receptors' (ERs) expression in the first trimester human placenta. Using an in vitro model of chorionic villous explants, we then compared the effects of p-NP and 17beta-estradiol (17beta-E2). Finally, a trophoblast-derived choriocarcinoma cell line, BeWo, was used as a model of trophoblast cell differentiation. Our results showed that the first trimester placenta expresses three ER-alpha isoforms of 67, 46, and 39 kDa and one ER-beta isoform of 55 kDa. Immunohistochemistry revealed the expression of ER-alpha in the villous cytotrophoblast, whereas ER-beta was mainly expressed by the syncytiotrophoblast. Treatment of explant cultures with p-NP (10(-9)M) and 17beta-E2 (10(-9)M) significantly increased beta-hCG secretion and cell apoptosis but did not modify ER expression. After 72 h of exposure, hormone release was significantly higher in p-NP- than 17beta-E2-treated explant cultures. By this time, cleavage of caspase-3 was evident in cultures treated with 17beta-E2 and p-NP. In BeWo cells, a caspase-3 band of 20-16 kDa was evident after 1 h of treatment with p-NP and after 24 h of treatment with 17beta-E2 or forskolin. These findings suggest that the human trophoblast may be highly responsive to p-NP and raise concern about maternal exposure in early gestation.

Homeodomain (HDM) proteins encoded by homeobox (HBX) genes represent a large family of transcriptional factors that control differentiation and development in certain cell types. DLX4 is a member of Distal-less (DLX) family of HBX genes. Recent studies have demonstrated that abnormal expression of DLX4 is present in several types of human tumors, such as breast cancer, leukemia and colon cancer. In the present study, we investigated DLX4 mRNA and protein expression in both normal placental tissues and human choriocarcinoma cell lines. Also, using RNA interference (RNAi) technique, we knocked down the expression of DLX4 and examined apoptosis in JEG-3 cells. Our studies demonstrated that DLX4 RNAi inhibited DLX4 mRNA expression and decreased DLX4 protein mass specifically and effectively, potentially enhancing apoptosis. Moreover, we examined expression of caspase-3 and caspase-8, and found that both caspases were increased after DLX4 knockdown. However, DLX4 RNAi did not influence Bax expression in JEG-3 cells. In conclusion, this study suggests that DLX4 may be involved in the survival of human choriocarcinoma cells, which may be mediated by the inhibition of apoptosis. The detailed mechanism needs further investigation.

Exposure to inorganic arsenic has been associated with various forms of cancer, nervous system pathogenesis, and vascular diseases, as well as reproductive and developmental toxicity. Here, the effect of inorganic arsenic on placental JAR choriocarcinoma cells was assessed. The nuclear protein levels of the CNC transcription factor Nrf2 were strongly induced in the presence of arsenic. Dosage response experiments showed that 0.5 microM of arsenic is sufficient to augment Nrf2 levels. The expression of the Nrf2 dimerization partners MafG and MafK appeared not to be modulated by arsenic, whereas MafF protein levels were slightly increased. Arsenic also induced the binding of endogenous Nrf2/small Maf DNA-binding complexes to a stress response element (StRE) recognition site. In addition, arsenic caused oxidative stress in the choriocarcinoma cell model as evidenced by an increase in intracellular H2O2 levels. Expression of the enzyme heme oxygenase-1 (HO-1), a known Nrf2 target gene, was upregulated by exposure of JAR cells to arsenic. These results suggest that Nrf2/small Maf heterodimers may play an important role in the response to arsenic-mediated stress in placental cells.

Pax proteins are transcriptional regulators that control a variety of developmental decisions in vertebrates. During development, the paired-box gene 8 (PAX8) is expressed in the thyroid, kidney, and several areas of the central nervous system. It is also expressed in the adult thyroid gland, in which it mediates TSH-induced modulation of the expression of important genes, such as those encoding thyroglobulin, thyroperoxidase, and the sodium/iodide symporter (NIS). Thus far, placental expression of PAX8 has been described only in mice. In the present study, we show that PAX8 is also expressed in the human placenta at term. In an in vitro model of placental cancer, the JAR choriocarcinoma cell line, human chorionic gonadotropin (hCG) increased levels of PAX8 mRNA and protein, and gel retardation assays indicated that the up-regulation of PAX8 protein expression is associated with an increase in its DNA-binding activity. The effects of hCG were mimicked by forskolin, indicating that they are cAMP dependent. Levels of mRNA for the Wilms' tumor 1 (WT1) and NIS genes were increased in JAR cells by hCG treatment, whereas overexpression of PAX8 increased only levels of WT1 mRNA. In cells transfected with PAX8-specific small interfering RNA, the stimulatory effects of hCG on WT1 mRNA levels were abolished, but hormonal enhancement of NIS mRNA levels was unchanged. These findings indicate that, in JAR cells, hCG activates a cAMP-dependent pathway that can up-regulate WT1 expression through PAX8.

Serotonin (5-hydroxytryptamine, 5-HT) has diverse physiological functions and acts as a mitogen in a variety of cell types, including bovine placental cells. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Previous studies have reported the presence of 5-HT(2) binding sites in human placental trophoblastic cells, but this has never been confirmed at the molecular level. In this study, we demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the human choriocarcinoma cell lines JEG-3 and BeWo as well as in normal human placental tissue. DNA sequencing has confirmed that the 5-HT(2A) receptor present in these cell lines and tissues is identical to the human 5-HT(2A) receptor found in platelets and in the cerebral cortex. This receptor was localized by immunofluorescence on the plasma membrane, in JEG-3 and BeWo cells. Furthermore, MTT proliferation assays revealed a positive effect of 5-HT on the proliferation of JEG-3 and BeWo cells. These results suggest that 5-HT constitutes a potent mitogen for neoplastic placental cells.

Objective: To isolate a choriocarcinoma suppressor gene and analyze its structure and function., Study Design: We constructed a polymerase chain reaction-based subtracted fragmentary cDNA library between normal placental villi and choriocarcinoma cell line CC1. A novel homeobox gene, designated NECC1 (not expressed in differ choriocarcinoma clone 1), is included in this library. We analyzed the structure and function of NECC1 with molecular biology., Results: NECC1 comprises an open reading frame of 219 nucleotides encoding 73 amino acids and contains a homeodomain as a consensus motif. NECC1 is located on human chromosome 4q11-q12, and its expression is ubiquitous in the brain, placenta, lung, smooth muscle, uterus, bladder, kidney and spleen. Normal placental villi abundantly expressed NECC1, but all choriocarcinoma cell lines examined and most surgically removed choriocarcinoma tissue samples failed to express it. We transfected this gene into choriocarcinoma cell lines and observed remarkable alterations in cell morphology and suppression of in vivo tumorigenesis. Induction of chorionic somatomammotropin hormone 1 by NECC1 transfection suggested differentiation of choriocarcinoma cells to syncytiotrophoblast-like cells., Conclusion: Our results suggest that loss of NECC1 expression is involved in malignant conversion of placental trophoblasts.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that has structural similarity to TSH. At the time of the peak hCG levels in normal pregnancy, serum TSH levels fall and bear a mirror image to the hCG peak. This reduction in TSH suggests that hCG causes an increased secretion of T4 and T3. Women with hyperemisis gravidarum often have high hCG levels that cause transient hyperthyroidsm. In the vast majority of such patients, there will be spontaneous remission of the increased thyroid function when the vomiting stops in several weeks. When there are clinical features of hyperthyroidism, it is be reasonable to treat with antithyroid drugs or a beta-adrenergic blocker, but treatment is rarely required beyond 22 weeks of gestation. Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors, either hydatiditform mole or choriocarcinoma. The diagnosis of hydatidiform mole is made by ultrasonography that shows a 'snowstorm' appearance without a fetus. Hydatidiform moles secrete large amounts of hCG proportional to the mass of the tumor. The development of hyperthyroidism requires hCG levels of >200 U/ml that are sustained for several weeks. Removal of the mole cures the hyperthyroidism. There have been many case reports of hyperthyroidism in women with choriocarcinoma and high hCG levels. The principal therapy is chemotherapy, usually given at a specialized center. With effective chemotherapy, long-term survival exceeds 95%. A unique family with recurrent gestational hyperthyroidism associated with hyperemesis gravidarum was found to have a mutation in the extracellular domain of the TSH receptor that made it responsive to normal levels of hCG.

Objective: Parathyroid hormone-related peptide (PTHrP) have been found to be expressed in a variety of human tumors. Parathyroid hormone-related peptide is known as the major mediator of humoral hypercalcemia of malignancy, may also regulate placental calcium flux, uterine contraction and fetal tissue development. The purpose of this study is to evaluate the expression of PTH/PTHrP receptor in choriocarcinoma JAR cell line., Methods: This study was carried out at the Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia, between November 2002 and August 2003. Choriocarcinoma JAR cell line treated for 12 and 72 hours with epidermal growth factor, (EGF) (20ng/ml), estradiol, E2 (10(-8) M), dexamethasone, (DEX) (10(-8) M) or 1,25 dihydroxycholecalciferol, 1,25 (DHCC) (10(-8) M). We investigated the expression of parathyroid hormone (PTH)/PTHrP receptor in JAR cell line with these treatments compared with untreated JAR cells. The PTH/PTHrP receptor expression were detected with 3.3nM 125I-PTHrP-34Tyrosine., Results: The expression of the receptors at 12 hours were increased following exposure to EGF, E2 or DEX, whereas 1,25 DHCC inhibited the receptor expression. In further experiments at 72 hours with the same treatments, the receptors expression were remarkably increased with EGF, E2 or DEX, whereas, 1,25 DHCC inhibited the receptor expression in these cells., Conclusion: These data suggested that in JAR cells, The EGF, E2 and DEX upregulated the PTH/PTHrP receptor expression, whereas the 1,25 DHCC down-regulated the PTH/PTHrP receptor, and the 1,25 DHCC may play an important role as antiproliferative drug for choriocarcinoma.

The trophoblast, an important component of the mammalian placenta, has several essential biological roles in the maintenance of pregnancy. First, trophoblast cells must attach to the uterine endometrium, and then they must invade to a depth at which the vascular network exists. Here, we investigated the effects of epidermal growth factor (EGF) on alpha2 integrin expression, adhesiveness to collagen, and invasive activity using human BeWo choriocarcinoma cells. EGF induced the expression of alpha2 integrin mRNA and protein, as shown by Northern blotting, Western blotting, and flow cytometry. Human chorionic gonadotropin (hCG) secretion was enhanced by the addition of EGF, which suggests that the BeWo cells functionally differentiated similarly to normal trophoblasts. EGF also dose-dependently stimulated the invasiveness of BeWo cells. Antibody against alpha2 integrin inhibited this effect, suggesting that it may be mediated by an increase of cell surface integrin. EGF had no effect on the adhesiveness of BeWo cells to collagen, whereas it stimulated the chemokinetic activity in a dose-dependent manner. The increase of chemokinetic activity was suppressed by antibody against alpha2 integrin. These results suggest that EGF may induce alpha2 integrin expression in trophoblast cells, thereby enhancing their invasiveness into the endometrium via an increase of their chemokinetic activity.

The mechanisms of endocrine and immune system disorders in normal and disturbed pregnancy as well as in chorionocarcinoma, hydatid mole, metastatic and non-metastatic chorionocarcinoma have been investigated. Relevant disorders were chiefly caused by the endocrine function of the thymus and gonadotropins from the trophoblast, which made a powerful impact on the periphery of hormonal and immunological homeostasis. Unlike normal pregnancy, gestational trophoblastic disease involved changes in the nature, direction and degree of correlations between endocrine formations, which in turn determined the pathogenetic pattern of the disease.

Endothelin (ET) secretion and expression of both ET-A and ET-B receptor subtypes have been found in a number of primary cancers. The present study tested (1) whether choriocarcinoma cells and their nonmalignant counterpart, the trophoblast, secrete ET-1 and express ET-A and ET-B receptors; (2) whether ET-1 secretion and receptor mRNA levels are regulated by the same factors in nonvascular tissues as in vascular tissues; and (3) whether such regulation is similar in malignant and nonmalignant cells. All cells secreted ET-1 in similar amounts (approximately 0.8 fmol/10(6) cells per 24 h) and secretion was unaffected by culture and treatment. Whereas ET-B accounted for almost all (>98%) ET receptor transcripts in the choriocarcinoma cells, the trophoblasts expressed about 20% ET-A receptor mRNA. During control cultures, ET-B mRNA levels rose in choriocarcinoma, with the greatest relative increase (6-fold; P < 0.05 vs 0 h) in BeWo, whereas in trophoblasts, ET-A mRNA transiently changed after 24 and 48 h. Treatment with dexamethasone and glucose did not alter the mRNA levels in all cells. Insulin induced changes (P < 0.05) in ET-B mRNA levels in BeWo (+90 and +60% after 24 and 48 h, respectively) and JEG-3 (-70%), but not in JAR and trophoblast cells. We conclude that malignant transformation affects the responsiveness of the endothelin receptor system to external stimuli and that the regulation of the endothelin system differs in vascular and nonvascular tissues.

Intraplacental choriocarcinoma is very rare, and is usually found only after maternal and fetal metastatic disease is identified. The purpose of this case report is to review the incidence and findings of intraplacental choriocarcinoma. A term placenta was investigated because the newborn was born with severe anemia (Hb 3.0 g/dL). A 2 cm nodule was noted on the surface of the amniotic membrane and grossly resembled an infarction. The tumor was examined microscopically with immunohistochemical staining for the alpha- and beta-human chorionic gonadotropin (alpha-hCG, beta-hCG) subunits, human placental lactogen (hPL) and Ki-67. Microscopically, the tumor consisted of necrotic areas with proliferation of atypical trophoblastic cells and destruction of the villi and capillaries. The cells were positive for the alpha-hCG, beta-hCG subunits, hPL and Ki-67, consistent with intraplacental choriocarcinoma. The mother and newborn were investigated for the presence of metastatic disease. Computed tomography scans and magnetic resonance imaging of the mother and infant were negative for metastatic disease. Choriocarcinoma, limited only to the placenta with no evidence of metastatic disease is very rare. Primary intraplacental choriocarcinoma may frequently be overlooked or missed, and choriocarcinoma may possibly arise in the placenta more often than in retained or persistent trophoblast following pregnancy.

We report on a short-statured boy in whom therapy with recombinant human growth hormone was initiated at the age of 9.7 years for assumed idiopathic growth hormone deficiency. On recombinant human growth hormone, height improved from -1.9 (standard deviation score) to -0.9 within 1 year, and the patient entered puberty spontaneously at 10.7 years. At 11.6 years he showed low morning cortisol and thyroxine levels, but was otherwise well. He showed an inconspicuous growth, and puberty progressed adequately until the age of 13.4 years, when he developed signs of an increased intracranial pressure, and a suprasellar choriocarcinoma was diagnosed. This case confirms the fact that beta chorionic gonadotrophin secreting tumours will not be diagnosed by the characteristic clinical manifestation of gonadotrophin-independent puberty if they occur at a time when normal puberty is expected. Particularly, it raises the question of how often the CNS should be re-evaluated by magnetic resonance imaging in children with growth hormone deficiency and normal initial neuroradiological imaging, when they develop additional hormonal deficiencies but no other clinical symptoms of an intracranial process., (Copyright 2001 S. Karger AG, Basel)

In gene therapy, tumor cells expressing herpes simplex virus thymidine kinase (HSV-tk) are sensitive to ganciclovir (GCV) and HSV-tk positive cells exposed to GCV are lethal to adjacent HSV-tk negative cells. This phenomenon has been called the bystander effect, and the gap junction is thought to mediate it. In this study, sensitivity to GCV and bystander effect in a human choriocarcinoma cell line, BeWo, transfected with HSV-tk were investigated. Furthermore, the effect of 8-bromo-cAMP on bystander effect and connexin40 gene transcription were examined. HSV-tk positive cells were sensitive to GCV at the concentration of 10 micrograms/ml in a time-dependent manner. The growth of HSV-tk negative cells was inhibited when the population of cultured cells contained more than 10% HSV-tk positive cells and 8-bromo-cAMP enhanced bystander effect. 8-bromo- cAMP increased connexin40 mRNA expression and gap junctional intercellular communication.

The 5'-flanking region from -78 to +9 in the HSD17B1 gene serves as a promoter, and an HSD17B1 silencer element is located in position -113 to -78. In the present studies, we have characterized three regulatory elements in the proximal 5'-flanking regions of the gene, using electrophoretic mobility shift assays and reporter gene analysis. First, nuclear factors recognized by antibodies against Sp1 and Sp3 were found to bind the Sp1 motif in the region from -52 to -43. Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3 cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a substantial role in the complete functioning of the HSD17B1 promoter. Second, the binding of AP-2 to its motif in the region from -62 to -53 led to reduced binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can repress the function of the HSD17B1 promoter by preventing binding to the Sp1 motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind their cognate sequence in the HSD17B1 silencer region, and mutations introduced into the GATA-binding site increased transcriptional activity to the level seen in constructs not containing the silencer element. Thus, GATA-3 seems to prevent transcription in the constructs, and hence, the GATA motif also may operate as a negative control element for HSD17B1 transcription.

The controlled invasiveness of the trophoblast is based on the balance between invasive properties at implantation and the differentiation program of the developing placenta. During placental development in rats a switch of connexin gene expression has been observed in parallel to the switch from the invasive to the differentiated phenotype of trophoblast cells. To investigate the role of connexin expression for trophoblast invasion, proliferation, and differentiation, we studied one rat trophoblast (HRP-1) and one rat choriocarcinoma cell line (Rcho-1). The choriocarcinoma cells were characterized by expression of cx31 and a lack of E-cadherin, corresponding to the invasive trophoblast in vivo, whereas HRP-1 cells expressed cx43, normally found in the spongiotrophoblast and in late giant cells, and E-cadherin. Upon retinoic acid treatment, Rcho-1 cells irreversibly lost cx31 expression, accompanied by a loss of functional coupling. No changes in regard to connexin expression and cell-cell communication could be observed in HRP-1 cells. In addition, treatment of Rcho-1 cells with retinoic acid for 7 days upregulated expression of cx43 transcript, but no protein could be found. Proliferation was clearly reduced and the mean volume of cells doubled from Day 4 to Day 7 of retinoic acid treatment in Rcho-1 cells, while both parameters were not affected in HRP-1 cells. Both cell lines showed a similar invasion rate using a Matrigel invasion assay, and invasion was equally suppressed upon retinoic acid treatment. Thus the different connexin expression appears more likely to play a role in regulating proliferation and differentiation along the multilineage pathway than invasiveness of rat trophoblast cells.

Objective: Our purpose was to determine whether human choriocarcinoma cells express autocrine motility factor receptor (AMF-R) and to study its function in this tumor system., Study Design: The expression and localization of AMF-R were compared in choriocarcinoma and normal placental trophoblasts using both cell lines and tissue sections. In addition, migratory properties of choriocarcinoma cells and normal placental cells was determined., Results: Using immunofluorescence and immunoperoxidase staining, we have detected the expression of AMF-R in choriocarcinoma cells with receptor clustering on the cell surface, while term placenta cells expressed AMF-R less intensely with no receptor clustering. In choriocarcinoma tissues, AMF-R was strongly expressed in malignant cytotrophoblasts cells while adjacent normal villous trophoblast cells and necrotic regions were weakly or negatively stained. Choriocarcinoma cells responded to AMF-R stimulation with increased cell motility, while term placental cells were unresponsive., Conclusion: Human choriocarcinoma cells express functional cell surface AMF-R in vitro and in choriocarcinoma tissue suggesting that this receptor may play an important role in cancer cell motility.

Although choriocarcinoma following a term pregnancy is an uncommon disease, it is important to identify these patients early. The time interval between delivery and onset of treatment is a crucial factor influencing prognosis. We present a case where the woman was delivered of a stillborn infant and histological examination of the placenta at first did not reveal the true diagnosis of choriocarcinoma. Due to persistent vaginal bleeding curettage was performed 11 weeks later establishing choriocarcinoma. In this particular case it was then possible to review the original material and to confirm the diagnosis in this specimen. The case is extraordinary since the placenta harbouring the primary tumour had been macroscopically and microscopically described and the material could be reviewed. Secondly the case underlines the diagnostic difficulties when examining a mature placenta.

Widened arterial pulse pressure is often found with conditions having exaggerated diastolic runoff flow, such as aortic insufficiency and arteriovenous malformation. We report a 17-year-old boy with metastatic testicular choriocarcinoma and a widened pulse pressure. Ultrasonography and Doppler analysis revealed significant runoff flow into multiple large hepatic metastases. After chemotherapy, there was diminution in his metastases, a narrowing of the pulse pressure, and no evidence of runoff flow in the liver. These cardiovascular findings have not been previously described in association with metastatic vascular malignancies.

Factors and cells of placental origin have been considered to be important in mediating local active immunosuppression that regulates maternal immune reactivity to aid fetal survival. In this context, we investigated the immunosuppressive capabilities of supernatants from human trophoblast-derived choriocarcinoma cell lines (HCS). We have previously reported the inhibitory effects of HCS on proliferative responses of T-lymphocytes both in vitro and in vivo (L. Krishnan, E. Menu, G. Chaouat, G. P. Talwar, and R. Raghupathy, Cell. Immunol. 138, 113, 1991; L. Krishnan, R. Kinsky, G. Chaouat, G.P. Talwar, and R. Raghupathy, Cell. Immunol. 150, 376, 1993). We now show that HCS also suppresses LPS-induced proliferation of murine lymphocytes but does not inhibit the constitutive proliferation of lymphoma cell lines and B cell hybridomas indicating that HCS has no inhibitory effects on terminally differentiated or transformed cells. Furthermore, we have succeeded in isolating and partially characterizing the HCS-derived suppressor factor (HCSf) from culture supernatants of a human choriocarcinoma cell line JEG-3. Purification of the factor has been accomplished by sequential fractionation on anion-exchange, gel filtration, and reverse-phase HPLC columns. The suppressor factor is a low-molecular-weight compound in the range of 5-6 kDa, composed predominantly of hydrophilic amino acids. Protease digestion of the factor revealed that the peptide moiety in HCSf is important for its inhibitory activity. HCSf mediates a dose-dependent suppression of proliferative responses of both human and murine lymphocytes.

To examine the hypothesis that nutritional signals regulate trophoblast cell function, JEG-3 choriocarcinoma cells were treated with drugs that stimulate peroxisome proliferator-activated receptors (PPARs). These receptors are thought to mediate in part the effects of lipidic nutrients on gene expression. Because PPARs are modulated by interactions with retinoid-X receptors, we also examined the actions of the peroxisome proliferators in the presence of retinoids. Clofibric acid, a known peroxisome proliferator, suppressed JEG-3 cell growth in association with increases in the tumor suppressor p53 protein and its messenger RNA (mRNA). It reduced CG secretion and CG alpha and CG beta mRNAs in growing cells. However, clofibric acid did not induce peroxisome proliferation in the JEG-3 cells, as assessed by electron microscopy and immunostaining for catalase, a peroxisomal enzyme, or alter levels of mRNAs for peroxisomal proteins, sterol carrier protein-X/sterol carrier protein-2 and acyl-Coenzyme-A oxidase. The mitochondrial cholesterol side-chain cleavage enzyme, cytochrome P450scc, was modestly increased in some experiments. All-trans-retinoic acid and 9-cis-retinoic acid increased CG secretion and CG alpha and CG beta mRNAs, but clofibric acid blunted these stimulatory effects. WY 14,643, another peroxisome proliferator, also reduced CG gene expression without increasing mRNAs encoding peroxisomal proteins or altering P450scc mRNA. The mRNA for a human PPAR, NUC1, was demonstrated in JEG-3 cells, and NUC1 mRNA was shown to be upregulated by 8-bromo-cAMP. We conclude 1) that JEG-3 cells express a PPAR and are subject to regulation by PPAR stimulators; 2) that PPAR stimulation in JEG-3 cells does not promote peroxisome proliferation; and 3) that peroxisome proliferators and retinoids differentially regulate JEG-3 cell endocrine activities. We suggest from these findings that JEG-3 cells possess mechanisms to respond to nutrient cues.

Placental trophoblasts are epithelial cells which undergo physiological fusion and generate syncytia. In this study, a placental choriocarcinoma cell line JAR was infected with two enveloped viruses, Parainfluenza type 3 (P3) or vesicular stomatitis virus (VSV). Both viruses possess a fusion glycoprotein known to be able to induce polykaryon formation in nonpolarized cells. The P3 virus fusion protein was localized on the apical as well as on the basal plasma membrane domains of the infected JAR cells. Infection of the JAR cell monolayer with P3 virus, whose fusion protein is active at pH 7.0, resulted in syncytia formation. Furthermore, the actin ring structure surrounding individual cells disappeared during the P3 virus induced cell-cell fusion. On the contrary, the VSV glycoprotein was found preferentially on the apical plasma membrane domain. To activate the VSV fusogen, the cells were subjected to pH 5.0. However, no syncytia formation peculiar to VSV-infected fibroblasts was observed, and the actin ring structures remained intact. We conclude that in JAR cells the VSV fusion protein exhibits a polarized expression while the P3 virus fusion glycoprotein is distributed between the two membrane domains. Our results suggest that an apically situated fusogen is not sufficient to mediate cell-cell fusion of JAR cells.

Cytotrophoblasts (from term placentae) and cells from the choriocarcinoma cell line JAr were cultivated either separately or in co-culture for 72 h. RNA was isolated from the cell cultures and Northern blots were developed using equal amounts of RNA. The RNA was hybridized with cDNA probes for CG alpha, CG beta and hPL. Corresponding m-RNAs were detected in the three RNAs except for hPL m-RNA which was absent from JAr cells RNA. The abundance of CG alpha and CG beta m-RNA in the RNA of the co-culture was higher than their accumulative abundances in the RNAs from cytotrophoblasts and JAr cells cultured alone and the abundance of hPL m-RNA in the RNA of the co-cultures was as high as that in the RNA from cytotrophoblasts cultured alone. On the basis of previous findings (Hochberg et al, 1991), it can be assumed that the cytotrophoblasts in the co-cultures are responsible for the increase in hormonal m-RNA production. It could be calculated that the abundances of the CG alpha, CG beta and hPL m-RNAs in the RNA which originated in the cytotrophoblast nuclei were 20, 100 and 10-fold higher respectively in the co-culture compared to those in the culture of cytotrophoblasts. This effect is limited to certain genes only as the concentration of the 92kD collagenase m-RNA and uPA (urokinase type plasminogen activator) m-RNA, which are both produced in cytotrophoblasts to a much higher extent than in JAr cells, and are not increased by cultivating the cytotrophoblasts with JAr cells in co-culture.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with 'high' affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with 'low' affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the 'NBMPR-sensitive' component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus 'NBMPR-insensitive'. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of 'erythrocyte-like' transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transporter elements is uncertain.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating PRL levels in two physiological models: 1) pregnant rats expressing (day 8) or not expressing (days 11 and 16) PRL surges, and 2) ovariectomized rats transplanted with rat choriocarcinoma cells, which secrete functional placental lactogen-I. Over a 4-min period between 0900 and 1400 h, rats were administered either vehicle or 5-HT (20 micrograms/6 microliters) through lateral ventricular cannulae. Plasma PRL levels were determined by RIA. NSD 1015 (25 mg/kg intraarterial), a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, was injected 20 min after initiation of ventricular infusion. Ten min later, the stalk-median eminence (SME) was dissected. The rate of DOPA accumulation, determined by measuring DOPA levels in the SME by HPLC, was used as an index of tyrosine hydroxylase catalytic activity, indicating tubero infundibular dopamine neuronal activity. In day-8 pregnant rats 5-HT reduced DOPA accumulation to 57% of vehicle-injected controls and increased circulating PRL levels 13-fold. In contrast, on days 11 and 16 of pregnancy 5-HT did not alter DOPA accumulation in the SME or plasma PRL levels. In nonpregnant rats ovariectomized for 24 h, 5-HT decreased DOPA accumulation in the SME to 43% of vehicle-infused controls and increased PRL levels approximately 26-fold. However, in nonpregnant rats with rat choriocarcinoma cells, 5-HT produced no changes in either DOPA accumulation in the SME or in circulating PRL levels. The inability of 5-HT to reduce tyrosine hydroxylase activity after mid-pregnancy may account for the lack of a PRL response. Placental lactogens secreted at midpregnancy, particularly placental lactogen-1, may induce this loss of 5-HT effect.

A 33-year-old highly parous woman developed severe abdominal pain and signs of circulatory collapse 10 months after tubal sterilisation in the absence of symptoms of pregnancy. A ruptured ectopic pregnancy sited interstitially in the right tube and extending into the myometrium and parametrium was found at laparotomy. Histopathologic examination revealed an ectopic pregnancy consisting of choriocarcinoma--a rare but life-threatening combination in a sterilised woman.

Hyperthyroidism is a well recognized complication of gestational trophoblastic tumours (GTT) and may be due to high circulating concentrations of human chorionic gonadotrophin (hCG) or its variants. We have studied 24 clinically euthyroid women with GTT. Eight were biochemically hyperthyroid with low or undetectable serum thyrotrophin (TSH) and had a mean serum hCG of 361.2 x 10(3) IU/l compared to 76.2 x 10(3) IU/l in the other patients (P less than 0.01). Purified hCG stimulated iodide uptake into FRTL-5 cells with 25 x 10(3) IU/l being equivalent in potency to 1 mU/l of thyrotrophin (TSH). Sixteen out of the 24 sera (67%) stimulated iodide uptake when applied to the cells at a 1:10 dilution. Sera from all eight hyperthyroid patients contained thyroid stimulating activity. The mean hCG concentration in the 16 stimulatory sera was 238.2 x 10(3) IU/l compared to 37.1 x 10(3) IU/l in the other eight sera (P less than 0.01). Six men with hCG-secreting testicular tumours were biochemically euthyroid although three of their sera stimulated iodide uptake into FRTL-5 cells. In human thyroid cells the mean cAMP production over 4 h with sera from five healthy controls was 54.2 +/- 1.81 pmol/mg cell protein compared to 67.0 +/- 3.8 pmol/mg protein with sera from five choriocarcinoma patients (P less than 0.02). Serum from patients with gestational trophoblastic tumours contains a thyroid stimulating activity which may be hCG and whose presence correlates with hyperthyroidism.

The authors report a case of hyperthyroidism induced by a metastatic choriocarcinoma (retroperitoneal, lungs, vertebral and epidural localisations) in a 49 year old man. Tachycardia and possibly weight loss were the only clinical signs of thyrotoxicosis. Chemotherapy led to a remission of hyperthyroidism and tumoral syndrome for 4 months. Neoplasia relapsed and circulating levels of thyroid hormones were increased during the final stages. This is the fifth male reported case of choriocarcinoma associated with hyperthyroidism. Many previous studies suggest that hyperthyroidism is induced by HCG thyroid stimulating activity.

Benign and malignant neoplasms arising in and around the pelvic structures may cause urinary obstruction by mechanical compression of the bladder or proximal urethra. These neoplasms less commonly cause neurogenic voiding dysfunction. Herein we describe the urodynamic abnormalities in a young man with massive involvement of the true pelvis with choriocarcinoma. Destruction of the pelvic plexus produced deficits of parasympathetic, sympathetic and somatic innervation of the bladder and urethra. The value of urodynamic evaluation as a neurologic test of thoracolumbar and sacral cord function is demonstrated.

Trophoblastic cells with their strictly controlled propensity for invasive growth hereby bear limited resemblance to malignant cells. Therefore, detailed description of molecular characteristics of this cell type in comparison to histologically related tumor cells may aid in defining physiologically relevant differences. In view of the potential importance of selective protein-carbohydrate interactions in recognitive processes, labelled neoglycoproteins were employed to evaluate systematically the presence and distribution of sugar receptors in tumor cells of chorionepithelioma and in trophoblastic cells of an early stage of gestation. Control reactions in glycohistochemistry, involving prolonged incubation, pH variation and use of nucleotides excluded the possibility that glycosidases or glycosyltransferases govern the sugar-specific binding to the tissue sections. Pronounced differences were detected in the expression of receptors (lectins) for alpha- and beta-glucosides as well as for N-acetylgalactosamine and N-acetylglucosamine. Further differences were revealed by probing both types of tissue with lactosylated, fucosylated, xylosylated and sialylated carrier protein. Upon developmental maturation of the normal cells in placenta the extent of binding of the neoglycoproteins decreased. The glycohistochemical differences between malignant and normally developing trophoblastic cells were found to be reflected in the alteration of expression of certain endogenous lectins, as ascertained by affinity chromatography and gel electrophoretic analysis. Consequently, we assume that the transient presence of certain endogenous lectins during early stages of gestation and their differential expression in relation to tumor cells of chorionepithelioma may be relevant for the special invasive and adhesive behavior of human trophoblastic cells.

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.

The antitumor properties of antibodies to the beta-subunit of hCG (hCG beta) coupled to tetanus toxoid (tt) were studied in hamsters implanted with the BeWo strain of human choriocarcinoma. After active immunization with hCG beta-tt mixed in complete Freund's adjuvant, hamsters synthesized large quantities of antibodies which bound whole hCG, as measured in vitro by the binding of radiolabeled hCG to plasma. Scatchard binding analysis indicated that the total high affinity hCG-binding capacity of hCG beta-tt plasma was approximately 130 microgram/ml. However, these antibodies were devoid of any antitumor activity. Choriocarcinoma implanted into hamsters during the peak times of [125I]iodo-hCG-binding activity by plasma did not show any change in tumor volume over a 20-day period of tumor growth compared to tumors in hamsters immunized with tt alone. Active immunization with hCG beta-tt did not alter the percentage of tumors taking, the length of lag phase of tumor growth, or the initial day of grossly detectable tumor necrosis. These results suggest that active immunization with hCG beta-tt will not alter the growth of transplantable choriocarcinoma.

Six choriocarcinoma cell lines that secrete chorionic gonadotropin (hCG) in tissue culture were investigated for the production of thyroid stimulators. In the mouse thyrotropin bioassay, no thyroid-stimulating activity was found in cell culture media even after 30-fold concentration by the method of Bates. Further, concentrates of culture media did not react in a porcine thyrotropin radioimmunoassay in which hCT cross-reacted completely. Thyroid function was also assessed in hamsters bearing hCG-secreting human choriocarinoma transplants. Even though the thyroid-stimulating factor present in commercial urinary hCG preparations caused an increase in PB131I in the hamster, the presence of the choriocarcinoma had no effect on PB131I. From these results, it appears that human choriocarcinoma cell lines maintained in tissue culture and human choriocarcinoma serially transplanted in the hamster are not suitable models for the study of the secretion of a thyroid-stimulating factor by trophoblastic tissues.

Various aspects of thyroid hormone economy were examined in 15 patients with molar pregnancy and in 5 patients with choriocarcinoma. None of the patients with molar pregnancy was clinically thyrotoxic, though serum thyroxine (T4) was increased in 13 and free T4 concentrations were above normal in four of ten in whom measurements were made. In 2 patients with elevated serum T4 levels and in one patient with normal serum T4 levels, the daily rate of T4 disposal was increase, and most of 131I derived from 131I-T4 was excreted into urine in two patients in whom estimation of urinary excretion was made. Serum total triiodothyronine (T3) concentrations closely paralleled those of serum T4, but T3/T4 ratios were lower than those previously found in patients with toxic diffuse goiter of Graves' disease. Unresponsiveness to exogenous thyrotropin-releasing hormone (TRH) was observed in 5 patients with molar pregnancy whose serum total T4 and T3 levels were increased. Human chorionic gonadotropin (hCG) concentrations in serum ranged from 0.5 to 2830 IU/ml and were less than 200 IU/ml in 36% of the patients, possibly because molar pregnancy was diagnosed by ultrasonography, rather than high titers of urinary or serum hCG. Serum thyroid stimulating activity measured by the McKenzie bioassay closely paralleled values for hCG (r:+0.94). All thyroid abnormalities disappeared quickly after removal of the mole. In choriocarcinoma, no abnormality of thyroid function was found. These results suggest that: 1) some patients with molar pregnancy display a marked elevation of serum T4, T3, and free T4 concentrations, probably as a result of the action of a thyroid stimulator which is closely related to serum hCG or may be hCG itself; 2) T4 production rates are increased in these patients, and the pituitary responds to TRH in a manner similar to that found in thyrotoxicosis. Despite this, frank clinical thyrotoxicosis is usually absent, possibly because of relatively low serum T3/T4 ratios, possibly because of the limited duration of thyroid hyperfunction, and possibly because of other factors that for the present remain unknown.

The influence of methotrexate (MTX), dibutyryl cyclic AMP, and actinomycin D on production of human chorionic gonadotropin (HCG) in normal first trimester human placental organ cultures was compared. Actinomycin D (10(-8) to 10(-6) M) elevated HCG production by as much as 3.5-fold in normal placenta, and a 2-fold increase in HCG levels was obtained by treatment with dibutyryl cyclic AMP (1 mM) and theophylline (1 mM). The combination of dibutyryl cyclic AMP (1 mM) plus theophylline (1 mM) and actinomycin D (10(-8) M) additively enhanced HCG production by 4.5-fold. In contrast, HCG levels in normal placental organ cultures were unaffected by MTX (10(-8) to 10(-5) M) despite several differing treatment regimens. The JAr line of human choriocarcinoma cells, on the other hand, exhibited an 8-fold increase in HCG levels following MTX exposure (10(-7) M). Incorporation of selected radiolabeled precursors of the de novo and salvage pathways of DNA synthesis was evaluated to assess potential metabolic alterations underlying the differential HCG response of these cultures to MTX. Deoxyuridine incorporation into DNA was decreased similarly in both normal and malignant placenta following MTX exposure. However, deoxycytidine incorporation was inhibited by MTX in normal placental cultures but was elevated by as much as 4-fold in JAr cultures exposed to MTX. Thymidine incorporation into DNA was increased in both groups in the presence of MTX; however, thymidine incorporation was more profoundly stimulated (5-fold) in normal placenta than in JAr cultures (2.5-fold). These data indicate dissimilar utilization of the de novo and salvage pathways of DNA synthesis by these cultures which may explain their differential responsiveness to MTX.