epidermal-growth-factor and Choriocarcinoma

Malignant germ-cell tumours arise from a neoplastic precursor, the carcinoma in situ, and develop into seminomas and/or non-seminomas (embryonal carcinomas, teratomas, yolk-sac tumours and choriocarcinomas). Based on histological and clinical findings, it has been postulated that seminomas can eventually transform into non-seminomas. Here, we used the cell line TCam-2 as model for seminomas and interrogated their differentiation potential. We demonstrate that TCam-2 cells are able to differentiate into mixed non-seminomatous lineages after supplementing the media with TGF-β1, EGF and FGF4. On a molecular level, the differentiation is initiated by repression of BMP/SMAD signalling. As a consequence, BLIMP1, a molecule known to inhibit the differentiation of murine primordial germ cells, is down-regulated and differentiation-inhibiting histone modifications are lost. The appearance of multinucleated giant cells and the expression of marker genes indicate that cells differentiate predominantly into extra-embryonic choriocarcinoma-like cells. This is most likely due to the presence of components of the Hippo pathway, TEAD4 and YAP1. These molecules have been described to trigger extra-embryonic fate determination in the murine system. This study supports the model that seminomas indeed have an intrinsic ability to transform into a non-seminoma. In addition, the data suggest that the transformation does not require an additional mutation, but can be triggered by changes in the tumour microenvironment.

Topics: Adaptor Proteins, Signal Transducing; Biomarkers; Bone Morphogenetic Protein Receptors; Cell Differentiation; Cell Line, Tumor; Choriocarcinoma; DNA-Binding Proteins; Epidermal Growth Factor; Fibroblast Growth Factor 4; Giant Cells; Histones; Humans; Male; Muscle Proteins; Neoplasms, Germ Cell and Embryonal; Polymerase Chain Reaction; Positive Regulatory Domain I-Binding Factor 1; Repressor Proteins; Seminoma; Signal Transduction; Smad Proteins; TEA Domain Transcription Factors; Testicular Neoplasms; Transcription Factors; Transforming Growth Factor beta1; Tumor Microenvironment

Human type II deiodinase is a master regulator of thyroid hormone activation in several tissues. In placenta, type II deiodinase mRNA levels and enzymatic activity are elevated only during the first trimester of pregnancy and then progressively decline. During this early stage, mitogens such as epidermal growth factor (EGF) have been shown to promote the proliferation of the trophoblast by acting through multiple mechanisms. Here we show that EGF modulates transcription of human type II deiodinase gene (Dio2) through distinct signaling pathways, leading to the assembly of a heterogeneous transcription factor complex. Gene expression and deiodination assays have shown that EGF promptly induces a short-lived Dio2 mRNA and enzymatic activity. The induction is mediated by ERK and p38 kinases, as demonstrated by selective inhibition or overexpression of different mitogen-activated kinases. Reporter assays of mutant constructs indicate that EGF-induced transcriptional activity on Dio2 promoter is mediated by the cAMP response element (CRE) and does not involve the activating protein 1 site. With functional and biochemical approaches, we have demonstrated that the EGF stimulation culminates with the assembly and recruitment over the Dio2 CRE of a composite complex, which consists of c-Jun, c-Fos, and CRE-binding protein. These results further support the hypothesis that placental iodothyronine metabolism is critical during early pregnancy.

Topics: Cell Line, Tumor; Choriocarcinoma; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Humans; Iodide Peroxidase; Iodothyronine Deiodinase Type II; JNK Mitogen-Activated Protein Kinases; Placenta; Pregnancy; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Thyroid Hormones; Transcription, Genetic; Uterine Neoplasms

The development of the human placenta involves a complex process of tightly regulated proliferation and invasion by extravillous trophoblast into the uterine decidua. Inadequate placentation is a feature of intrauterine growth restriction and other gestational pathology. There is some evidence that T(3) plays a role in the regulation of these processes and that T(3) may act synergistically with epidermal growth factor (EGF). The aim of this study was to define the expression of thyroid hormone receptors in extravillous trophoblast, elucidate the effects of T(3) on both proliferation and differentiation of human trophoblast cells of varying origins, and define the potential interaction between EGF and T(3) on these processes. Using immunohistochemistry, specific thyroid hormone receptor isoforms were localized in extravillous trophoblast in first- and second-trimester placental bed biopsies, indicating potential sensitivity to T(3). In studies of human trophoblast-derived cell lines and primary cultures of cytotrophoblast cells in vitro, T(3) and EGF exerted an antiproliferative effect on an extravillous-like cell line (SGHPL-4) but stimulated proliferation in JEG-3 choriocarcinoma cells. EGF enhanced survival of nonproliferative term primary cytotrophoblast cells and significantly enhanced invasion of fibrin gels by SGHPL-4 cells, an effect attenuated by T(3). Both T(3) and EGF also significantly enhanced SGHPL-4 motility. These results suggest that EGF and T(3) may act synergistically to regulate both proliferation and differentiated function of human trophoblast.

Topics: Cell Division; Cell Line, Transformed; Cell Movement; Choriocarcinoma; Epidermal Growth Factor; Female; Fibrin; Gels; Humans; In Vitro Techniques; Neoplasm Invasiveness; Pregnancy; Thyroid Hormone Receptors alpha; Thyroid Hormone Receptors beta; Triiodothyronine; Trophoblasts; Uterine Neoplasms

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.. 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.. 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.. 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.

Topics: Calcitriol; Cell Division; Cell Line, Tumor; Choriocarcinoma; Dexamethasone; Down-Regulation; Epidermal Growth Factor; Estradiol; Female; Humans; Parathyroid Hormone; Pregnancy; Pregnancy Complications, Neoplastic; Receptor, Parathyroid Hormone, Type 1; Tumor Cells, Cultured; Up-Regulation; Uterine Neoplasms

The aim of this study was to examine the invasiveness of first trimester trophoblasts according to the secretion profile of MMP-2 and -9 at different gestational stages, and to test the similarity between primary trophoblast cell-culture and the JAR choriocarcinoma cell-line.. First trimester trophoblasts were divided into two groups: 6-8 weeks (early) and 9-12 w (late) of gestation. The two trophoblast groups and JAR cells were cultured in medium, with various concentrations of forskolin and Epidermal Growth Factor (EGF). Proteolytic activity was detected by zymography and invasiveness was assessed by Matrigel invasion assay. Student's T-test was used for statistical analysis.. In 6-8 w trophoblast, proMMP-2 was only slightly dominant over proMMP-9 (53.2% vs. 46.8% respectively), whereas in 9-12 w, proMMP-9 was clearly dominant over proMMP-2 (61.7% vs.38.3% respectively). In JAR cells proMMP-2 was strongly dominant (90.2% vs.9.8% respectively). In JAR cells forskolin significantly increased proMMP-2 and -9 secretion (128.5% +/- 12 and 183.2% +/- 27.9 of control, respectively). EGF had a dual effect on JAR cells: at 8 ng/ml both proMMP-2 and -9 were increased (133.5% +/-15 and 223.9% +/- 32.4 of control, respectively) while at 80 ng/ml both proMMP-2 and -9 were decreased (65.1% +/- 18.3 and 66.6% +/- 37 of control, respectively). Forskolin significantly increased both proMMP-2 and -9 secretion in 6-8 w and 9-12 w trophoblasts (125.9% +/- 6.3,128.4% +/- 6.4; 169.7% +/- 20.3, 120.3% +/- 4.5 of control, respectively). EGF also significantly increased both proMMP-2 and -9 secretion in 6-8 w and 9-12 w trophoblasts (141.22% +/- 14.8, 138.8% +/- 10.3; 168.3% +/- 18.2, 117.3 +/- 3.8 of control, respectively). Both forskolin and EGF increased trophoblast cells invasiveness in all groups. The invasive ability of trophoblast cells, induced by forskolin, was reduced by MMP-2 antibody in: JAR cells, 6-8 w and 9-12 w trophoblasts. Likewise trophoblast invasion induced by EGF was reduced by MMP-2 antibody in all groups. However the invasive ability induced by forskolin or EGF was inhibited by MMP-9 antibody only in trophoblasts from 9-12 w.. First trimester trophoblasts express differential gelatinase secretion profile according to the gestational week. In JAR and early trophoblasts (6-8 w) MMP-2 is the main gelatinase and the key enzyme in trophoblast invasion. Thereafter in late first trimester trophoblasts (9-12 w), both MMP-2 and -9 participate in trophoblast invasion.

Topics: Cell Line, Tumor; Cells, Cultured; Choriocarcinoma; Colforsin; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Placenta; Pregnancy; Pregnancy Trimester, First; Trophoblasts

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.

Topics: Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Chemotaxis; Choriocarcinoma; Collagen; Epidermal Growth Factor; Female; Humans; Integrin alpha2; Neoplasm Invasiveness; RNA, Messenger; Uterine Neoplasms

Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotein alpha subunit gene expression by EGF. Using 5' deletion mutagenesis, we have delineated the primary EGF/PKC responsive region of the equine alpha subunit gene to be located between -2039 to -2032 base pairs upstream of the transcriptional start site. The sequence within this region contains an activator protein 1 (AP-1)-like response element (TGAATCA) and is similar to a consensus AP-1 (TGAC/GTCA) response element. This element appeared to preferentially interact with a c-fos/JunD heterodimer. Stimulation by EGF induced the binding of c-fos and JunD to this element and subsequently elevated promoter activity. In conclusion, an EGF/PKC/MAPK signal transduction pathway regulates equine glycoprotein alpha subunit gene expression through a distinct regulatory element(s) that lies between -2039 to -2032 of the equine glycoprotein alpha subunit promoter in trophoblasts and involves an AP-1 complex.

Topics: Animals; Base Sequence; Binding Sites; Choriocarcinoma; DNA; Epidermal Growth Factor; Gene Expression Regulation; Glycoprotein Hormones, alpha Subunit; Horses; Humans; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Kinase C; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-raf; Response Elements; Signal Transduction; Transcription Factor AP-1; Transfection; Trophoblasts; Tumor Cells, Cultured

The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone alpha subunit gene, a subunit of chorionic gonadotropin. Studies examined the effects of EGF and the adenylate cyclase activator forskolin on the expression of a transfected alpha subunit reporter gene in a human choriocarcinoma cell line (JEG3). At maximal doses, administration of EGF resulted in a 50% increase in a subunit reporter activity; forskolin administration induced a fivefold activation; the combined actions of EGF and forskolin resulted in synergistic activation (greater than eightfold) of the alpha subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to mediate EGF-forskolin-induced synergistic activation. The combined actions of EGF and forskolin resulted in potentiated activation of extracellular signal-regulated kinase (ERK) enzyme activity compared with EGF alone. Specific blockade of ERK activation was sufficient to block EGF-forskolin-induced synergistic activation of the alpha subunit reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated protein kinase inhibitor did not influence activation of the alpha reporter. However, overexpression of c-Jun N-terminal kinase (JNK)-interacting protein 1 as a dominant interfering molecule abolished the synergistic effects of EGF and forskolin on the alpha subunit reporter. CRE binding studies suggested that the CRE complex consisted of CRE binding protein and EGF-ERK-dependent recruitment of c-Jun-c-Fos (AP-1) to the CRE. A dominant negative form of c-Fos (A-Fos) that specifically disrupts c-Jun-c-Fos DNA binding inhibited synergistic activation of the alpha subunit. Thus, synergistic activation of the alpha subunit gene induced by EGF-forskolin requires the ERK and JNK cascades and the recruitment of AP-1 to the CRE binding complex.

Topics: Choriocarcinoma; Chorionic Gonadotropin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Synergism; Enzyme Activation; Epidermal Growth Factor; Genes, Reporter; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mutagenesis; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Response Elements; Transcription Factor AP-1; Transcriptional Activation; Tumor Cells, Cultured

The treatment of highly purified nuclear membranes isolated from JEG-3 human choriocarcinoma cells with epidermal growth factor (EGF) resulted in an increase of the receptor autophosphorylation and the phosphorylation of several other proteins, including 44-kDa, 34-kDa and 24-kDa proteins, and a decrease in the phosphorylation of a 40-kDa protein. The kinetics of phosphorylation and the use of RG-13022, a selective inhibitor of EGF-receptor kinase activity, suggested that receptor activation was necessary for the phosphorylation response of the other proteins. Tyr was exclusively phosphorylated in the EGF receptor and 24-kDa proteins, Tyr and Thr were phosphorylated in the 44-kDa protein, and Ser was phosphorylated in the 34-kDa protein and dephosphorylated in the 40-kDa protein. The molecular size, Thr/Tyr phosphorylation, immunoprecipitation and enzymatic activity towards myelin basic protein suggested that the 44-kDa protein was a mitogen-activated protein (MAP) kinase. The EGF treatment not only increased phosphorylation but also catalytic activity of MAP kinase and these increases were prevented by the addition of RG-13022. In summary, this report demonstrates that target cell nuclei contain EGF receptors, which use the MAP kinase signaling pathway.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cell Fractionation; Choriocarcinoma; Epidermal Growth Factor; Humans; Kinetics; Membrane Proteins; Molecular Weight; Nuclear Envelope; Phosphorylation; Tumor Cells, Cultured

Pregnancy in the rat and mouse is characterized by a developmental switch in expression at midgestation between the related placental lactogens I and II (PLI, PLII) suggesting that these proteins have important but different roles. The factors that control this differential gene expression are poorly understood. In this paper we describe experiments which investigate the effects of EGF and TGFalpha on rPLI and rPLII mRNA levels in the rat choriocarcinoma cell line, Rcho. This cell line has been widely used as a model system for studying genes expressed in the rodent placental giant cell. We find that in these cultures both growth factors produce a two fold increase in endogenous rPLI mRNA levels, and a two fold decrease in rPLII mRNA levels. Using DNA transfection assays we have tested the effects of TGFalpha on the expression of hybrid rPLI and rPLII 5'flanking/luciferase reporter constructs in Rcho cells. The transfected rPLI/luciferase reporter constructs produce a similar two fold increase in reporter expression to that seen for the endogenous mRNA, suggesting that EGF/TGFalpha positively regulate rPLI mRNA levels by effects on gene transcription. Unlike the endogenous gene, however, the rPLII/ luciferase constructs show a five fold increase in rPLII-directed luciferase expression in the presence of TGFalpha. The effect of EGF/TGFalpha on placental rPLII mRNA levels appears to involve negative response element(s) located elsewhere in the gene to those regions tested.

Topics: Animals; Choriocarcinoma; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Luciferases; Placental Lactogen; Rats; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

In order to elucidate the regulation of placental growth, we have characterized the expression of proliferating cell nuclear antigen (PCNA), apoptotic DNA fragmentation and bcl-2 protein in human placenta during pregnancy. PCNA and bcl-2 protein expression were examined by immunohistochemical techniques, while the occurrence of apoptotic DNA fragmentation was assessed by in situ analysis of DNA 3'-end labeling method. Both PCNA expression and apoptotic DNA fragmentation were found in cytotrophoblasts (C-cells), being most abundant in early placenta, less abundant in midterm placenta and least abundant in term placenta. In contrast, bcl-2 protein expression was found in syncytiotrophoblasts (S-cells), being least abundant in early placenta, less abundant in midterm placenta and most abundant in term placenta. These data indicate that early placenta is characterized by the highly proliferative activity of C-cells associated with the increased occurrence of apoptosis, whereas term placenta is characterized by the abundant expression of bcl-2 protein in S-cells. Furthermore, effects of EGF on the proliferative activity and differentiated function of trophoblasts were investigated using an organ culture system. Explants of trophoblastic tissues were cultured with or without EGF, in the presence or absence of 10(-8)M triiodo-L-thyronine (T3) in a serum-free condition. In 4-5 week placentas, EGF and EGF receptor were almost exclusively localized in C-cells, and EGF augmented the proliferative activity of C-cells without affecting the ability to secrete hCG and hPL. By contrast, in 6-12 week placentas, EGF and EGF receptor were predominantly localized in S-cells, and EGF stimulated hCG and hPL secretion without affecting the proliferative activity of C-cess. The addition of T3 (10(-8)M) resulted in an increased secretion of immunoreactive EGF by cultured placental explants. These findings suggest that EGF acts as a local factor in regulating early placental growth and function in synergy with thyroid hormone. On the other hand, progesterone selectively inhibited pleise hCG (alpha, beta) mRNAs expression and decreased hCG secretion in normal placental tissues, whereas choriocarcinoma did not respond to progesterone. This suggests that inhibitory regulation of hCG synthesis in choriocarcinoma is different from normal placenta. It was also found that in molar trophoblasts and choriocarcinoma cells PCNA expression was high, but both bcl-2 protein and apoptotic signal

Topics: Cell Differentiation; Cell Division; Choriocarcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hydatidiform Mole; Immunohistochemistry; In Vitro Techniques; Placenta; Pregnancy; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Trophoblasts; Uterine Neoplasms

Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.

Topics: 17-Hydroxysteroid Dehydrogenases; Breast Neoplasms; Choriocarcinoma; Cyclic AMP; Drug Synergism; Epidermal Growth Factor; Female; Humans; Isoenzymes; Pregnancy; RNA, Messenger; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin transporter expressed in JAR human placental choriocarcinoma cells was investigated. Treatment of the cells with ATA for 16 h led to a significant stimulation of the serotonin transporter activity. This effect was not observed, however, when the treatment was done for 1-2 h. The stimulatory effect was associated with an increase in the maximal velocity of the transport process with no significant change in the Michaelis-Menten constant. Northern blot hybridization revealed that ATA treatment caused a marked increase in the steady-state levels of serotonin transporter-specific transcripts. Treatment of the cells with ATA was found to increase tyrosine phosphorylation of a 180-kDa protein. The phosphotyrosine content of a protein of a similar molecular size increased dramatically when the cells were exposed to epidermal growth factor (EGF), suggesting that this protein may be the EGF receptor. Treatment of the cells with EGF for 24 h could reproduce the stimulatory effects of ATA on the serotonin transporter activity, the maximal velocity of the transport process, and the steady-state levels of the transporter-specific mRNAs. Genistein, a tyrosine kinase inhibitor, was able to block the stimulatory effect of ATA and EGF. It is concluded that EGF increases the serotonin transporter expression in JAR cells and that the neuroprotective compound ATA produces similar effects on the transporter most likely by activating the EGF receptor through tyrosine phosphorylation.

Topics: Aurintricarboxylic Acid; Biological Transport; Blotting, Northern; Carrier Proteins; Choriocarcinoma; Epidermal Growth Factor; Gene Expression; Genistein; Growth Inhibitors; Humans; Isoflavones; Kinetics; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Neuroprotective Agents; Phosphorylation; Placenta; RNA, Messenger; Serotonin Plasma Membrane Transport Proteins; Tumor Cells, Cultured; Tyrosine

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B(0) (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657-18661, 1996). The presence of the amino acid transport system B(0) (ATB(0)) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB(0) in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na(+)-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB(0). Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB(0) activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB(0) activity and on ATB(0) mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB(0) activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB(0) activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B(0) and that the expression of the system in these cells is stimulated by EGF.

Topics: Alanine; Amino Acid Transport Systems; Amino Acid Transport Systems, Basic; Amino Acid Transport Systems, Neutral; Aurintricarboxylic Acid; Biological Transport; Carrier Proteins; Choriocarcinoma; Cloning, Molecular; Epidermal Growth Factor; ErbB Receptors; Female; Genistein; HeLa Cells; Humans; Isoflavones; Leucine; Phosphorylation; Placenta; Tumor Cells, Cultured; Tyrosine; Uterine Neoplasms

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Topics: Adolescent; Adult; Choriocarcinoma; Epidermal Growth Factor; Female; Humans; Hydatidiform Mole; Hydatidiform Mole, Invasive; Immunohistochemistry; Male; Middle Aged; Placenta; Pregnancy; Reference Values; Uterine Neoplasms

Expression of the ovine P-450 side-chain cleavage enzyme gene (CYP11A1) is stimulated by epidermal growth factor (EGF) through a pathway that involves c-Jun in JEG-3 placental cells. Growth factor signaling involves ras-dependent and ras-independent signaling pathways, which in turn regulate gene transcription through related but distinct mitogen-activated protein kinase pathways (MAPKs) including the extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs). We investigated the intracellular signaling pathways governing EGF induction of the CYP11A1 promoter. EGF stimulation of the CYP11A1 promoter (4-fold) was reduced 60% by a dominant negative mutant of ras (N17), and 30-40% by antisense ras. EGF induced both ERK and SAPK activity in JEG-3 cells. EGF-induced CYP11A1 promoter activity was reduced 60% by the MEK1 inhibitor PD098059 and 50% by a dominant negative mutant of the ERK-specific regulator MEK1. In contrast, dominant negative mutants of the SAPK-specific activator, SEK1, induced a further increase in EGF-induced CYP11A1 promoter activity. Constitutively active mutants of ras (V12 or L61) increased CYP11A1 promoter activity 6- to 8-fold. Deletion of the EGF response element (EGF-RE) between -92 and -77 bp reduced ras induction by 60%; however, a residual 3-fold induction remained through the proximal -77 bp. Mutation of the EGF-RE AP-1-like sequence in the context of the native promoter reduced CYP11A1 promoter activation by ras 60%. The EGF-RE sequence was sufficient for 6-fold activation by ras in the context of an heterologous thymidine kinase promoter. Candidate transcription factor targets (c-Jun, c-Ets-2) for the ras-signaling cascade were examined for their effects on CYP11A1 promoter activity. Overexpression of c-Jun induced the CYP11A1 promoter through the EGF-RE; however, c-Ets-2 activation of the CYP11A1 promoter (12-fold) required the proximal ras-responsive promoter sequences that are distinct from the EGF/MEK/c-Jun-responsive element. Induction of the CYP11A1 promoter by EGF involves a ras/MEK1/AP-1-dependent pathway that is distinct from induction by ras/c-Ets-2.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cholesterol Side-Chain Cleavage Enzyme; Choriocarcinoma; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation; Genes, jun; Genes, ras; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins; Repressor Proteins; Sequence Deletion; Signal Transduction; Trans-Activators; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

We examined four choriocarcinoma cell lines, NaUCC-1, NaUCC-3, NaUCC-4 and BeWo, for the presence of epidermal growth factor (EGF) by enzyme immunoassay and reverse transcription and polymerase chain reaction, and for EGF receptor (EGFR) by 125I-EGF binding assay. Specific EGF binding and EGF proteins were detected in these four choriocarcinoma cell lines. On the cell lines examined, NaUCC-4 had the greatest EGF binding capacity (18 x 10(5) sites/cell) and the highest amount of immunoreactive EGF (142 pg/ml). These results prompted us to assess the significance of EGF/EGFR autocrine mechanism in NaUCC-4 cells. Low doses of exogenous EGF stimulated 3H-thymidine incorporation, and monoclonal antibodies against EGF or EGFR dose-dependently inhibited 3H-thymidine incorporation. On the other hand, these antibodies did not significantly affect hCG production. These results suggested that EGF might function in an autocrine manner to stimulate proliferation rather than differentiation of NaUCC-4 choriocarcinoma cells.

Topics: Adult; Cell Division; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hydatidiform Mole; Pregnancy; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Uterine Neoplasms

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

Topics: Blotting, Western; Cell Membrane; Cell Nucleus; Choriocarcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Immunosorbent Techniques; Microscopy, Immunoelectron; Nuclear Envelope; Phosphorylation; Phosphotyrosine; Placenta; Pregnancy; Receptors, LH; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured; Tyrosine

The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-alpha cDNA expression vector. The vector directs the synthesis of antisense hCG-alpha subunit mRNA which would then bind to sense hCG-alpha subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis. The transfection with pRSV-antisense hCG-alpha cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-alpha and -beta subunit mRNA levels. The decrease of hCG-beta subunit transcripts was unexpected and it was not due to contamination of antisense hCG-alpha cDNA construct with hCG-beta sequence. The transcription of hCG-alpha and -beta subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steady-state hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector.

Topics: Biomarkers, Tumor; Cell Division; Choriocarcinoma; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; DNA, Antisense; DNA, Complementary; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Glycoprotein Hormones, alpha Subunit; Humans; Neoplasm Proteins; Peptide Fragments; Receptors, LH; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Uterine Neoplasms

We examined whether epidermal growth factor (EGF), interleukin (IL)-1, IL-6 and cAMP analogs, which regulate mouse placental lactogen II secretion, affect rat placental lactogen (rPL)-II secretion using a rat choriocarcinoma cell line, Rcho-1. EGF, IL-1 and IL-6 did not affect rPL-II secretion, but 8-bromo-cAMP and forskolin inhibited rPL-II secretion by the 8th day of culture. The effects were dose dependent and the lowest concentrations of 8-bromo-cAMP and forskolin that significantly inhibited rPL-II secretion were 125 and 5 mumol/l, respectively. Cholera toxin also inhibited rPL-II secretion. The reverse hemolytic plaque assay for rPL-II indicated that the cells releasing rPL-II in the culture were giant cells and that 8-bromo-cAMP decreased the number of rPL-II-releasing cells. Western blot analysis of rPL-II yielded a single band at approximately 24.5 K, and 8-bromo-cAMP treatment significantly reduced the band intensity. Northern blot analysis of rPL-II indicated that 8-bromo-cAMP also reduced rPL-II gene expression. These findings suggest that the increase of intracellular cAMP accumulation results in inhibition of rPL-II secretion by decreasing rPL-II gene expression and inhibiting giant cell differentiation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blotting, Northern; Blotting, Western; Cholera Toxin; Choriocarcinoma; Colforsin; Cyclic AMP; Epidermal Growth Factor; Interleukin-1; Interleukin-6; Placental Lactogen; Rats; Tumor Cells, Cultured

a rare case of primary choriocarcinoma of the lung in a male is described with immunohistochemistry for human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and EGF-receptor. The extragonadal trophoblastic origin of this pulmonary carcinoma was definitely confirmed by an autopsy examination, and hCG-production and hCG-positive staining of the tumor cells. Furthermore, the tumor cells clearly expressed EGF and its receptor which play an important role in the proliferation and differentiation of normal and neoplastic trophoblasts of the uterus. Our present case suggests that EGF may act in an autocrine manner in the tumor cells of primary pulmonary choriocarcinoma.

Topics: Aged; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Fatal Outcome; Humans; Immunohistochemistry; Lung Neoplasms; Male

Analysis of clinical data has implicated ethanol (EtOH) as an embryotoxic agent and as an agent that disrupts normal placental structure and function. Because epidermal growth factor (EGF) is an important regulator of placental function, we have studied the effects of EtOH on EGF-induced hormone secretion using JEG-3 choriocarcinoma cells that serve as a model for trophoblast cells. EtOH at physiological (5-100 mM) concentrations modulated effects of EGF in a time and dose-dependent manner. EGF-induced P4 secretion was increased by 20-100 mM EtOH after a 2-day pretreatment of cells with EtOH, but not after a 6-day pretreatment. Preincubation with 50 mM EtOH doubled the P4 responses to 50 and 100 ng/ml EGF. Although a 2- or 4-day preincubation of cells with 10-50 mM EtOH increased the secretion of E2 in response to 20 ng/ml EGF, a 6-day preincubation inhibited the secretory response to EGF. Pretreatment of cells with 10-50 mM, but not 100 mM EtOH for 2 to 6 days enhanced the human chorionic gonadotropin (hCG) secretory response to EGF. At 50 mm EtOH, the secretion of hCG in response to EGF was increased 2-fold. EtOH also increased basal hCG secretion in a dose-dependent manner between 10-50 mM EtOH. These results suggest that EtOH may modulate EGF-stimulated hormone secretion from cells of placental origin. Such alterations, if they occur in vivo, may impact on the function of the placenta and could potentially explain the pathophysiology of alcohol toxicity during pregnancy.

Topics: Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Dose-Response Relationship, Drug; Epidermal Growth Factor; Estradiol; Ethanol; Female; Fetal Alcohol Spectrum Disorders; Humans; Placental Hormones; Pregnancy; Progesterone; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms

17 beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) is a steroidogenic enzyme that catalyzes the reversible interconversion of estrone and estradiol. In this study, we investigated the roles of epidermal growth factor (EGF) and tumor growth factor-alpha (TGF alpha) in the regulation of 17HSD type 1 gene expression and catalytic activity in cultured JAR, JEG-3, and BeWo choriocarcinoma cells. EGF and TGF alpha increased 17HSD type 1 protein concentrations in JAR and JEG-3 cells, as measured by time-resolved immunofluorometric assay, and 17HSD catalytic activity, as determined by production of estradiol from estrone. These increases were accompanied by parallel increases in concentrations of the 1.3-kilobase messenger RNA coding for 17HSD type 1 in these cells. EGF receptor tyrosine kinase activity inhibitors, tyrphostins, inhibited EGF action in JEG-3 cells, indicating that tyrosine kinase activity is needed for stimulation of the 17HSD type 1 gene. Treatment with 8-bromo-cAMP or phorbol 12-myristate 13-acetate increased the amount of 17HSD type 1 protein. Furthermore, phorbol 12-myristate 13-acetate potentiated the stimulatory effect of EGF. These results suggest that EGF and/or TGF alpha may play an important role in 17HSD type 1 regulation and, consequently, in estrogen production in the human placenta.

Topics: 17-Hydroxysteroid Dehydrogenases; 8-Bromo Cyclic Adenosine Monophosphate; Catechols; Choriocarcinoma; Epidermal Growth Factor; Humans; Nitriles; Protein-Tyrosine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

The influence of epidermal growth factor, insulin-like growth factors I and II, insulin, transforming growth factor beta 1 and transferrin on the growth of a postgestational rat choriocarcinoma was examined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. The cell line was cultured in RPMI 1640 medium supplemented with fetal calf serum, beta-mercaptoethanol, glucose, sodium pyruvate and antibiotics. The experiments were done in media supplemented with 10% (optimal) or 3% (suboptimal) fetal calf serum. Among the different growth factors tested, only epidermal growth factor and to a certain extent insulin had a growth-promoting effect by themselves. The other growth factors had either an additive effect in the presence of epidermal growth factor or no effect at all. The cytotrophoblast cells expressed both epidermal growth factor and transferrin receptors whereas the more differentiated giant cells expressed only transferrin receptors.

Topics: Animals; Cattle; Cell Differentiation; Cell Division; Choriocarcinoma; Colorimetry; Epidermal Growth Factor; ErbB Receptors; Fetal Blood; Growth Substances; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Rats; Receptors, Transferrin; Tetrazolium Salts; Thiazoles; Transferrin; Transforming Growth Factor beta; Trophoblasts; Tumor Cells, Cultured

Cytochrome P-450scc (P-450scc) 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-450scc 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-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc 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-450scc 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-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc 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-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.

Topics: Adrenodoxin; Analysis of Variance; Blotting, Northern; Cholera Toxin; Cholesterol; Cholesterol Side-Chain Cleavage Enzyme; Choriocarcinoma; Cyclic AMP; Cycloheximide; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Nucleic Acid Hybridization; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In this study we examined the expression of EGF receptor mRNA after EGF administration in hCG producing tumor (choriocarcinoma). We transplanted the tissue of choriocarcinoma into female nude mice and investigated the effects of EGF on the growth of tumors, the binding activity of EGF receptor and the expression of EGF receptor mRNA in the tumor tissues. Two doses of EGF 5.0 micrograms, 50 micrograms and phosphate buffered saline as a control were injected subcutaneously every day for four weeks. Removed tumors were used for immunocytochemical studies and EGF receptor mRNA investigations. HCG and EGF receptors were detected immunocytochemically in the tumor. The low dose EGF employed stimulated the tumor growth while the high dose EGF inhibited the tumor growth compared with that of the control group. The binding activity of EGF receptor and the expression of EGF receptor mRNA also changed in accordance with the stimulation or inhibition of tumor growth. The growth of hCG producing tumor by EGF administration appeared to be dependent upon the binding activity of EGF receptor and the expression of EGF receptor mRNA.

Topics: Animals; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Transplantation; Pregnancy; RNA, Messenger

We examined the presence and characteristics of epidermal growth factor (EGF) receptors in hCG producing tumors (CC-2-JCK) transplanted in female nude mice. We also examined the in vivo effects of EGF on tumor growth. Specific receptors with apparent dissociation constants of 3.89 x 10(-10) and 1.0 x 10(-9) M and binding capacities of 5.96 x 10(-10) and 1.52 x 10(-9) M/mg protein for EGF have been identified in the hCG-producing tumor. [125I]EGF binding to the tumor tissues was time, temperature, and tissue weight dependent and specific. EGF and transforming growth factor-alpha (TGF alpha) competed for [125I]EGF binding, with 50% of the bound [125I]EGF displaced by approximately 0.52 nM EGF and 3.10 nM TGF alpha. TGF beta competed for [125I]EGF binding slightly. Five micrograms of EGF caused an increase in the rate of tumor growth, while 50 micrograms EGF strongly inhibited tumor growth. The concentration of [125I]EGF binding in the tumor treated with low doses of EGF was high, and that in the tumor treated with high doses of EGF was low. These changes in EGF binding were attributable to the changes in the number of high affinity EGF receptors with no significant alteration in binding affinity. In conclusion, the existence of high concentrations of EGF receptors with high affinity and specificity to EGF was demonstrated in an hCG-producing tumor transplanted in nude mice and appeared to be correlated with tumor growth.

Topics: Animals; Binding, Competitive; Cell Division; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Pregnancy; Transplantation, Heterologous; Uterine Neoplasms

Binding of epidermal growth factor (EGF) to its receptor was evaluated in the four cultured choriocarcinoma cell lines BeWo, NaUCC-1, NaUCC-2, and NaUCC-3. Also, the effect of the anti-tumor drugs actinomycin-D (Act-D) and methotrexate (MTX) on the EGF receptor binding was investigated in these cell lines. Incubation of these cells with [125I]EGF at 37 degrees C resulted in a higher binding than that at 22 degrees C or at 4 degrees C. These bindings were saturable during 30- to 60-min incubation, and were specific and reversible. Scatchard analysis showed that the maximal number of receptor binding sites was 2.89 X 10(3)/cell in BeWo cells, 2.04 X 10(3)/cell in NaUCC-1 cells, 1.84 X 10(3)/cell in NaUCC-2 cells, and 1.01 X 10(3)/cell in NaUCC-3 cells. Preincubation with Act-D or MTX for 24 hr decreased the number of receptor binding sites (26%-53%) and slightly increased the receptor binding affinities. Combination of the two drugs resulted in a further diminution of EGF receptor binding sites in BeWo, NaUCC-1, and NaUCC-3 cells, respectively, but reversed the Act-D effect in NaUCC-2 cells. These results indicated that choriocarcinoma tissue is rich in EGF receptors, that the anti-tumor drugs Act-D and MTX diminish the receptor binding sites in the tissue, and suggest that MTX might induce a drug resistance to Act-D in some choriocarcinoma tissue.

Topics: Choriocarcinoma; Dactinomycin; Epidermal Growth Factor; ErbB Receptors; Humans; Methotrexate; Tumor Cells, Cultured

Previous studies using arachidonic acid and preferential inhibitors of the arachidonic acid pathway have implicated the lipoxygenase system in choriogonadotropin (hCG) secretion by JEG-3 cells. Presently, JEG-3 cells are used in order to examine the effect of lipoxygenase products on hCG secretion. Results show that 30 microM 15-hydroxyeicosatetraenoic acid (15-HETE) induces an approximately 3-fold increase in basal hCG secretion, while 5-HETE, 12-HETE, and leukotriene LTA4 have no significant effect. In addition, 15-HETE potentiates the stimulation of hCG secretion induced by 3 nM epidermal growth factor (EGF), but has no significant effect on the stimulation of hCG induced by 22 nM tetradecanoylphorbol acetate (TPA). The present study further implicates the arachidonic acid pathway in the control of hCG secretion and documents that the effect of EGF can be rate-limited by a product of the lipoxygenase system.

Topics: Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxygenase; Phorbol Esters; Pregnancy; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Neoplasms

The Jar choriocarcinoma cell line was used as an in vitro placental cell model to determine the effects of polypeptide growth factors on hCG beta secretion. Epidermal and fibroblast growth factor (FGF) treatment of serum-free cultures stimulated hCG beta secretion 2.5- and 4.0-fold over basal serum-free control levels within a 15-h incubation period. Insulin-like growth factor I, nerve growth factor, and transforming growth factor-beta had no significant effect on hCG beta secretion. FGF at concentrations as low as 0.125 ng/ml significantly elevated medium hCG beta levels without increasing cell number or total cellular protein. FGF stimulation of secretion was not detectable until 2 h of treatment. Intracellular hCG beta remained constant (23%) relative to total hCG beta (cell plus medium) as total hCG beta increased 3-fold, suggesting that FGF stimulated de novo hCG beta synthesis. Insulin significantly augmented the FGF-induced hCG beta stimulation without stimulating hCG beta production itself.

Topics: Choriocarcinoma; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Insulin; Insulin-Like Growth Factor I; Kinetics; Nerve Growth Factors; Peptide Fragments; Transforming Growth Factors; Tumor Cells, Cultured

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) regulate hormone production in several endocrine cells cultures. We have previously found that 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C activator, potentiates the cAMP-mediated secretion of human CB (hCG) in cultured human choriocarcinoma cells. We have now studied whether EGF and IGF-I modify cAMP-mediated hCG secretion in JEG-3 cells, which possess high affinity receptors to these growth factors. EGF, TPA, and cholera toxin (CT), an activator of adenylate cyclase, stimulated the secretion of hCG in a concentration-dependent manner during a 24-h culture period. The maximal effective concentrations of EGF (10 ng/ml), TPA (10 ng/ml), and CT (1.0 ng/ml) exerted 2.3-, 2.4-, and 3.9-fold increase over unstimulated level, respectively. EGF and TPA potentiated the effect of CT on hCG secretion from 3.9- to 7.8-fold and from 3.9- to 14.8-fold, respectively. By contrast, IGF-I was ineffective. During a 24-h culture, EGF and TPA potentiated the effect of CT on cAMP accumulation 1.4-fold and 1.3-fold over the production of CT-treated cells. Time-course studies indicated that these effects on cAMP and hCG were detectable at 3 h and 6 h, and they continued to increase up to 48 h and 72 h, respectively. When added alone, EGF and TPA increased cAMP production y 2.0-fold and 2.5-fold over controls at 24 h. Again, IGF-I was ineffective. Moreover, EGF and TPA potentiated the effect of 8-bromo-cAMP (on hCG production to a similar extent than they did to CT-stimulated hCG production. The binding of [125I]iodo-EGF to the cells was not altered by a 48-h CT-treatment whereas the binding of [125I]iodo-IGF-I was increased by 2.1-fold above untreated cells. Our data show that both EGF and TPA potentiated the effect of CT on hCG secretion in JEG-3 cells, whereas IGF-I had no effect. Although EGF and TPA facilitated CT-stimulated cAMP accumulation, their site of action on cAMP-mediated hCG production is distinct from the adenylate cyclase or EGF-receptor level since EGF and TPA potentiated the hCG secretion stimulated by 8-bromo-cAMP and an increase in cAMP production did not alter the binding properties of EGF-receptor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Binding, Competitive; Cholera Toxin; Choriocarcinoma; Chorionic Gonadotropin; Cyclic AMP; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin-Like Growth Factor I; Kinetics; Somatomedins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The effects of cholera toxin (CT), which stimulates adenylate cyclase, 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on progesterone (P) and estradiol (E2) secretion by human choriocarcinoma JEG-3 cells were studied. During a 48 h incubation, CT, TPA and EGF stimulated P production in a concentration-dependent manner, whereas IGF-I was without effect. CT (1.0 ng/ml), TPA (10 ng/ml) and EGF (10 ng/ml) stimulated P production maximally 4.3-, 3.3- and 2.3-fold over basal, respectively. When added together with CT, TPA and EGF stimulated P production 10.0- and 5.0-fold over basal production showing that the effects of CT plus TPA were more than additive but those of CT plus EGF less than additive. Time-course studies indicated that the effects were detectable at 12 h, and continued to increase up to 48 h. The conversion of added dehydroepiandrosterone sulfate (DHEAS) to E2 was stimulated by CT and TPA and inhibited by IGF-I in a concentration-dependent manner. By contrast, EGF had no effect. The maximal responses in E2 production were 3.2- and 2.0-fold over unstimulated cells by CT (1.0 ng/ml) and TPA (10 ng/ml), respectively. When both agents were added together, their effects on E2 production were additive with 5.5-fold increase over unstimulated cells. IGF-I (30 ng/ml) inhibited maximally basal and CT-stimulated E2 production by 33% and 42%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cholera Toxin; Choriocarcinoma; Epidermal Growth Factor; Estradiol; Female; Humans; Insulin-Like Growth Factor I; Phorbol Esters; Placental Hormones; Pregnancy; Progesterone; Somatomedins; Tumor Cells, Cultured; Uterine Neoplasms

Epidermal growth factor (EGF) binds specifically (Ka = 4 X 10(9) M-1; 1.3 X 10(11) receptors/mg cellular protein) to JEG-3 cells, which respond in the succeeding 24 h by a 400% increase in hCG secretion without a significant change in cell number. Since JEG-3 cells store less than 2% of the 24-h hCG secretion, a significant increase in hCG in the culture medium reflects increased hCG biosynthesis. It is observed that a tumor promoter, phorbol myristate acetate (PMA), binds specifically to the cells (Ka = 5 X 10(8) M-1; 3.9 X 10(11) receptors/mg), reduces (33%) the affinity but not the number of EGF receptors, and stimulates hCG to the same extent as does EGF. The relative potencies of PMA (100%), phorbol dibutyrate (25%), and nonesterified phorbol (no effect) to stimulate hCG parallel their known tumor-promoting activities. Since phospholipids and fatty acids have been implicated in the mechanism of action of EGF and PMA, the effects of arachidonic acid (AA) and inhibitors of its metabolism were studied. The addition of AA had no effect on basal or PMA-stimulated hCG secretion, but it potentiated the effect of EGF by 200%. Indomethacin (a cyclooxygenase inhibitor) had an effect similar to that of AA. Nordihydroguairetic acid (a cyclooxygenase and lipoxygenase inhibitor) reduced by 90% basal and EGF- or PMA-stimulated hCG secretion. These results indicate that the AA pathway can influence the effects of EGF and PMA on hCG secretion and suggest an intermediary role for the lipoxygenase system.

Topics: Arachidonic Acid; Arachidonic Acids; Caenorhabditis elegans Proteins; Carrier Proteins; Catechols; Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Indomethacin; Masoprocol; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pregnancy; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug; Receptors, Immunologic; Tetradecanoylphorbol Acetate; Uterine Neoplasms

Both 12-O-tetradecanoyl-phorbol-1- acetate and teleocidin B stimulated the secretion of human chorionic gonadotropin by cultured choriocarcinoma cells. These tumor promoters also stimulated production of progesterone in the cells. However, the 2 tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor that also had a stimulatory effect on production of these hormones.

Topics: Alkaloids; Animals; Carcinogens; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Kinetics; Lyngbya Toxins; Male; Mice; Phorbols; Pregnancy; Progesterone; Tetradecanoylphorbol Acetate; Uterine Neoplasms

A new human functional tumor cell line, designated as T3M-3, has been established from a xenotransplanted choriocarcinoma grown in nude mice. One of the biggest problems of the in vitro culture of these tumor cells using the xenotransplanted tumors had been the dense contamination of fibroblasts of host nude mouse origin. In the present study, these fibroblasts were completely removed by incubating the cells with antiserum raised against nude mouse spleen cells. The cell line established from the remaining tumor cells has been successfully propagated in vitro for as long as 4 years. These cells show the morphology of epithelioid cells containing a prominent nucleus with one or two large nucleoli. The cells grow in a monolayered sheet with the population-doubling time of 19 hr. The cells show perfect tumor takes when they are reinoculated into nude mice. Chromosomal analysis revealed that the cell is a human aneuploid one with a hypotriploid mode. These cultured cells maintained well the function of secreting large amounts of human chorionic gonadotropin, progesterone, and estrogen. The secretion of human chorionic gonadotropin and progesterone by these cells is enhanced by stimulation with tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin B, or with epidermal growth factor in a dose-and time-dependent manner. Interestingly, however, the tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor, indicating that the receptors for these reagents in T3M-3 cells are not shared by epidermal growth factor.

Topics: Animals; Carcinogens; Cell Division; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Culture Media; Epidermal Growth Factor; Female; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Pregnancy; Uterine Neoplasms

The cultured human choriocarcinoma JEG-3 cells secrete biologically active HCG and free HCG alpha-subunit. When compared with the alpha-subunit dissociated from HCG obtained either from pregnancy urine or JEG-3 cells, free alpha-subunit has a larger molecular weight, is more acidic and is non-functional, lacking the property to recombine with the HCG beta-subunit. The understanding of the biochemical differences observed between free alpha-subunit and alpha-subunit found in HCG is important and should help to unravel the biosynthesis of gonadotrophins. Two proteins which bind to the cell membrane, epidermal growth factor and concanavalin A, are capable of stimulating JEG-3 cell secretion. Epidermal growth factor stimulates the secretion of HCG while concanavalin A stimulates both HCG and HCG alpha-subunit secretion. Amphotericin B, an antifungal agent commonly used in tissue cultures, which also affects the cell membrane, was shown to stimulate HCG and HCG alpha-subunit secretions. The use of these agents should contribute to the understanding of membrane-related events which lead to the secretion of HCG and alpha-subunit.

Topics: Amphotericin B; Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Concanavalin A; Epidermal Growth Factor; Female; Humans; Pregnancy; Uterine Neoplasms

The ability of epidermal growth factor (EGF) to modulate the secretion of human chorionic gonadotropin (hCG) in both normal and malignant placental cells was compared. Receptors for EGF were present on the JAr line of choriocarcinoma cells and were localized to the trophoblast cells of normal placental organ cultures as detected by immunofluorescence. Despite the presence of EGF receptors, the normal placenta did not respond to EGF by significantly increasing its levels of hCG production. The JAr line of choriocarcinoma exhibited a 2-fold increase in hCG secretion after the addition of EGF. EGF stimulated growth in the JAr cells, as measured by the protein content of the cultures, but did not elevate the incorporation of [methyl-3H]thymidine in either the JAr cells or placental organ cultures.

Topics: Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Humans; Organ Culture Techniques; Placenta; Pregnancy; Uterine Neoplasms

Epidermal growth factor (EGF) stimulates the production of progesterone by JEG-3, a clonal strain of human choriocarcinoma cells. Stimulation occurs in a time and dose-dependent manner. In addition, EGF increases [14C]-acetate incorporation into [14C]-cholesterol in JEG-3 cells, and this may constitute its mechanism of action in enhancing progesterone synthesis.

Topics: Animals; Cell Line; Choriocarcinoma; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Kinetics; Mice; Peptides; Pregnancy; Progesterone; Uterine Neoplasms

Epidermal growth factor (EGF) binds to JEG-3 cells, a tissue culture line of human choriocarcinoma. EGF also stimulates secretion of human chorionic gonadotropin (hCG) and to a lesser extent the secretion of free hCG-alpha.

Topics: Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Culture Media; Epidermal Growth Factor; Female; Humans; Peptide Fragments; Peptides; Pregnancy; Uterine Neoplasms