Nitric oxide inhibits polyamine-induced apoptosis in the human extravillous trophoblast cell line SGHPL-4

BACKGROUND: Polyamines are regulators of proliferation and differentiation in mammalian cells. They are also known to regulate cell survival and apoptosis, although their precise function varies between cell types. We have investigated the effect of polyamines on the apoptosis of human extravillous trophoblasts. METHODS: Using the extravillous trophoblast-derived cell line SGHPL-4 we performed time-lapse microscopy studies to evaluate the induction of apoptosis following exposure to polyamines. RESULTS: The polyamines spermine, and to a lesser extent spermidine, were able to induce apoptosis in extravillous trophoblasts. The induction of apoptosis occurred rapidly and was accompanied by DNA fragmentation and morphological changes consistent with the onset of apoptosis. Apoptosis was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk, although no activity was detected using assays for caspase-2, -3, -6, -8 or -9 activity. We demonstrated that an oxidation product of spermine accounted for the induction of apoptosis and implicated the formation of hydrogen peroxide as this oxidation product. We have also demonstrated that exposure to nitric oxide inhibited the onset of spermine-induced apoptosis. CONCLUSIONS: Spermine and spermidine induce apoptosis in extravillous trophoblasts following their oxidation and the production of hydrogen peroxide. Nitric oxide is able to inhibit this apoptosis.     

Intermediate filaments of human trophoblast and choriocarcinoma cell lines

Summary Human term placenta and two human choriocarcinoma cell lines were studied immunohistochemically and by immunoblotting with monoclonal antibodies to keratin polypeptides and vimentin. Four keratin polypeptides (40, 45, 52, 54 K) were detected in both normal and malignant trophoblastic cells. The presence of the 54 K keratin polypeptide distinguishes the benign and malignant trophoblastic cells from human embryonal carcinoma cells and a yolk sac carcinoma cell line.     

Establishment of a choriocarcinoma model from immortalized normal extravillous trophoblast cells transduced with HRASV12

Gestational choriocarcinoma is a malignant trophoblastic tumor. The development of novel molecular-targeted therapies is needed to reduce the toxicity of current multiagent chemotherapy and to treat successfully the chemoresistant cases. The molecular mechanisms underlying choriocarcinoma tumorigenesis remain uncharacterized, however, and appropriate choriocarcinoma animal models have not yet been developed. In this study, we established a choriocarcinoma model by inoculating mice with induced-choriocarcinoma cell-1 (iC3-1) cells, generated from HTR8/SVneo human trophoblastic cells retrovirally transduced with activated H-RAS (HRASV12). The iC3-1 cells exhibited constitutive activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and developed into lethal tumors in all inoculated mice. Histopathological analysis revealed that the tumors consisted of two distinct types of cells, reminiscent of syncytiotrophoblasts and cytotrophoblasts, as seen in the human choriocarcinoma. The tumors expressed HLA-G and cytokeratin (trophoblast markers) and hCG (a choriocarcinoma marker). Comparative analysis of gene expression profiles between iC3-1 cells and parental HTR8/SVneo cells revealed that iC3-1 cells expressed matrix metalloproteinases, epithelial-mesenchymal transition-related genes, and SOX3 at higher levels than parental trophoblastic cells. Administration of SOX3-specific short-hairpin RNA decreased SOX3 expression and attenuated the tumorigenic activity of iC3-1 cells, suggesting that SOX3 overexpression might be critically involved in the pathogenesis of choriocarcinoma. Our murine model represents a potent new tool for studying the pathogenesis and treatment of choriocarcinoma.     

Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.     

Cyclic AMP enhances the expression of an extravillous trophoblast marker,melanoma cell adhesion molecule,in choriocarcinoma cell JEG3 and human chorionic villous explant cultures

Human trophoblasts consist of two main cell lineages, villous trophoblasts (VT) and extravillous trophoblasts (EVT). To identify the molecules which are involved in EVT differentiation, we have raised a monoclonal antibody (mAb) designated CHL1, by immunizing a mouse against human chorion laeve which is composed of EVT. By immunohistochemical analysis, the CHL1 antigen was found to be expressed on the majority of EVT but not on VT in addition to its expression on endothelial and myometrial cells. A subsequent cDNA panning method revealed that the CHL1 antigen was identical to melanoma cell adhesion molecule (MCAM, Mel-CAM, S-endo 1 or MUC18/CD146), which has been previously reported as one of the EVT markers. MCAM expression on JEG3 cells, a human choriocarcinoma-derived cell line, was significantly enhanced when they were co-cultured with isolated human decidual tissue. Various cytokines and growth factors that were reportedly present in decidual tissue failed to increase MCAM expression in JEG3 cells, but decidua-induced MCAM expression in JEG3 cells was attenuated by the addition of protein kinase A inhibitor H89. In addition, cAMP, which is known to stimulate differentiation of VT, enhanced MCAM expression in JEG3 cells. Its promoting effect on MCAM expression was also observed in human chorionic villous explant cultures. These findings suggest that a cAMP-dependent intracytoplasmic signalling pathway is involved in the differentiation mechanism of human EVT.     

Effects of interleukin-6 on extravillous trophoblast invasion in early human pregnancy

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(?) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.     

Immunological studies of trophoblast antigens: no evidence for human leucocyte antigen (HLA) linkage

Parental disparity for trophoblast-lymphocyte crossreactive (TLX) antigens may promote successful pregnancy. A TLX antigen system has been defined on peripheral blood lymphocytes by heteroantisera. More recently, we have reported additional activity against antigens on B lymphocytes alone termed trophoblast-B lymphocyte crossreactive (TBX) antigens. In the present study we have investigated ten TLX sera in order to determine if their target antigens are linked to the human leucocyte antigen (HLA) gene complex. The sera showed no selective activity when tested against target B lymphocytes from ten normal donors. Cytotoxic activity of TLX antisera against peripheral blood lymphocytes from six normal donors was not reduced when the class I HLA antigens of the target cells were blocked with a monoclonal antibody (PA 2.6). Similarly the cytotoxic activity of both TBX antisera against B lymphocytes from six normal donors was not decreased when class II HLA antigens were blocked by a monoclonal antibody (FMC 4). Within a family the cytotoxic activity of the TLX antisera was absorbed equally by lymphocytes from siblings who shared neither HLA haplotype. Antibody content in TLX and TBX antisera is not directed toward the classically defined HLA class I or class II antigens and is not linked to the HLA gene complex.     

Evaluation of a monoclonal antibody, BC-1, which identifies an antigen expressed on the surface membrane of human extravillous trophoblast.

This paper describes a new Mab BC-1 which is directed at a cell surface antigen expressed by extravillous cytotrophoblast and not by villous cytotrophoblast, so it is useful for distinguishing between the two populations. The antigen recognised by BC-1 is trypsin-resistant, which allows it to be used for flow cytometric analysis of living trophoblast isolated by enzymic disaggregation. However, BC-1 is not trophoblast-specific but cross-reacts with some other tissues, in particular endothelial cells and peripheral blood monocytes. The nature of this antigen has not been established.     

The isolation and characterization of a population of extravillous trophoblast progenitors from first trimester human placenta

BACKGROUND: It is widely accepted that most if not all villous cytotrophoblasts from term placentae are committed to differentiate into syncytiotrophoblast, but that early in gestation villous cytotrophoblasts are bipotential and capable of differentiating into either extravillous trophoblasts (EVTs) or syncytiotrophoblast. In contrast, our previous work has suggested that two separate populations of cytotrophoblast exist in the first trimester, one committed to EVT differentiation and the other to syncytiotrophoblast differentiation. In this work, we have isolated and characterized the population of 'EVT progenitors'. METHODS: First trimester villous explants were cultured for 10 days then subjected to sequential trypsinization. Viable cells that adhered to Matrigel following trypsinization were cultured for up to 5 days and characterized by immunohistochemistry. RESULTS: The isolation protocol yielded >90% cytokeratin positive trophoblasts, which expressed markers characteristic of EVT progenitors. Over 5 days of culture, these isolated putative EVT progenitors did not syncytialize, but approximately 20% differentiated into HLA-G positive EVTs. CONCLUSIONS: It is likely that the isolated putative EVT progenitors are the population of EVT progenitors previously identified in vivo. The characteristics of these isolated putative EVT progenitors provides further evidence for separate progenitors of EVT and syncytiotrophoblast in the first trimester.     

The BeWo cell line derived from a human placental choriocarcinoma is permissive for respiratory syncytial virus infection

Virus Genes - The respiratory syncytial virus (RSV) is the main pathogen associated with upper respiratory tract infections during early childhood. Vertical transmission of this virus has been...     

Effects of anti-phospholipid antibodies on a human trophoblast cell line (HTR-8/SVneo)

Antibodies to phospholipids (aPL) have been shown to adversely affect trophoblast invasion in vivo and in vitro. HTR-8/SVneo cells derived from first trimester of pregnancy extravillous trophoblast were studied. Matrigel invasion assay, cytochemistry and cell-based enzyme-linked immunosorbant assay (ELISA) with aPL or normal IgG was used. Our data show that aPL at 100 microg/ml decrease invasiveness of HTR-8/SVneo cells to 60% of control (p<0.01), and this was also shown for primary cytotrophoblast (to 15.5% of control, p<0.001). aPL treatment caused a significant decrease in integrin alpha(1), alpha(5), and beta(1) proteins (86%, 84%, and 87%, respectively). We conclude that HTR-8/SVneo cell culture is a suitable model to study mechanisms of action of aPL on trophoblast, which in HTR-8/SVneo cells inhibit invasion by decreasing integrins alpha(5), alpha(1), and beta(1).     

Discovery of a stable cell line antigen in human internal organs

A cell membrane antigen was found in stable cell lines of epithelial origin, identical to an antigen in homogenates of the human gastric mucosa. The antigen was detected by means of antiserum against extracted membrane antigens of HeP-2 cells, absorbed by a mixture of homogenates of human lung, liver, and papillomas of the human larynx and breast. The antigen described was found in some of the adenocarcinomas of the stomach that were tested. It differs from Gold's carcinoembryonic antigen and from the secretory component of IgA, it is not the structural antigen of the D-type primate oncornavirus produced by HeP-2 cells and, evidently, it is not coded by the genome of that virus.Department of Etiology and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 690–693, December, 1977.