Human Choriocarcinoma Cell Line BeWo Research Articles

Sirtuin 1 is a potential therapeutic candidate gene for fetal growth restriction via insulin-like 4

Objective Insufficient placental development causes various obstetric complications, including fetal growth restriction (FGR). The Sirtuin 1 (SIRT1) and insulin-like 4 (INSL4) protein-coding genes have been demonstrated to play an important role in placental development. However, no treatment for FGR is available due to placental dysfunction. Therefore, this study aimed to examine the potential of the SIRT1–INSL4 axis as a treatment candidate for FGR caused by insufficient placental development. Methods Twenty patients were enrolled, including 10 with FGR and 10 full-term controls. FGR and control placental samples were collected. Quantitative real-time polymerase chain reaction, immunohistochemical analysis, and western blotting were used to analyze INSL4 and SIRT1 expression. An in-vitro loss-of-function approach with the human choriocarcinoma cell line BeWo was applied for functional analyses of SIRT1 in placental development. BeWo cells were differentiated into syncytiotrophoblasts by silencing SIRT1 using small interfering RNA. SIRT1 activator was added during differentiation of SIRT1-knockdown BeWo cells into syncytiotrophoblasts. Results The FGR samples had lower INSL4 and SIRT1 mRNA and protein expression levels than the control samples. Immunohistochemistry showed that both SIRT1 and INSL4 were expressed mainly in syncytiotrophoblasts. In-vitro analyses showed that SIRT1 knockdown decreased INSL4 expression; however, SIRT1 activator restored SIRT1 expression in SIRT1-silenced BeWo cells. Conclusions SIRT1 and INSL4 are downregulated in the placenta of FGR, and INSL4 is regulated by SIRT1. These findings indicate that the SIRT1–INSL4 axis may be a potential therapeutic target for FGR.

Factors Involved in miRNA Processing Change Its Expression Level during Imitation of Hypoxia in BeWo b30 Cells.

One of the main complications of pregnancy and causes of maternal and perinatal mortality is preeclampsia. The pathophysiology of preeclampsia is associated with the development of placenta and fetal hypoxia and secretion of a number of effective molecules. The human choriocarcinoma cell line BeWo b30 is often used as a model of the placental barrier. It was shown that oxyquinoline derivatives can mimic hypoxia by suppressing HIF-prolyl hydroxylases and the accumulation of HIF-1α. This effect also leads to a change in the expression of microRNAs and their target genes. However, with hypoxia in cells, not only the level of individual miRNAs but also the ratio of miRNA isoforms (isomiRs) can change, presumably due to inaccuracies in the work of the Drosha and Dicer enzymes. In this work, we showed a change in the expression of the factors involved in the maturation of miRNAs when simulating hypoxia in BeWo b30 cells with an oxyquinoline derivative, which may be one of the causes for the change in the ratio of miRNA isoforms.

MiR-34a-5p regulates viability, invasion and apoptosis of placental trophoblastic cells via modulating CDK6 and PI3K/AKT pathway

To investigate the roles of microRNA (miR)-34a-5p and cyclin-dependent kinase (CDK) 6 in the regulation of cell viability, apoptosis and invasion of human placental trophoblastic cells and the relationship between miR-34a-5p and CDK6. We examined the expression of miR-34a-5p using RT-qPCR in cultured human trophoblast HTR-8/Svneo cells and human choriocarcinoma cell lines BeWo and JEG-3HTR-8/Svneo. HTR-8/Svneo cells transfected with a miR-34a-5p-mimic, the miR-34a-5p-inhibitor, or pcDNA-CDK6 along with the mimic group were analyzed for changes in cell proliferation using MTT assay; the apoptosis of the cells were assessed by detecting caspase 3 activity and cleaved caspase 3 protein expression, and the cell invasion was evaluated using Transwell assay. Western blotting was used to determine the protein levels of CDK6, cleaved caspase 3, and MMP-9 in the cells. The interaction between CDK6 and miR-34a-5p analyzed using a luciferase reporter assay. Transfection with the miR-34a-5p mimic significantly reduced the viability (P=0.000), suppressed the invasion (P=0.049), enhanced the cell apoptosis (P=0.018), down-regulated the expressions of MMP-9 (P=0.004) and CDK6 (P=0.014), and up-regulated caspase 3 activity (P=0.018) and cleaved caspase 3 expression (P=0.003) in cultured HTR-8/Svneo cells. CDK6 was confirmed as one of the target gene of miR-34a-5p. Transfection with pcDNA-CDK6 significantly reversed the effects of miR- 34a-5p overexpression on the cell viability (P=0.000), apoptosis (P=0.015), and invasion (P=0.046). Treatment of the cells with insulin-like growth factor 1 (IGF-1), an activator of the PI3K/AKT pathway, also significantly attenuated the effects of miR-34a- 5p overexpression on the cell viability (P=0.011), apoptosis (P=0.004), and invasion (P=0.002). miR-34a-5p promotes apoptosis and inhibits the viability and invasion of human placental trophoblastic cells by down-regulating CDK6 and inactivating the PI3K/AKT pathway.

Ламинин 521 изменяет экспрессию генов SNAI1, ZNF708 и GRN в клетках BeWo b30 и создает физиологические условия для плацентарного барьера

A human choriocarcinoma cell line BeWo b30 imitates cytotrophoblast cells and thus allows to model the placental barrier. Since these cells can form multilayered structures, a method to control the number of cells based on the MTS test has been developed. It was found that during the cell growth on a laminin-521-containing extracellular matrix a significant change in the transepithelial electrical resistance (TEER) and the electrical capacitance of the cell monolayer is observed as compared to the cultivation without the extracellular matrix. The transcriptome analysis of the cells revealed that in the presence of laminin-521, the expression of the ZNF708 gene decreases and that of the SNAI1 and progranuline gene GRN increases as compared to the data obtained without the extracellular matrix. This may indicate that in the first case the cells were prepared to fuse into syncytiotrophoblast. A change in the expression of five microRNA genes and one snoRNA gene was also observed. The above mentioned effects can be associated with the used laminin. BeWo b30, placenta, barrier, microRNA, laminin 521 (laminin-11), SNAI1 The study was supported by the Ministry of Education and Science of the Russian Federation in the framework of the Federal Targeted Program for Research and Development in Priority Areas of Advancement of the Russian Scientific and Technological Complex for 2014-2020 (RFMEFI58817X0007).

Impedance Spectroscopy and Transcriptome Analysis of Choriocarcinoma BeWo b30 as a Model of Human Placenta

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.

Активация HIF-1α снижает экспрессию гена-хозяина микроРНК hsa-miR-603 KIAA1217 и повышает экспрессию гена-мишени CCND1 в клетках BeWo b30

The model of the placental barrier based on the human choriocarcinoma cell line BeWo b30 allows studying the effect of hypoxia on trophoblast cells. The effect of the oxyquinoline derivative inhibiting HIF-prolyl hydroxylases was studied on this model. Inhibition of these enzymes leads to an increase in the HIF-1α subunit in the cytoplasm, mimicking the cell response to hypoxia. Incubation of the cells with the drug at a concentration of 10 uM for 24 h did not affect the paracellular transport, but reduced the transport of glucose through the cell barrier. The transcriptome analysis after the exposure with oxyquinoline derivative revealed a decreased expression of the KIAA1217 gene and its intronic gene MIR603, which encodes microRNA hsa-miR-603. The expression of the target gene of this miRNA, CCND1 encoding cyclin D1, after oxyquinoline derivative exposition increased significantly, which may indicate a potential microRNA-mRNA regulatory mechanism in the response of trophoblast cells to hypoxia. BeWo b30, placenta, hypoxia, oxyquinoline, barrier, microRNA, cyclin The study was performed with the equipment of the «Postgenomic and Metabolomic Methods of Study in Molecular Biology» Common Use Center (BioClinicum Scientific and Technical Center). The study was supported by the Ministry of Education and Science of the Russian Federation in the framework of the Federal Targeted Program for Research and Development in Priority Areas of Advancement of the Russian Scientific and Technological Complex for 2014-2020 (Project no. RFMEFI58817X0007).

Upregulation of Angiogenic Factors via Protein Kinase C and Hypoxia-induced Factor-1α Pathways under High-glucose Conditions in the Placenta.

Abnormal glucose metabolism during pregnancy is an established risk factor for preeclampsia (PE). Disruption of the balance between placental angiogenic factors is linked to PE pathophysiology. We examined whether hypoxia-induced factor-1α (HIF-1α) and protein kinase Cβ (PKCβ) are involved in the regulation of placental angiogenic factors under high-glucose conditions in vitro. The human choriocarcinoma cell lines BeWo and JEG-3, and the human trophoblast cell line HTR-8/SVneo were cultured with 10 and 25 mmol/L glucose [control glucose group (CG) and high-glucose group (HG), respectively]. We examined the changes in HIF-1α, soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) expression in the CG and HG by real-time PCR and ELISA. PKC activation was also measured by ELISA. The expressions of HIF-1α, sFlt-1, PlGF, and VEGF were significantly higher in the HG than in the CG. PKC activity was significantly increased in the HG. High glucose affected the expression of angiogenic factors in choriocarcinoma cells via the PKCβ and HIF-1α pathways, suggesting their involvement in PE pathogenesis.

Regulation of hypoxia-inducible factor-1α, regulated in development and DNA damage response-1 and mammalian target of rapamycin in human placental BeWo cells under hypoxia

IntroductionHypoxia-inducible factor-1 (HIF-1), regulated in development and DNA damage response-1 (REDD1) and mammalian target of rapamycin (mTOR) are crucial mediators of many metabolic processes in various cell types under hypoxia. The involvement and regulation of these three factors underlying trophoblasts' response to hypoxia remains to be determined. MethodsSpecific siRNAs were applied to inhibit the expression of the corresponding genes and to investigate the roles of HIF-1α in modulating REDD1/mTOR and REDD1 in regulating mTOR/HIF-1α in the human choriocarcinoma cell line BeWo under normoxia and hypoxia. ResultsExposure of BeWo cells to 1% oxygen (compared with 21% oxygen) led to a remarkable increase of both HIF-1α and REDD1 and an obvious decrease of mTOR at both the mRNA and protein levels. Interference of HIF-1α expression by siRNA resulted in an apparent reduction of REDD1 parallel with that of HIF-1α during normoxia and hypoxia. Additionally, the hypoxia-induced REDD1 increase was blocked through loss of HIF-1α, and the downregulation of mTOR in hypoxia could be partly obstructed by HIF-1α-siRNA transfection. Separately, the modulation effect of REDD1 was confirmed in an experiment demonstrating that the hypoxia-induced decrease of mTOR was inhibited by REDD1 knockdown, which was measured by changes in both the mRNA and protein levels. The disruption of REDD1 expression also led to increased accumulation of HIF-1α under both normoxia and hypoxia. DiscussionThe HIF-1α-REDD1-mTOR pathway was involved in the response to hypoxia in BeWo cells. Hypoxia-induced REDD1 upregulation is mediated by a HIF-1α-dependent pathway. Disruption of REDD1 blocked the effects of hypoxia on suppressing mTOR and resulted in additional accumulation of HIF-1α in BeWo cells.

Progranulin shows cytoprotective effects on trophoblast cells in vitro but does not antagonize TNF-α-induced apoptosis.

The glycoprotein progranulin directly binds to TNF-receptors and thereby can antagonize the inflammatory effects of TNF-α. Here we analyzed the impact of both cytokines on cytotoxicity and viability of trophoblast cells. Isolated villous first trimester human trophoblast cells and the human choriocarcinoma cell line BeWo were treated with recombinant human progranulin and TNF-α. Analyses were performed by LDH- and MTT-assay and measurement of caspase-8-activity. Progranulin treatment showed some cytoprotective effects on isolated trophoblast cells. However, TNF-α-induced apoptosis was not antagonized by addition of progranulin. Effects were similar, but more pronounced in BeWo cells. The cytoprotective activity of progranulin on trophoblast cells in vitro was only weak and of doubtful biologic relevance. It was not able to antagonize TNF-α. Future studies should focus on possible paracrine activities of progranulin.

PP010. Does aldosterone participate in placental angiogenesis via PLGF?

Angiogenic signals are a vital signal of placental integrity. Aldosterone has recently been shown to enhance placental growth factor (PlGF) expression in the peripheral vasculature [1] and to promote trophoblast growth [2]. The plgf gene possesses a functional mineralocorticoid receptor responsive element in the promoter region. Thus, we hypothesized that aldosterone adapts placental angiogenesis to trophoblast growth by secreting PlGF. The human choriocarcinoma cell line BeWo and first and third trimester human primary trophoblasts cells were subjected to several syncytialization signals. Upon visual confirmation, the cultured cells were subjected to either control conditions, the known stimulator forskolin, and increasing amounts of aldosterone (10(-9) to 10(-6)M) with and without the competitive aldosterone receptor blocker spironolactone. After 6 and 24h of incubation, RNA and protein were extracted. PlGF transcripts were quantified by Taqman PCR normalized to several housekeeping genes. Protein expression was quantified by ELISA. PlGF mRNA expression increased 3-fold with forskolin in BeWo cells. In this cell line, aldosterone could slightly stimulate PlGF production. In non-syncytialized primary human first trimester trophoblasts, aldosterone did not exert a specific effect. In contrast, the term primary human trophoblasts did respond with a 2.5-fold increase after incubation with aldosterone (10(-7)M) in the presence of forskolin to allow forming a syncytial layer. PlGF protein was already slightly upregulated following 6h of incubation with aldosterone. We concluded that aldosterone does regulate PlGF expression in specified conditions during pregnancy. Inappropriately low aldosterone levels such as in preeclampsia might such not only compromise plasma volume and trophoblast growth but also placental vascularization and systemic PlGF availability. These observations merit further investigation.

Oxidative stress stimulates α-tocopherol transfer protein in human trophoblast tumor cells BeWo

α-Tocopherol transfer protein (α-TTP) has been identified as the major intracellular transport protein for the antioxidant vitamin E (α-tocopherol). Expression of α-TTP on the reproductive system has been described both in mouse uterus and lately in the human placenta. The aim of this study was to clarify if placental expression of α-TTP can be modified by substances causing oxidative reactions. The human choriocarcinoma cell line BeWo was, therefore, treated with two known pro-oxidants. α-TTP expression was determined with immunocytochemistry and evaluated by applying a semiquantitative score. The presence of pro-oxidants in BeWo cells induced α-TTP expression. We thus hypothesize that stimulation of α-TTP expression by oxidative stress, as this was induced by pro-oxidants, could be part of an antioxidant process occurring in the placenta in the aim of enhancing the supply of α-tocopherol. This process could occur both in normal pregnancies, as well as in pregnancy disorders presented with intensified oxidative stress. In that view, this model is proposed for further oxidative stress studies on trophoblast and placenta, on the grounds of clarifying the role of α-tocopherol in pregnancy physiology and pathophysiology.

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

The Mechanism of Carrier-Mediated Transport of Folates in BeWo Cells: The Involvement of Heme Carrier Protein 1 in Placental Folate Transport

The aim of this study was to elucidate the mechanism of folate transport in the placenta. A study of folate was carried out to determine which carriers transport folates in the human choriocarcinoma cell line BeWo, a model cell line for the placenta. We investigated the effects of buffer pH and various compounds on folate uptake. In the first part of the study, the expression levels of the mRNA of the folate receptor alpha (FRalpha), the reduced folate carrier (RFC), and heme carrier protein 1 (HCP1) were determined in BeWo cells by RT-PCR analysis. Folate uptake into BeWo cells was greater under an acidic buffer condition than under a neutral one. Structure analogs of folates inhibited folate uptake under all buffer pH conditions, but anion drugs (e.g., pravastatin) inhibited folate uptake only under an acidic buffer condition. Although thiamine pyrophosphate (TPP), a substrate of RFC, had no effect on folate uptake, hemin (a weak inhibitor of folate uptake via HCP1) decreased folate uptake to about 80% of the control level under an acidic buffer condition. Furthermore, kinetic analysis showed that hemin inhibited the low-affinity phase of folate uptake under an acidic buffer condition. We conclude that pH-dependent folate uptake in BeWo cells is mediated by at least two carriers. RFC is not involved in folate uptake, but FRalpha (high affinity phase) and HCP1 (low affinity phase) transport folate in BeWo cells.

EXPRESSION AND FUNCTIONAL ACTIVITY OF BREAST CANCER RESISTANCE PROTEIN (BCRP, ABCG2) TRANSPORTER IN THE HUMAN CHORIOCARCINOMA CELL LINE BEWO

1. Breast cancer resistance protein (BCRP, ABCG2) is a drug efflux transporter that is believed to affect the drug disposition of several drugs and xenobiotics. In the present study, we evaluated the localization and functional expression of BCRP in the human choriocarcinoma cell line BeWo, an in vitro model of the human trophoblast, and compared it with the expression of P-glycoprotein (MDR1, ABCB1) as the most widely studied placental transporter. In addition, the expression of BCRP at the mRNA level was compared with that of MDR1 in the human term placenta. 2. Western blotting analysis revealed high endogenous expression of BCRP protein in BeWo cells. Using indirect immunofluorescence microscopy, we found that the BCRP transporter appears to be localized predominantly at the apical plasma membrane. Functional studies showed a significant effect of the BCRP inhibitors GF120918 (5 micromol/L) and Ko143 (1 micromol/L) on mitoxantrone accumulation and, thus, confirmed efflux activity of BCRP in BeWo cells. 3. Using absolute mRNA quantification with real-time reverse transcription-polymerase chain reaction, we found high expression of BCRP in BeWo cells, whereas no transcript of MDR1 (P-glycoprotein), the most extensively studied drug transporter, was detected. 4. In the human placenta, BCRP was localized predominantly in the syncytiotrophoblast layer; however, immunopositivity for the BXP-21 antibody was also observed in fetal vessels of the chorionic villi. The number of BCRP transcripts in the human term placenta was found to be more than 10-fold higher compared with the expression of MDR1. 5. In conclusion, we suggest that BeWo cells could serve as a suitable in vitro model to study trans-trophoblast transport of BCRP substrates and that placental BCRP can play an important role in preventing the accumulation of potentially toxic xenobiotics in the trophoblast cells.

Functional and placental expression analysis of the human NRF3 transcription factor.

Members of the Maf protooncogene and cap'n' collar families of basic-leucine zipper transcription factors play important roles in development, differentiation, oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basic-leucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/MAFG heterodimer recognizes nuclear factor-erythroid 2/Maf recognition element-type DNA-binding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We found that NRF3 transcripts are present in placental chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocarcinoma cell lines BeWo and JAR, derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3-specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF-alpha in JAR cells. Our functional studies suggest that human NRF3 is a potent transcriptional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development.

Co-expression of Fas (APO-1, CD95)/Fas ligand by BeWo and NJG choriocarcinoma cell lines

Objective Fas (CD95) is a transmembrane protein of the tumor necrosis factor receptor superfamily that induces apoptosis in susceptible cells on crosslinking by its ligand (FasL). The Fas loss of function and concurrent expression of its ligand (FasL) have been associated with malignant phenotype. In this study, we sought to investigate the hitherto undescribed expression of Fas and FasL on the immortalized human choriocarcinoma cell lines BeWo and NJG. Methods Receptor and ligand expression was demonstrated using specific antibodies and multiple techniques including immunocytochemistry, confocal immunofluorescence microscopy, flow cytometry, immunoblots, and reverse transcription polymerase chain reaction (RT-PCR). Results Data from this study indicate that human choriocarcinoma cell subtypes co-express both Fas and FasL. A specific cytoplasmic and membranous pattern of immunoreactivity was noted that was further confirmed at mRNA transcripts by RT-PCR. In addition, we provide evidence using flow cytometry that the Fas receptors are downregulated. The mean fluorescence intensities for NJG and BeWo were 1.47 ± 0.5 and 1.59 ± 0.4, while that for Fas-positive Jurkat cells was 25.6 ± 3.1. Conclusions To our knowledge, this is the first report on the identification and constitutive co-expression of Fas and FasL in BeWo and NJG choriocarcinoma cells. Choriocarcinoma cells evade immune attack by downregulating the Fas receptor and by killing lymphocytes through expression of FasL. Taken together, our investigations suggest that the Fas/FasL system may represent a mechanism by which malignant trophoblasts become resistant to apoptosis, escape immune surveillance, and metastasize.

Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen

Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo.

We have used Northern blot analysis to detect mRNA from class I HLA genes in the human choriocarcinoma cell line BeWo, which has been previously shown to express an atypical HLA class I molecule, in the absence of HLA A and B. Hybridization was seen with three class I DNA probes, the strongest being seen with the probe pC800, which corresponds to an 800-bp section of the Cw3 gene. We have made cDNA libraries from BeWo cells and screened for positive clones by using class I DNA probes. Of the clones isolated, we determined the complete sequence of one and partial sequence of five shorter clones. They all code for an identical C locus-related product, which does not correspond to published C locus sequences.

Gamma-interferon enhances expression of Class I MHC antigens in the weakly HLA+ human choriocarcinoma cell line BeWo, but does not induce MHC expression in the HLA- choriocarcinoma cell line Jar.

PHA-activated lymphocyte supernatants and high doses of affinity-purified human gamma-interferon enhance the expression of apparently normal Class I histocompatibility antigens in a malignant human trophoblast cell line that expresses low amounts of these antigens under normal culture conditions. Another human choriocarcinoma cell line, Jar, which is normally HLA-, did not respond to this treatment. This system provides a model in which to study further the regulation and effects of MHC antigen expression in cells of trophoblastic origin.