Cultured Choriocarcinoma Cells Research Articles

LncRNA Tuna is activated in cadmium-induced placental insufficiency and drives the NRF2-mediated oxidative stress response.

Cadmium (Cd) is a toxic heavy metal found throughout the environment and one of the top ten toxicants of major public health concern identified by the World Health Organization. In utero Cd exposure causes fetal growth restriction, malformation, and spontaneous abortion; however, the mechanisms by which Cd impacts these outcomes are poorly understood. Cd accumulates in the placenta, suggesting that these negative outcomes may be a consequence of disrupted placental function and placental insufficiency. To understand the impact of Cd on gene expression within the placenta, we developed a mouse model of Cd-induced fetal growth restriction through maternal consumption of CdCl2 and performed RNA-seq on control and CdCl2 exposed placentae. The top differentially expressed transcript was the Tcl1 Upstream Neuron-Associated (Tuna) long non-coding RNA, which was upregulated over 25-fold in CdCl2 exposed placentae. Tuna has been shown to be critical for neural stem cell differentiation. However, within the placenta, there is no evidence that Tuna is normally expressed or functional at any developmental stage. To determine the spatial expression of Cd-activated Tuna within the placenta, we used in situ hybridization as well as placental layer-specific RNA isolation and analysis. Both methods confirmed the absence of Tuna expression in control samples and determined that Cd-induced Tuna expression is specific to the junctional zone. Since many lncRNAs regulate gene expression, we hypothesized that Tuna forms part of the mechanism of Cd-induced transcriptomic changes. To test this, we over-expressed Tuna in cultured choriocarcinoma cells and compared gene expression profiles to those of control and CdCl2 exposed cells. We demonstrate significant overlap between genes activated by Tuna overexpression and genes activated by CdCl2 exposure, with enrichment in the NRF2-mediated oxidative stress response. Herein we analyze the NRF2 pathway and show that Tuna increases NRF2/NRF2 both at the transcript and protein levels. Tuna drives increased NRF2 target gene expression, a result that is abrogated with the use of an NRF2 inhibitor, confirming that Tuna activates oxidative stress response genes through this pathway. This work identifies the lncRNA Tuna as a potential novel player in Cd-induced placental insufficiency.

In vitro study of the effects of DC electric fields on cell activities and gene expression in human choriocarcinoma cells

ABSTRACT Physiological electric fields (EFs), as one of the environmental cues influencing both normal and tumor cells, have profound effects on tumor cell malignancy potential. The cellular responses to EFs by choriocarcinoma cells and their underlying mechanisms are unknown. In this study, the migration/motility, cell cycle progression and proliferation of choriocarcinoma cells in electric field culture showed that choriocarcinoma cells migrated cathodally in an applied EF, and EF stimulation influenced cell cycle progression through G2/M arrest and therefore induced a reduction in cellular proliferation. The transcriptome of choriocarcinoma cells subjected to EF stimulation (150 mV/mm) was analyzed using RNA sequencing (RNA-Seq), and the results were verified by reverse transcription quantitative polymerase chain reaction. A Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that ErbB and HIF-1 signaling pathways that are involved in cell migration/motility, cell cycle progression and proliferation were significantly altered in cells treated with an EF of 150 mV/mm compared with control cells, and in addition, the downstream pathways of these signaling pathways such as AKT and P42/P44 MAPK (ERK1/2) showed primary activation by Western blotting. This study’s results suggest that an applied EF is an effective cue in regulating cellular phenotypes of choriocarcinoma cells and that transcriptional analysis contributes to the understanding of the mechanism of EF-guided cell functions.

Suppression of choriocarcinoma invasion and metastasis following blockade of BDNF/TrkB signaling

Brain-derived neurotrophic factor (BDNF) acts through its cognate receptor tyrosine kinase-B (TrkB) to regulate diverse physiological functions in reproductive and other tissues. In normal and malignant trophoblastic cells, the BDNF/TrkB signaling promotes cell growth. Due to the highly malignant nature of choriocarcinoma, we investigated possible involvement of this system in choriocarcinoma cell invasion and metastasis. We demonstrated that treatment of cultured choriocarcinoma cells, known to express both BDNF and TrkB, with a soluble TrkB ectodomain or a Trk receptor inhibitor K252a suppressed cell invasion accompanied with decreased expression of matrix metalloproteinase-2, a cell invasion marker. In vivo studies using a tumor xenograft model in athymic nude mice further showed inhibition of cell invasion from tumors to surrounding tissues following the suppression of endogenous TrkB signaling. For an in vivo model of choriocarcinoma metastasis, we performed intravenous injections of JAR cells expressing firefly luciferase into severe combined immunodeficiency (SCID) mice. Treatment with K252a inhibited metastasis of tumors to distant organs. In vivo K252a treatment also suppressed metastatic tumor growth as reflected by decreased cell proliferation and increased apoptosis and caspases-3/7 activities, together with reduced tissue levels of a tumor marker, human chorionic gonadotropin-β. In vivo suppression of TrkB signaling also led to decreased expression of angiogenic markers in metastatic tumor, including cluster of differentiation 31 and vascular endothelial growth factor A. Our findings suggested essential autocrine/paracrine roles of the BDNF/TrkB signaling system in choriocarcinoma invasion and metastasis. Inhibition of this signaling could serve as the basis to develop a novel therapy for patients with choriocarcinoma.

Effects of intermittent high glucose on BeWo choriocarcinoma cells in culture

The aim of this study was to investigate the cellular effects of intermittent high glucose on the human BeWo placental choriocarcinoma cell line, used as a model of the effects of glucose fluctuation in diabetic pregnancies. BeWo cells were subjected to three different glucose conditions for 48 h: 7 mmol/L (control), 42 mmol/L (high glucose), or 7 and 42 mmol/L glucose (intermittent, alternated every 6 h). Cell viability was assessed using cell counts, a cell proliferation assay, and a cell viability assay. Apoptosis was also studied using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and by immunocytochemistry of fractin, the N-terminal fragment of actin, which can distinguish apoptotic from necrotic cells. Furthermore, the expression of the well-known survival factors of trophoblast cells, heparin-binding epidermal growth factor-like growth factor and leptin, was evaluated by real-time polymerase chain reaction and Western blot analyses. Intermittent high-glucose conditions significantly decreased cell viability and enhanced apoptosis compared with control or continuous high-glucose conditions. Furthermore, the expression of heparin-binding epidermal growth factor-like growth factor, but not that of leptin, was significantly increased under intermittent high-glucose conditions compared to its expression under either control or continuous high-glucose conditions. These data indicate that intermittent high glucose is more deleterious to BeWo cells than continuous high-glucose conditions. Although further in vitro and in vivo study is necessary, excess fluctuation of glucose levels in the placental circulation might be involved in the impairment of placental development leading to the placental dysfunction.

Effects of anti‐β2‐glycoprotein I antibody on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cell line

Anti-β2-glycoprotein I (anti-β2-GPI) antibody has been detected in cases of recurrent abortion and intrauterine death. Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are growth factors that accelerate villous proliferation. Soluble VEGF receptor-1 (sVEGFR1) is a soluble receptor for PlGF and suppresses the function of PlGF. In order to study the pathological mechanism of anti-β2-GPI antibody, we evaluated the effects of anti-β2-GPI antibody on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cells. The sera were taken from six anti-β2-GPI antibody-positive and six anti-β2-GPI antibody-negative patients with recurrent miscarriages. Choriocarcinoma cells (JEG-3) were cultured in 24-well plates. After each serum and isolated immunoglobulin G (IgG) were added to the culture medium, the PlGF, VEGF and sVEGFR1 in the culture medium were measured by ELISA 24 h later. When the isolated IgG was added to culture medium, only the levels of PlGF were measured. The anti-β2-GPI antibody-positive sera and isolated IgG significantly suppressed the production of PlGF from JEG-3 cells more strongly than the anti-β2-GPI antibody-negative sera. The suppressive effects were not changed by serum inactivation. There was no significant difference in the values of the XTT assay and the production of VEGF and sVEGFR1 between the antibody-positive and antibody-negative sera. Anti-β2-GPI antibody-positive sera and IgG suppress the production of PlGF from JEG-3 cells. We suggest that the anti-β2-GPI antibodies may suppress PlGF production from trophoblasts and cause the failure of placenta formation and function.

Abstract 1596: Human choriocarcinomas: Placental growth factor-dependent preclinical tumor models

Abstract Choriocarcinomas are an aggressive form of cancer that develops in the uterus from tissue that would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and the tumors are highly angiogenic. Human placental growth factor (hPlGF) is an angiogenic growth factor that promotes the growth of blood vessels in the placenta. hPlGF levels can be higher under pathological conditions such as cancer. Some human tumor specimens have higher PlGF levels than normal tissue, thus hPlGF is a target for anti-angiogenic therapy. The human choriocarcinoma cell lines BeWo, JAr, and JEG-3 were evaluated as preclinical models of hPlGF overexpression. The levels of hVEGF-A and hPlGF secreted by the choriocarcinomas and 11 human and mouse cancer cell lines in culture were measured by ELISA. The choriocarcinomas secreted higher levels of hPlGF and lower levels of hVEGF-A than 10/11 other cancer cell lines. The hPlGF serum levels in mice bearing subcutaneous choriocarcinoma tumors correlated with tumor volume and reached >2,000 pg/ml. By comparison, no hPlGF was detected in the serum of mice bearing H460 non-small lung carcinoma, HT29 colon carcinoma or MOLM-13 acute myeloid leukemia xenograft tumors. Although choriocarcinoma cells secreted both hPlGF and hVEGF-A in culture, serum levels of hVEGF-A in mice bearing choriocarcinoma xenografts were undetectable or <50 pg/ml at the time points serum was collected indicating that angiogenic factor secretion by the cells in culture was not the same in vivo. JEG-3 tumors grew faster than JAr tumors following the subcutaneous implantantion of 1×106 cells into nude mice. BeWo tumors grew the slowest and had the highest serum PlGF levels. Immunohistochemical methods confirmed expression of hPlGF in the choriocarcinoma xenografts. Studies involving double immunofluorescence on the xenograft tumors are ongoing to investigate endothelial cell and pericyte interactions and stromal involvement in the tumors. sFLT01, a novel fusion protein that neutralizes PlGF and VEGF-A, was added to choriocarcinoma cells in culture and administered to mice bearing choriocarcinoma xenografts in efficacy studies. Exposure of JAr and JEG-3 cells in culture to sFLT01 resulted in a phenotypic change whereby the cells produced less hPlGF and no significant hVEGF-A. In vivo, sFLT01 (25 mg/kg, IP injection, 2x per week) slowed tumor growth in JAr and JEG-3 tumors and increased median survival by 5-30 days compared to vehicle controls. These results indicate that human choriocarcinoma xenograft models are a valuable research tool for evaluating anti-angiogenic agents that target hPlGF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1596. doi:10.1158/1538-7445.AM2011-1596

P114 Effect of preeclampsia sera on mRNA expression of soluble Endoglin and HO-1 from cultured choriocarcinoma cells

P114 Effect of preeclampsia sera on mRNA expression of soluble Endoglin and HO-1 from cultured choriocarcinoma cells

Inhibition of Brain-Derived Neurotrophic Factor/Tyrosine Kinase B Signaling Suppresses Choriocarcinoma Cell Growth

Brain-derived neurotrophic factor (BDNF) signals through its receptor tyrosine kinase (Trk)B to regulate the development trophoblast cells during peri- and postimplantation periods. Possible involvement of this signaling system in malignant human trophoblastic cell growth has not been investigated. Here, we found the expression of BDNF and neurtropin-4/5 together with TrkB in human trophoblastic choriocarcinoma cells. Treatment of cultured choriocarcinoma cells with a soluble TrkB ectodomain or a Trk receptor inhibitor K252a suppressed cell proliferation and increased apoptosis associated by the disruption of mitochondrial functions, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using these specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase and ERK pathways in mediating BDNF actions. Based on PCR array analyses to identify changes in expression profiles of cell cycle- and apoptosis-related genes in cultured choriocarcinoma cells, we found that suppression of endogenous TrkB signaling led to decreases in key proproliferation cell cycle genes and increases in two inhibitory cell cycle genes together with the up-regulation of several proapoptotic genes. In vivo studies in athymic nude mice bearing choriocarcinoma cell tumors further demonstrated that treatment with K252a, but not K252b, suppressed tumor growth accompanied by decreased cell proliferation, reduced levels of a tumor marker, human chorionic gonadotropin-beta, and increased levels of apoptosis and caspase-3/7 activities. Thus, autocrine signaling of the BDNF/TrkB system is important for human choriocarcinoma cell growth, and inhibition of BDNF/TrkB signaling in these cells could provide a novel therapy for patients with choriocarcinoma.

Resistance to Fas-mediated cell death in BeWo and NJG choriocarcinoma cell lines: implications in immune privilege

Objective An immune privileged site occurs when the allogenic tissue grafts have the propensity for prolonged survival in the host tissue. In this context, the survival and proliferation of malignant trophoblasts in the gravid uterus are currently unclear. In a previous study, we documented that Fas and FasL are coexpressed in choriocarcinoma [Gynecol. Oncol. (2003)]. This study was conducted to examine the role of the Fas/FasL pathway in immune privilege of BeWo and NJG choriocarcinoma cells in culture. Methods The ability of anti-Fas mAb (CH-11) to sensitize choriocarcinoma cell lines to Fas-mediated cytotoxicity was assessed by MTT assays. Coculture experiments with Fas-sensitive Jurkat cells were used to demonstrate functional FasL from choriocarcinoma. RT-PCR was used to assess the expression of cFLIP. Results The mean cell viability of BeWo and NJG cells declined to about 58 and 63% compared to controls after 72 h of culture in the presence of anti-Fas mAb (CH-11) while the Fas-sensitive Jurkat cells showed viability of only 10%. This resistance to Fas-mediated apoptosis in choriocarcinoma cells is reversed in the presence of cycloheximide (0.5 μg/ml) which further decreased the viability to 36 and 32%, respectively, at a dose of 300 ng/ml ( P < 0.05). The observed resistance to Fas-mediated apoptosis therefore could be attributed to the short-lived endogenous inhibitor, cFLIP as demonstrated by the RT-PCR technique. In coculture experiments, FasL from choriocarcinoma cells induced apoptosis in the Fas-sensitive Jurkat cells, thereby indicating the capacity to evade immune attack. Conclusion Decreased sensitivity to Fas-mediated apoptosis and counterattacking the lymphocytes may impart immune privilege in these malignant trophoblasts for prolonged survival in the host.

Formation and degradation of dihydrotestosterone by recombinant members of the rat 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4 isomerase family

The structures of cDNA clones encoding four members of the rat 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4 isomerase (3β-HSD) family were characterized. The rat type I, type II and the novel type IV are genuine NAD +/H-dependent 3β-HSD isoenzymes. On the other hand, the liver-specific type III protein is a specific 3-keto-reductase (3-KSR) that catalyzes the conversion of 5α-androstane-3-one-17β-ol (DHT) and 5α-androstane 3,17-dione (A-dione) into their 3β-hydroxy metabolites. The aim of the present study was to further characterize the enzymatic properties of rat types I, III and IV, especially their role in the formation and degradation of DHT after transient expression in intact human HeLa cervical carcinoma, JEG-3 choriocarcinoma or SW-13 adrenal cortex adenocarcinoma cells in culture. The expressed type III 3-KSR in intact HeLa cells catalyzed the reduction of DHT into 3β-diol, whereas expression of type I 3β-HSD in these cell lines had no significant effect on the basal conversion of DHT into 3β-diol, but it did increase the formation of DHT from 3β-diol. A-dione is the predominant product obtained when DHT and 5α-androstane-3β,17β-diol (3β-diol) are used as substrates in intact JEG-3 and SW-13 cells transfected with rat type I 3β-HSD. Furthermore, this predominant 17β-HSD activity was also observed in SW-13 cells transfected with the novel rat type IV 3β-HSD. The predominance of this ‘secondary’ 17β-HSD activity is also reflected in HeLa cells transfected with type I 3β-HSD by the deduced predominant pathway 3β-diol → DHT → 5α-androstane- 3α, 17β-diol (3α-diol) → androsterone (ADT), in which formation of 3α-diol was due to endogenous 3α-HSD activity of HeLa cells, whereas the other reactions are catalyzed by the type I 3β-HSD isoenzyme. This observation thus demonstrates that rat type I 3β-HSD may also catalyze the conversion of 3α-diol into ADT through its intrinsic 17β-HSD activity. The predominant metabolic pathways observed in the present study could be attributed to preponderant bioavailability of NAD + and NADPH in the intact transfected cells used.

Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells

Cytochrome P-450 scc ( p450 scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450 scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12- O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450 scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450 scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450 scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450 scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450 scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-1) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450 scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3′, 5′-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450 scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.

Effects of adenine nucleotides on choriogonadotropin α and β subunit synthesis

The α subunit of human chorionic gonadotropin (CG) contains a discrete cAMP response element in the 5′ flanking region of the gene. Since cAMP also stimulates the synthesis of the CGβ subunit the presence of a cAMP cis element in the CGβ gene was examined. Deletion mutants bearing various lengths of CGβ 5′ region in front of the chloramphenicol acetyl transferase (CAT) gene were transfected in placental tumor cells. No discrete cAMP response element could be identified. Unexpectedly we also observed that AMP and adenosine not only stimulated CAT activity driven by CGβ promoter sequences but also enhanced synthesis of CGα and β subunits in cultured choriocarcinoma cells. GMP, CMP, guanosine, and cytosine were inactive at comparable concentrations. These data suggest that the response of the CGα and β genes to the non-cyclic adenine derivatives occurs by a mechanism that differs from cAMP.

Insulin-Like Growth Factor (IGF) Binding Protein from Human Decidua Inhibits the Binding and Biological Action of IGF-I in Cultured Choriocarcinoma Cells**

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells. We conclude that decidual 34 K IGF-BP inhibits the cellular binding and biological action of IGFs in JEG-3 cells. Our data show that JEG-3 cells represent a cell type that can produce IGF, but not IGF-BPs. These cells may thus provide a useful model system for a better understanding of autocrine growth regulation mediated by the IGFs.

Mechanism of methotrexate-sensitivity of choriocarcinoma cells in culture.

Four cell lines established from choriocarcinoma were compared for sensitivity to methotrexate (MTX). In this paper, we have compared the relative gene copy number of dihydrofolate reductase (DHFR), the target enzyme of methotrexate (MTX), in order to clarify whether or not amplification of the gene is involved in the relative resistance to MTX observed for one of the cell lines, designated NaUCC‐1, which is 4‐ to 5‐fold more resistant to MTX as compared with the other cell lines and exhibits a reduced uptake of [3H]MTX. Neither dot blot nor Southern blot hybridization revealed any significant difference in the gene copy number among the four cell lines. Therefore, the resistance to MTX of the NaUCC‐1 line is explained by a reduced uptake of the drug, rather than amplification of the target gene.

Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors.

The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, we examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia.

Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast

Estrogen synthetase (aromatase) is present in large amounts in human term placenta. However, the localization of aromatase within the cellular structure of the placental villus is obscure. By immunocytochemical techniques using antibodies that separately recognize each component of the aromatase cytochrome P-450 enzyme system, the fraction of term placental trophoblast cells in primary culture expressing each aromatase component antigen increased from 20% in fresh mononucleated cells to about 65% for multinucleated giant cells after 72 h. In contrast, about 80% of human choriocarcinoma cells in continuous culture (JAr line) expressed each aromatase component antigen. The fraction of trophoblast cells in primary culture containing human chorionic gonadotropin increased from about 14% in fresh mononucleated cells to about 45% after 72 h and was about 30% in the choriocarcinoma cells. Fibroblast cells in culture, derived from trypsin-treated placental villi, contained aromatase activity, albeit much lower than term placental trophoblast cells. Aromatase specific activity in these placental fibroblasts did not change following growth with dibutyryl cAMP plus theophylline for 72 h.

Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.

Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells.

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.

Immunobiology of complete molar pregnancy and gestational trophoblastic tumor.

The unique curability of gestational trophoblastic tumors may in part be attributable to a host immunologic response. The occurrence of rapidly progressive and fatal choriocarcinoma may be favored by histocompatibility between patients and their partners. However, histocompatibility is not a prerequisite for the development and persistence of gestational choriocarcinoma. The expression of HLA by choriocarcinoma cells in culture is enhanced following incubation with gamma-interferon and this may be of both biologic and clinical significance. Complete molar pregnancy is a complete allograft because all molar chromosomes are of paternal origin. Patients with complete mole are sensitized to paternal HLA antigen which is expressed in molar tissue. Other polymorphic antigen systems including trophoblast-leukocyte common antigens and placental-type alkaline phosphatase are also expressed in molar tissue. We have studied the immunopathology of the molar implantation site to investigate possible humoral and cellular immune responses. The relationships among normal placenta, complete mole and choriocarcinoma are not clearly understood. The pattern of expression of oncofetal antigens in these three gestational tissues may be used to assess trophoblastic differentiation. In studies to date, molar trophoblast has the same pattern of expression of oncofetal antigens as normal placental trophoblast. We will review recent advances in our understanding of the immunobiology of gestational trophoblastic disease and suggest new directions for further research.

Purification, from cultured human choriocarcinoma cells, of a 75000-Mr protein reacting with antibodies to a synthetic peptide based on a cloned human endogenous provirus nucleotide sequence

We previously detected in cultured choriocarcinoma cells a 75000-Mr polypeptide defined by immunoblotting with antibody to a synthetic peptide Sp23 (Cys-Glu-Asn-Pro-Ser-Gln-Phe-Tyr-Glu-Asp-Leu) based on a cloned human endogenous proviral nucleotide sequence. On immunohistological staining, anti-Sp23 stains antigen(s) in the syncytiotrophoblasts of first-trimester placentas and in renal-cell adenocarcinoma tissues. The present report describes purification to homogeneity of the protein from cultured choriocarcinoma cells. The procedure involves extraction with non-ionic detergent and h.p.l.c. using, sequentially, gel-permeation, anion-exchange and reverse-phase columns. The yield was 110 micrograms/g of total choriocarcinoma-cell protein. The results indicate that the purified protein is a monomeric and relatively hydrophilic molecule of Mr 75000.