Choriocarcinoma Cell Line Research Articles

Overexpression of NANOG in Gestational Trophoblastic Diseases: Effect on Apoptosis, Cell Invasion, and Clinical Outcome

Gestational trophoblastic disease includes choriocarcinoma, a frankly malignant tumor, and hydatidiform mole (HM), which often leads to the development of persistent gestational trophoblastic neoplasia and requires chemotherapy. NANOG is an important transcription factor that is crucial for maintaining embryonic stem cell self-renewal and pluripotency. We postulated that NANOG is involved in the pathogenesis of gestational trophoblastic disease. In this study, significantly higher NANOG mRNA and protein expression levels, by quantitative PCR and immunoblotting, respectively, were demonstrated in HMs, particularly those that developed persistent disease, when compared with normal placentas. In addition, significantly increased nuclear NANOG immunoreactivity was found by immunohistochemistry in HMs (P < 0.001) and choriocarcinoma (P = 0.002). Higher NANOG expression levels were demonstrated in HMs that developed persistent disease, as compared with those that regressed (P = 0.025). Nuclear localization of NANOG was confirmed by confocal microscopy and immunoblotting in choriocarcinoma cell lines. There was a significant inverse correlation between NANOG immunoreactivity and apoptotic index assessed by M30 CytoDeath antibody (P = 0.012). After stable knockdown of NANOG in the choriocarcinoma cell line JEG-3 by an shRNA approach, increased apoptosis was observed in relation to with enhanced caspases and poly(ADP-ribose) polymerase activities. NANOG knockdown was also associated with decreased mobility and invasion of JEG-3 and down-regulation of matrix metalloproteases 2 and 9. These findings suggest that NANOG is involved in the pathogenesis and clinical progress of gestational trophoblastic disease, likely through its effect on apoptosis, cell migration, and invasion.

Human trophoblast cells express the immunomodulator progesterone-induced blocking factor

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48 kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1–18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90 kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34 kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.

Up-Regulation of Placental Leptin by Human Chorionic Gonadotropin

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.

Relative involvement of three 17β-hydroxysteroid dehydrogenases (types 1, 7 and 12) in the formation of estradiol in various breast cancer cell lines using selective inhibitors

We investigated the relative involvement of three reductive 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms, namely types 1, 7 and 12, in the formation of potent estrogen estradiol (E2) in 10 human breast cancer cell lines (T-47D, MCF-7, ZR-75-1, CAMA-1, BT-20, BRC-17, BRC-31, BRC-32, BRC-36 and BRN-196) and also in 1 choriocarcinoma cell line (JEG-3) using selective inhibitors. In T-47D, BT-20 and JEG-3 cells, a 17beta-HSD1 inhibitor almost completely inhibited the formation of E2 at 1microM from 60nM of estrone (E1) (98%, 91% and 90%, respectively), whereas no significant inhibition of E2 formation was obtained using inhibitors of types 7 and 12. However, we obtained lower levels of inhibition (32%, 36% and 35% respectively using inhibitors of types 1, 7 and 12 at 10microM) in MCF-7 cells and even lower and variable levels of inhibition (15%, 23% and 18% respectively using inhibitors of types 1, 7 and 12 at 10microM) in ZR-75-1 cells. No inhibition of E2 formation was observed in CAMA-1 cells with a 17beta-HSD1 inhibitor at 1microM whereas inhibitors of types 7 and 12 inhibited 40% and 30% of E2 formation, respectively. In BRC and BRN cell lines, types 1, 7 and 12 17beta-HSDs were all involved in the formation of E2, but type 12 seemed to predominate. At 10microM, each inhibitor inhibited 10-50% of the formation of E2. Using MCF-7 and BRC-32 cell lines, a combination of the three inhibitors (3x10microM) does not fully inhibit the 17beta-HSD activity (65% and 75%). In addition to identify the relative importance of types 1, 7 and 12 17beta-HSDs in the formation of E2 in human breast cancer cell lines, our results show also a great variability between each cell line. In some cases the formation of E2 was completely inhibited, but this was not the result observed in other cell lines, suggesting the presence of another enzyme involved in the biosynthesis of E2.

Hypermethylation of SOX2 Gene in Hydatidiform Mole and Choriocarcinoma

This study investigated the expression and methylation profiles of SOX2, a stem cell-related transcription factor, in placentas and gestational trophoblastic disease. The methylation status of SOX2 promoter region in 55 hydatidiform moles, 4 choriocarcinoma, 23 first trimester, and 15 term placentas was evaluated by methylation-specific polymerase chain reaction. The methylated allele was found in 4.4% (1/23) of first trimester placentas, 26.7% (4/15) term placentas, and 56.4% (31/55) of hydatidiform moles and all choriocarcinoma samples and cell lines. A significant reduction in SOX2 messenger RNA expression was found in the hydatidiform moles (P = .027) when compared with that in the placentas. SOX2 messenger RNA expression was significantly correlated with SOX2 hypermethylation (P < .001). SOX2 expression was restored in choriocarcinoma cell lines following treatment to 5-Aza-2(')-deoxycytidine and/or Trichostatin A, demethylation and histone deacetylase inhibitors, respectively, and the response was synergistic. Epigenetic mechanisms may play important role on the transcriptional regulation of SOX2 and contribute to pathogenesis of gestational trophoblastic disease.

Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts

The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts. We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells. The effects of small-interfering (si) RNA-mediated stathmin knockdown on trophoblast proliferation and migration were measured by WST-1 and Transwell assays, respectively. Trophoblast differentiation was induced by dibutyryl (db)-cAMP treatment and evaluated by measuring human chorionic gonadotrophin beta (hCGbeta) and syncytin expression and cell fusion. We examined the effect of knockdown and induced stathmin expression on db-cAMP-induced differentiation. siRNA-induced silencing of stathmin expression had a marked inhibitory effect on BeWo, JEG-3 and HTR-8/SVneo cell migration and also suppressed their proliferation, albeit to a lesser extent. db-cAMP-enhanced hCGbeta and syncytin expression and cell fusion in BeWo cells was inhibited by stathmin knockdown. However, induced expression of stathmin reversed the hCGbeta and syncytin expression and cell fusion in the Tet-On BeWo cells. Suppression of stathmin expression also inhibited the migration of and hCGbeta production by first-trimester trophoblasts. Stathmin expression may be closely associated with early trophoblast migration and differentiation into syncytiotrophoblasts during placentation.

Specific tumour-associated methylation in normal human term placenta and first-trimester cytotrophoblasts

Human placentation displays many similarities with tumourigenesis, including rapid cell division, migration and invasion, overlapping gene expression profiles and escape from immune detection. Recent data have identified promoter methylation in the Ras association factor and adenomatous polyposis coli tumour suppressor genes as part of this process. However, the extent of tumour-associated methylation in the placenta remains unclear. Using whole genome methylation data as a starting point, we have examined this phenomenon in placental tissue. We found no evidence for methylation of the majority of common tumour suppressor genes in term placentas, but identified methylation in several genes previously described in some human tumours. Notably, promoter methylation of four independent negative regulators of Wnt signalling has now been identified in human placental tissue and purified trophoblasts. Methylation is present in baboon, but not in mouse placentas. This supports a role for elevated Wnt signalling in primate trophoblast invasiveness and placentation. Examination of invasive choriocarcinoma cell lines revealed altered methylation patterns consistent with a role of methylation change in gestational trophoblastic disease. This distinct pattern of tumour-associated methylation implicates a coordinated series of epigenetic silencing events, similar to those associated with some tumours, in the distinct features of normal human placental invasion and function.

Activated Stat3 expression in gestational trophoblastic disease: correlation with clinicopathological parameters and apoptotic indices

To assess the expression profile of the activated form of signal transducer and activator of transcription (Stat)3 in gestational trophoblastic disease (GTD) and correlate the findings with clinicopathological parameters. By immunohistochemistry, both cytoplasmic and nuclear expression of p-Stat3-Ser(727) was demonstrated in 88 trophoblastic tissues, including placentas and GTD. Nuclear immunoreactivity of p-Stat3-Ser(727) was significantly higher in hydatidiform mole (HM) (P < 0.001) and choriocarcinoma (P = 0.009) when compared with normal placentas. Placental site trophoblastic tumours (PSTT) and epithelioid trophoblastic tumours (ETT) also demonstrated higher nuclear p-Stat3-Ser(727) expression than their normal trophoblast counterparts. Higher p-Stat3-Ser(727) expression was confirmed in choriocarcinoma cell lines, JEG-3 and JAR, than in a normal trophoblast cell line, with both nuclear and cytoplasmic fractions demonstrated by immunoblotting. Spontaneously regressed HM showed significantly increased nuclear and cytoplasmic p-Stat3-Ser(727) immunoreactivity over those that developed gestational trophoblastic neoplasia (GTN) (P = 0.013, P = 0.039). There was a significant positive and inverse correlation between nuclear p-Stat3-Ser(727) immunoreactivity and apoptotic indices [terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling and M30 CytoDeath antibody] (P = 0.001, P < 0.001, Spearman's rho test) and Bcl-2 expression (P = 0.034), respectively. p-Stat3-Ser(727) plays a role in the pathogenesis of GTD, probably through the regulation of apoptosis. p-Stat3-Ser(727) immunoreactivity is a potential marker in predicting GTN in HM.

Differential transferrin expression in placentae from normal and abnormal pregnancies: a pilot study

BackgroundThe placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction.MethodsIn the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies.ResultsVariations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse).ConclusionThese observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.

Effects on human leukocyte antigen-G on proliferation and invasion of JEG-3 cell line

To investigate the effect of human leukocyte antigen-G (HLA-G) on the growth and invasion of JEG-3 cell line and the role of HLA-G in the onset and development of pre-eclampsia. The experiment was composed of three groups: groups of transfection, negative control and blank control, which corresponded to groups of HLA-G siRNA transfection, negative siRNA transfection and no transfection. HLA-G overexpressed choriocarcinoma cell line JEG-3 was used. The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA (siRNA). Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and western blot analysis. Changes of cell cycle, apoptosis, proliferation and invasion were respectively detected by methyl thiazolyl tetrazolium (MTT), flow cytometry assay and transwell test. (1) The mRNA and protein levels of HLA-G control group and blank control group were 0.0013 +/- 0.0014, 0.0163 +/- 0.0007 and 0.1923 +/- 0.0384, 0.2184 +/- 0.0153, respectively, which were both significantly different (P<0.05); the number of negative transfection group was 0.1606 +/- 0.0133 and 0.2020 +/- 0.0132, which had no significant difference compared with blank control group (P>0.05). (2) The integral absorbance (IA) values of the HLA-G transfection group and blank control group were 0.44 +/- 0.04 and 0.75 +/- 0.13 respectively, which was significantly different (P<0.01); the IA value of negative control group was 0.69 +/- 0.10, which was not significantly different compared with blank group (P>0.05). (3) The ratios of G2/M and S phase cells in transfection group were (10.9 +/- 2.2)% and (58.6 +/- 0.8)% respectively, significantly different compared with the blank control group [(15.4 +/- 1.9)% and (52.9 +/- 2.3)% respectively; P<0.01]. (4) The ratio of early apoptosis cells in transfection group [(14.5 +/- 2.7)%] was significantly increased compared with negative [(5.3 +/- 1.1)%] and blank control group [(4.7 +/- 0.6)% ; P<0.01]. (5) The invasion number of transfection group and blank control group were 121+/- 12 and 452 +/- 17 respectively, with a significant difference between them (P<0.01). HLA-G is probably involved in the onset of preeclampsia by regulating proliferation and invasion of trophoblast.

Endothelin (ET)‐1 and ET‐3 promote expression ofc‐fosandc‐junin human choriocarcinoma via ETBreceptor‐mediated Gi‐ and Gq‐pathways and MAP kinase activation

Endothelins (ETs) and their G protein-coupled receptors exert key physiological functions during normal and aberrant placental development. Trophoblast cells mediate the contact between the embryo and the mother, by establishing a transient organ, the placenta. Choriocarcinoma cells display many of the biochemical and morphological characteristics of in utero invasive trophoblast cells and may therefore be used as a suitable model to study epithelial tumour progression of foetal-derived cells. The present study aimed at investigating ET receptor-mediated activation of the mitogen-activated protein kinase (MAPK) pathway in human choriocarcinoma. Both JAR and Jeg-3 choriocarcinoma cell lines expressed ET receptor subtype B (ET(B)) but not ET(A) receptor transcripts. ET(B) receptor engagement by ET-1 and ET-3 resulted in a similar time- and concentration-dependent phosphorylation of p42/44 MAPK, also known as extracellular regulated kinase 1/2. Using specific pharmacological antagonists/inhibitors, we showed that ET-1/-3-mediated signal transduction by the ET(B) receptor is transmitted via G(i)- and G(q)-dependent pathways through activation of the Src (G(i)) and protein kinase C (G(q)) axis that converge at Ras/Raf, leading to downstream activation of p42/44. On a functional level, ET(B) engagement and subsequent phosphorylation of p42/44 resulted in enhanced transcription of the immediate early response genes c-fos and c-jun, a process commonly assumed to be mediated by the ET(A) receptor, and increased cell growth and relative cell area. As human choriocarcinoma cells secrete ETs, pharmacological antagonism of ETs and/or ET(B) receptor-mediated signal transduction could represent a likely target therapy for choriocarcinoma.

Oct4 is Epigenetically Regulated by Methylation in Normal Placenta and Gestational Trophoblastic Disease

Oct4 is a transcription factor that plays a crucial role in maintaining pluripotency of embryonic stem cells. Down-regulation of Oct4 is associated with the differentiation of trophectoderm cell lineage, from which the normal placenta derives. We investigated the methylation and expression status of Oct4 in normal placenta and gestational trophoblastic disease (GTD) as attempts to investigate the role of Oct4 in the pathogenesis of GTD. By methylation-specific PCR, we observed both methylated and unmethylated Oct4 alleles in all 25 first trimester and 10 term placentas while 33% (18/54) of hydatidiform moles, and two choriocarcinoma cell line (JEG3 and JAR), only displayed methylated Oct4 allele. By quantitative TaqMan real-time PCR, Oct4 mRNA was significantly reduced in hydatidiform moles ( P = 0.04), JEG3 and JAR ( P = 0.024) when compared with normal placentas. Oct4 methylation was significantly correlated with Oct4 mRNA expression in placenta and GTD ( P = 0.012). Hypermethylation in minimal promoter and exon 1 region of Oct4 were confirmed in JEG3 and JAR by bisulfite genomic sequencing. The Oct4 mRNA expression in JEG3 and JAR increased after treatment with 5-aza-2′-deoxycytidine and/or trichostatin A. Our findings suggest that Oct4 is down-regulated by hypermethylation in normal placenta and GTD and such process is important in pathogenesis of GTD.

Maintaining the integrity of trophoblast during growth of the placenta. Focus on “Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta”

maternal-fetal exchange of nutrients and respiratory gases occurs across the brush border of the trophoblast lining the intervillous space of the placenta. The trophoblast is syncytial in nature and has a high rate of turnover. Therefore syncytiotrophoblast is continually replenished from an

Modulation of Glucose Uptake in a Human Choriocarcinoma Cell Line (BeWo) by Dietary Bioactive Compounds and Drugs of Abuse

The aim of this work was to investigate the putative modulation of glucose uptake in trophoblast cells by several dietary compounds and by drugs of abuse. For this, the acute (26 min) and chronic (48 h) effect of these substances on the apical uptake of 3H-2-deoxy-D-glucose (3H-DG) by a human choriocarcinoma cell line (BeWo) was determined. 3H-DG apical uptake by BeWo cells was time dependent, displayed saturable kinetics (Vmax=1210+/-29 nmol mg protein(-1) 6 min(-1) and Km=13.4+/-0.5 mM) and was insulin-insensitive and cytochalasin B-sensitive (by up to 60%). Acutely, acetaldehyde (30-100 mM), resveratrol, xanthohumol, epigallocatechin-3-gallate (100 microM), chrysin and quercetin (10-100 microM) decreased 3H-DG apical uptake, whereas rutin, catechin (10-100 microM), epicatechin (100 microM) and ethanol (10 mM) increased it. Quercetin and xanthohumol seem to be non-competitive inhibitors of 3H-DG apical uptake, whereas both epigallocatechin-3-gallate and acetaldehyde decreased both the Km and Vmax values. Chronically, rutin and myricetin increased the apical uptake of 3H-DG both isolated (0.1-1 microM) and in combination (both at 1 microM), whereas theophylline (0.1-1 microM) and amphetamine, 3,4-methylenedioxymethamphetamine (0.25-1 microM) and Delta9-tetrahydrocannabinol (1 nM) decreased it. In conclusion, 3H-DG apical uptake by BeWo cells is differentially modulated by different compounds present in drinks and by drugs of abuse.

Stimuli through Toll‐like receptor (TLR) 3 and 9 affect human chorionic gonadotropin (hCG) production in a choriocarcinoma cell line

Toll-like receptors (TLR) are expressed mostly on mucosal innate immune cells which recognize microorganism-associated structures, the so called pathogen-associated molecular patterns (PAMP). Recently, possible roles of TLR in placental tissue, especially their role in protecting the conceptus from maternal infection, have been discussed, but the physiopathological roles of TLR in placental functions have yet to be examined. The choriocarcinoma cell line BeWo was used as a model of trophoblast stem cells. First, the expression of TLR 1-9 on BeWo cells was investigated using reverse transcriptase-polymerase chain reaction. Then, the cells were treated with TLR 1-9 agonists, using the human TLR 1-9 agonist kit (Pam3CKS4, HKLM, Poly (I:C), Escherichia coli K12 lipopolysaccharide (LPS), Salmonella typhimurium flagellin, FSL1, imiquimod, ssRNA 40, and ODN 2006). The concentration of human chorionic gonadotropin (hCG) in the cell culture supernatants was examined on ELISA. BeWo cells constitutively expressed TLR 1-9 mRNA in the presence or absence of forskolin. The TLR 3 agonist, Poly(I:C), and the TLR 9 agonist, CpG-DNA (ODN2006), upregulated hCG production but had minimal effects in the absence of forskolin. Stimulation with the other TLR agonists produced no remarkable increase, whether in the presence or absence of forskolin. TLR 3 and TLR 9 agonists upregulated hCG production in BeWo cells. The present observations suggest alternative roles of TLR signaling in the control of placental functions and blastocyst-endometrial dialogue, as well as in pathophysiology, including for infertility and ectopic pregnancy.

Characterization of VAR2CSA-deficient Plasmodium falciparum-infected erythrocytes selected for adhesion to the BeWo placental cell line.

BackgroundMalaria in pregnancy is characterized by accumulation of infected erythrocytes (IE) in the placenta. The key ligand identified as mediating this process is a Plasmodium falciparum erythrocyte membrane protein 1 family member, termed VAR2CSA. VAR2CSA appears to be the main ligand responsible for adhesion to chondroitin sulphate A (CSA). Whether other PfEMP1 molecules can also mediate placental adhesion, independent of CSA binding, is unclear.MethodsThe parasite line CS2 carrying a disrupted var2csa gene (CS2KO) was selected for adhesion to the BeWo choriocarcinoma cell line, which has been proposed as a model for placental malaria. The selected and control IE were tested for adhesion to placental sections and flow cytometry was used to measure recognition of IE by three serum sets from malaria-exposed men and women.ResultsWild-type CS2 adhere to BeWo and placental tissue via CSA. CS2KO IE were successfully selected for adhesion to BeWo, and adhered by a CSA-independent mechanism. They bound to immobilized ICAM-1 and CD36. BeWo-selected CS2KO bound at moderate levels to placental sections, but most binding was to placental villi rather than to the syncytiotrophoblast to which IE adherence occurs in vivo. This binding was inhibited by a blocking antibody to CD36 but not to ICAM-1. As expected, sera from malaria-exposed adults recognized CS2 IE in a gender and parity dependent manner. In one serum set, there was a similar but less pronounced pattern of antibody binding to selected CS2KO IE, but this was not seen in two others. One var gene, It4var19, was particularly abundant in the selected line and was detected as full length transcripts in BeWo-selected IE, but not unselected CS2KO.ConclusionThis study suggests that IE with characteristics similar to the CS2KO have a limited role in the pathogenesis of placental malaria. VAR2CSA appear to be the major ligand for placental adhesion, and could be the basis for a vaccine against pregnancy malaria.

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.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution.

Histone deacetylase inhibitors induce growth inhibition, cell cycle arrest and apoptosis in human choriocarcinoma cells

We investigated the effect of five histone deacetylase inhibitors (HDACIs) on the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of five HDACIs, and their effects on cell growth, cell cycle, apoptosis, and related measurements were investigated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays showed that the BeWo choriocarcinoma cell line was sensitive to the growth inhibitory effect of five HDACIs. Cell cycle analysis indicated that exposure to HDACIs decreased the proportion of cells in the S-phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Furthermore, HDACI treatment of this cell line increased acetylation of H3 and H4 histone tails. These results raise the possibility that HDACIs may prove particularly effective in the treatment of choriocarcinoma.

Analysis of the promoter region of human placenta-specific DSCR4 gene

The gene DSCR4 locates in the band q22.2 of human chromosome 21 and encodes a protein of 118 amino acids. Expression of DSCR4 is restricted to human placenta and placental choriocarcinoma cell lines BeWo and JEG3. The 5'-RACE method using RNA from human placenta indicated the major transcription start site at 93 nt upstream (nt -93) of the initiation codon. Transfection assay using a series of deletion constructs of the 5'-flanking region fused to the luciferase reporter gene identified three positive regions nt -2200 to -2088, nt -2064 to -1924, nt -810 to -632 and two negative regions nt -1923 to -1740, nt -631 to -425. The computer analysis predicted the presence of several cis-elements in these regions and the promoter assay using various mutants of consensus sequence identified two distinct cis-elements for OLF-1 and E47. The electrophoretic mobility shift assay (EMSA) using the extracts of DSCR4-expressing cells confirmed the binding of certain protein factors to these cis-elements. In fact, OLF-1-like transcription factor, EBF-3 and EBF-4 were detected in the DSCR4-expressing cell lines and human placenta. Based on these data, we postulated that transcription of DSCR4 gene is regulated positively by binding of OLF-1-like transcription factor and negatively by binding of E47-like transcription factor.