Choriocarcinoma Tissues Research Articles

CLIC1 Protein: A Candidate Prognostic Biomarker for Malignant-Transformed Hydatidiform Moles

The purpose of this study was to identify prognostic biomarkers indicating malignant transformation of hydatidiform moles (HMs). Two-dimensional gel electrophoresis-based proteomic approach was used to compare the protein profiles of complete benign moles (3 samples) with those of malignant-transformed moles (3 samples). Matrix-assisted laser desorption/ionization time of flight mass spectrometry was used to identify differentially expressed proteins. Western blot was used to verify the results of 2-dimensional gel electrophoresis, and immunohistology was used to explore the function of these proteins in gestational trophoblastic disease. Eighteen proteins, deregulated in the malignant-transformed group compared with the benign group (ratio ≥ 2; P < 0.05), were identified. A bioinformatic analysis indicated that most of these 18 proteins were involved in the processes of cell proliferation and cell survival. Among the 18 proteins, chloride intracellular channel protein 1 (CLIC1) was chosen for further study. Our results showed that the levels of CLIC1 expression in choriocarcinoma tissue were higher than in complete HM tissue (P < 0.01). Chloride intracellular channel protein 1 expression was increased in the tissues of malignant-transformed HMs compared with nontransformed HMs (P < 0.01). Our findings suggest that CLIC1 could be a potential new prognostic biomarker for hydatidiform mole that undergoes malignant transformation.

Genetic genesis of choriocarcinoma

To distinguish choriocarcinoma from gestational or non-gestational choriocarcinoma and also identify the causative pregnancy of gestational choriocarcinoma by the genetic origin through molecular genetic analysis. Twelve patients with choriocarcinoma, who had experienced surgery prior to chemotherapy were enrolled in this study. All 12 cases were diagnosed pathologically as choriocarcinoma. Peripheral venous blood samples and formalin-fixed paraffin-embedded blocks of choriocarcinoma tissue microdissected from haematoxylin and eosin-stained sections of tissue by microdissection method were available from the patient and (or) her husband. DNA was then prepared from the couples' blood samples and choriocarcinoma tissue by using standard techniques. PCR amplification and fluorescent microsatellite genotyping were performed by using DNA from the couples and captured choriocarcinoma tissues. The genetic contributions to the choriocarcinoma tissue were determined by comparing the fragments of genes from the choriocarcinoma tissue to those from blood samples of the couples. The primary lesion was ovary in 7 cases, but only 4 of them had the maternal contribution, indicating a non-gestational origin; the other three were gestational choriocarcinoma. The primary lesion was uterus in 5 cases, which were all gestational choriocarcinoma confirmed by genetic analyses. The causative pregnancies of the 8 cases with gestational choriocarcinoma were identified as androgenetic complete hydatidiform mole (AnCHM) in six cases and normal pregnancies in two cases, respectively. Microsatellite polymorphism analysis is a molecular approach for distinguishing the non-gestational choriocarcinoma from the gestational one, and also be used to identify the causative pregnancy of gestational choriocarcinoma.

Molecular Genetic Analyses of Choriocarcinoma

Background Choriocarcinoma is a highly malignant trophoblastic neoplasm. Most of them are gestational in origin, while non-gestational ones are exceedingly rare. The genetic origin, immunogenicity, sensitivity to chemotherapy and prognosis of these two kinds of conditions are quite different, so identification of these two kinds of choriocarcinoma is of great importance. The objective of this study is to distinguish choriocarcinoma as gestational or non-gestational and identify the causative pregnancy of gestational choriocarcinoma through molecular genetic analysis. Methods Twelve patients with choriocarcinoma, who had experienced surgery prior to chemotherapy, were enrolled in this study. DNA was prepared from blood samples from the patient and her partner using standard techniques. In order to prepare DNA from choriocarcinoma tissue, areas of choriocarcinoma were firstly microdissected from haematoxylin and eosin-stained sections. PCR amplification and fluorescent microsatellite genotyping were performed using DNA from the couples and captured tissue. The genetic contributions to the choriocarcinoma were determined by comparing the genotypes of the choriocarcinoma and that of the couples. Results Four of twelve cases had only a maternal contribution, indicating a non-gestational origin. The remaining eight cases were all gestational in origin and the causative pregnancies were identified as AnCHM (androgenetic complete hydatidiform mole) in six and normal pregnancies in two respectively. Conclusion Microsatellite polymorphism analysis is a molecular approach for distinguishing the non-gestational choriocarcinoma from the gestational one, and can also be used to identify the causative pregnancy of gestational choriocarcinoma. Antecedent pregnancy prior to choriocarcinoma is not always its causative pregnancy. Therefore, it is reasonable to identify the causative pregnancy by its genetic origin, instead of clinical impression.

Expression and Localization of StarD7 in Trophoblast Cells

The StAR-related lipid transfer (START) domain is defined as a motif of around 200 amino acids implicated in lipid/sterol binding. In a previous study, we identified the StarD7 transcript encoding one of the 15 family members with START domain present in the human genome. This transcript was found to be overexpressed in choriocarcinoma JEG-3 cells. In addition, we demonstrated that the recombinant StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface, showing marked surface activity and interaction with phospholipid monolayers, mainly with phosphatidylserine, cholesterol and phosphatidylglycerol. This study was undertaken to evaluate the expression and localization of StarD7 protein in trophoblastic samples. Here, we show for the first time the presence of StarD7 protein in human trophoblast cells. Western blot assays revealed a unique specific 34 kDa protein in JEG-3 cell line, choriocarcinoma tissue, complete hydatidiform mole, early and normal term placenta. Immunohistochemical data from early and normal term placentas and complete hydatidiform moles showed that this protein is abundant in the syncytiotrophoblasts, mainly at the apical side of the syncytium, with a weak and focal reaction in the cytotrophoblast cells. Furthermore, an increased StarD7 mRNA and protein expression, as well as a change in its sub-cellular localization was observed in in vitro differentiating cytotrophoblast isolated from normal term placenta. Taken together, these findings support and allow future studies to explore the possibility that StarD7 protein mediates transplacental lipid transport and/or is involved in syncytialization.

IGF-II regulates metastatic properties of choriocarcinoma cells through the activation of the insulin receptor

Choriocarcinoma is a highly malignant tumor that can arise from trophoblasts of any type of gestational event but most often from complete hydatidiform mole. IGF-II plays a fundamental role in placental development and may play a role in gestational trophoblastic diseases. Several studies have shown that IGF-II is expressed at high levels in hydatidiform moles and choriocarcinoma tissues; however, conflicting data exist on how IGF-II regulates the behaviour of choriocarcinoma cells. The purpose of this study was to determine the contribution of the receptors for IGF-I and insulin to the actions of IGF-II on the regulation of choriocarcinoma cells metastasis. An Immuno Radio Metric Assay was used to analyse the circulating and tissue levels of IGF-I and IGF-II in 24 cases of hydatidiform mole, two cases of choriocarcinoma and eight cases of spontaneous abortion at the same gestational age. The JEG-3 choriocarcinoma cell line was used to investigate the role of IGF-II in the regulation of cell invasion. We found that mole and choriocarcinoma tissue express high levels of IGF-II compared to first trimester placenta. Both IGF-I and IGF-II regulate choriocarcinoma cell invasion in a dose dependent manner but through a different mechanism. IGF-II effects involve the activation of the InsR while IGF-I uses the IGF-IR. The positive effects of IGF-II on invasion are the result of enhanced cell adhesion and chemotaxis (specifically towards collagen IV). The actions of IGF-II but not those of IGF-I were sensitive to inhibition by the insulin receptor inhibitor HNMPA(AM)3. Our results demonstrate that the insulin receptor regulates choriocarcinoma cell invasion.

Epidermal growth factor receptor is a marker for syncytiotrophoblastic cells in testicular germ cell tumors

Neutral endopeptidase (NEP)/CD10, a cell-surface peptidase degrading various bioactive peptides, is mainly present in syncytiotrophoblasts in the human placenta. However, the change in NEP expression upon trophoblast differentiation remains to be clarified. In the present study, we examined the expression of NEP in the differentiating trophoblast using the BeWo choriocarcinoma cell line as a model system. Under the normal culture conditions, NEP was very weakly expressed on most proliferating cytotrophoblastic BeWo cells, while a minority of the cell population (less than 5 per cent ), consisting of giant, multinucleated cells, clearly expressed NEP at the cell membrane. Treatment of BeWo cells with forskolin (FSK) for 48–72 h resulted in an 11− to 44-fold increase in the level of hCG secretion and induced cell fusion leading to the formation of multinucleated syncytiotrophoblasts, indicating functional and morphological differentiation. Fluorescence-activated cell sorting (FACS) analysis revealed that treatment with FSK significantly increased the cell-surface protein expression of NEP on differentiating BeWo cells. Consistently, there was a significant increase in the NEP enzymatic activity after FSK treatment. The level of hCG secretion from the FSK-treated cells was further enhanced when the cells were treated in the presence of the NEP inhibitor phosphoramidon. Immunohistochemical analysis of normal chorionic villi and choriocarcinoma tissues revealed the localization of NEP in syncytiotrophoblastic cells, as opposed to weak or negative staining in cytotrophoblastic cells. These data demonstrate that induction of choriocarcinoma cell differentiation is associated with an increase of NEP/CD10 expression at the cell surface, suggesting a role of this enzyme in regulating differentiated trophoblast functions such as hCG secretion. NEP/CD10 may also be a new cellular differentiation marker of both the normal and neoplastic trophoblast.

Expression of Placental Leucine Aminopeptidase and Adipocyte-Derived Leucine Aminopeptidase in Human Normal and Malignant Invasive Trophoblastic Cells

We recently identified two novel aminopeptidases, placental leucine aminopeptidase (P-LAP) and adipocyte-derived leucine aminopeptidase (A-LAP). Enzymatically, P-LAP degrades oxytocin, vasopressin, and angiotensin III, while A-LAP degrades angiotensin II and kallidin. In this study we investigated the expression and localization of P-LAP and A-LAP in human trophoblastic cells in the normal placenta (n = 26), gestational choriocarcinoma (n = 8), and placental site trophoblastic tumor (n = 3). On immunoblot analysis both P-LAP and A-LAP proteins were detected in normal placenta and five choriocarcinoma tissues, as well as in two choriocarcinoma cell lines. Immunohistochemical staining of normal placental tissues demonstrated that P-LAP was not only localized in villous syncytiotrophoblasts but also highly expressed in extravillous trophoblasts (EVTs) invading the decidua or maternal spiral arteries. The expression level of P-LAP on these invasive EVTs reached a maximum during the late first to second trimesters of pregnancy, and it decreased in the third trimester. Similarly, A-LAP was strongly expressed in EVTs invading the decidua or spiral arteries in the second trimester of pregnancy, while it was weakly or moderately expressed in villous cytotrophoblasts or EVTs located in the cell columns. These two aminopeptidases were more strongly expressed in all eight choriocarcinomas and three placental site trophoblastic tumors and mainly localized to the intermediate-type trophoblastic tumor cells invading the uterine myometrium or stromal vessels. In summary P-LAP and A-LAP were predominantly expressed in the invasive phenotype of EVTs during placentation, as well as in the invasive tumor cells of trophoblastic neoplasms. These results suggest the involvement of these aminopeptidases in invasiveness of both normal and malignant intermediate-type trophoblasts possibly through degradation of specific peptide substrates.

Enhancement of aminopeptidase A expression during angiotensin II-induced choriocarcinoma cell proliferation through AT1 receptor involving protein kinase C- and mitogen-activated protein kinase-dependent signaling pathway.

Angiotensin II (Ang II) is a bioactive peptide of the renin-angiotensin system, exerting its actions not only as a vasoconstrictor, but also as a growth promoter. In human placenta, type 1 Ang II receptors (AT(1)R) are predominantly expressed in trophoblasts, and we previously reported that aminopeptidase A (APA), a cell surface peptidase that converts Ang II to Ang III, is also expressed in both normal and neoplastic trophoblasts. However, the roles of Ang II and APA in trophoblast function remain to be clarified. In the present study we examined the effects of Ang II on proliferation and APA expression in trophoblast-like BeWo choriocarcinoma cells. Treatment of BeWo cells with Ang II significantly increased DNA synthesis in a dose-dependent manner. Ang II also enhanced APA mRNA and cell surface expression in BeWo cells analyzed by Northern blotting, flow cytometry, and enzyme activity assay. The Ang II-induced proliferation and APA up-regulation were blocked by the AT(1)R antagonist candesartan, but not by the AT(2)R antagonist PD123319. Furthermore, these Ang II effects were abolished by the protein kinase C inhibitor bisindolylmaleimide I and the MAPK inhibitor PD98059. Immunohistochemistry using choriocarcinoma tissues demonstrated that APA was expressed on the cell surface of AT(1)R-positive cytotrophoblastic cells in vivo. With these findings we demonstrate that Ang II stimulates the proliferation of trophoblastic cells via AT(1)R that are linked to protein kinase C /MAPK-dependent signaling pathways, and that the Ang II-degrading enzyme APA is up-regulated during Ang II-induced cell proliferation. These observations suggest the possible regulatory mechanism by the local renin-angiotensin system, especially the Ang II-AT(1)R-APA system, for the growth of human choriocarcinoma cells.

NECC1, a candidate choriocarcinoma suppressor gene that encodes a homeodomain consensus motif

We isolated a candidate choriocarcinoma suppressor gene from a PCR-based subtracted fragmentary cDNA library between normal placental villi and the choriocarcinoma cell line CC1. This gene comprises an open reading frame of 219 nt encoding 73 amino acids and contains a homeodomain as a consensus motif. This gene, designated NECC1 (not expressed in choriocarcinoma clone 1), is located on human chromosome 4q11–q12. NECC1 expression is ubiquitous in the brain, placenta, lung, smooth muscle, uterus, bladder, kidney, and spleen. Normal placental villi expressed NECC1, but all choriocarcinoma cell lines examined and most of the surgically removed choriocarcinoma tissue samples failed to express it. We transfected this gene into choriocarcinoma cell lines and observed remarkable alterations in cell morphology and suppression of in vivo tumorigenesis. Induction of CSH1 (chorionic somatomammotropin hormone 1) by NECC1 expression suggested differentiation of choriocarcinoma cells to syncytiotrophoblasts. Our results suggest that loss of NECC1 expression is involved in malignant conversion of placental trophoblasts.

Reduced expression of tissue inhibitor of metalloproteinase (TIMP)-2 in gestational trophoblastic diseases.

To elucidate the involvement of type IV collagenases [matrix metalloproteinase (MMP)-2 and MMP-9] and their tissue inhibitors (TIMP-1 and TIMP-2) in the development of gestational trophoblastic disease (GTD), we quantified their levels in hydatidiform mole and choriocarcinoma tissues using specific enzyme-linked immunosorbent assays, and the results were compared with those from normal first trimester placenta. Levels of pro-MMP-2 were increased in hydatidiform mole, and they were further elevated in choriocarcinoma. Levels of pro-MMP-9 in choriocarcinoma and those of TIMP-1 in both hydatidiform mole and choriocarcinoma were also increased. In contrast, TIMP-2 levels were markedly decreased in both hydatidiform mole and choriocarcinoma. Similar results were obtained by the tissue culture of first trimester placenta and hydatidiform mole. Gelatin zymography indicated that the levels of both pro- and activated forms of MMP-2 and MMP-9 were higher in hydatidiform mole and choriocarcinoma. The decreased expression of TIMP-2 in hydatidiform mole and choriocarcinoma was confirmed by Western blot, Northern blot and immunohistochemistry, with the decrease being more pronounced in choriocarcinoma. Taken together, the present study shows that both TIMP-2 mRNA and protein levels are markedly decreased in GTD and the imbalance of MMP-TIMP production, shifted toward greater MMP activity, may be involved in the pathogenesis of GTD.

Human chorionic gonadotropin (hCG) beta-core fragment is produced by degradation of hCG or free hCG beta in gestational trophoblastic tumors: a possible marker for early detection of persistent postmolar gestational trophoblastic disease

The present study was undertaken to investigate whether human chorionic gonadotropin (hCG) beta-core fragment (hCG beta cf) was directly produced by gestational trophoblastic tumors. Immunoreactivity of hCG beta cf was demonstrated in the extracts as well as in the culture media of hydatidiform mole tissues. It was also present in the extracts of choriocarcinoma tissues, and its molar concentration exceeded that of intact hCG. The presence of hCG beta cf was then confirmed by gel chromatography and Western blot analysis. Immunohistochemistry showed localization of hCG beta cf immunoreactivity to the syncytiotrophoblasts and scattered cells in the stroma of mole tissue, and to syncytiotrophoblastic cells in choriocarcinoma. Immunoreactivity of hCG beta cf was also detected in the sera of the patients with gestational trophoblastic disease, although the hCG beta cf/hCG ratio was less than one hundredth of that in the tissue extracts. Serial measurement of serum hCG beta cf levels after mole evacuation showed that they declined much more rapidly than those of hCG and became undetectable in the patients with subsequent spontaneous resolution, while hCG beta cf remained or became detectable before the rise of hCG was observed in the patients with subsequent persistent trophoblastic disease. Taken together, these results suggest that hCG beta cf is directly produced by gestational trophoblastic tumors, and monitoring of hCG beta cf in the serum after mole evacuation may be useful for early prediction of subsequent development of postmolar persistent trophoblastic disease.

Upregulation of Neutral Endopeptidase Expression and Enzymatic Activity during the Differentiation of Human Choriocarcinoma Cells

Neutral endopeptidase (NEP)/CD10, a cell-surface peptidase degrading various bioactive peptides, is mainly present in syncytiotrophoblasts in the human placenta. However, the change in NEP expression upon trophoblast differentiation remains to be clarified. In the present study, we examined the expression of NEP in the differentiating trophoblast using the BeWo choriocarcinoma cell line as a model system. Under the normal culture conditions, NEP was very weakly expressed on most proliferating cytotrophoblastic BeWo cells, while a minority of the cell population (less than 5 per cent ), consisting of giant, multinucleated cells, clearly expressed NEP at the cell membrane. Treatment of BeWo cells with forskolin (FSK) for 48–72h resulted in an 11− to 44-fold increase in the level of hCG secretion and induced cell fusion leading to the formation of multinucleated syncytiotrophoblasts, indicating functional and morphological differentiation. Fluorescence-activated cell sorting (FACS) analysis revealed that treatment with FSK significantly increased the cell-surface protein expression of NEP on differentiating BeWo cells. Consistently, there was a significant increase in the NEP enzymatic activity after FSK treatment. The level of hCG secretion from the FSK-treated cells was further enhanced when the cells were treated in the presence of the NEP inhibitor phosphoramidon. Immunohistochemical analysis of normal chorionic villi and choriocarcinoma tissues revealed the localization of NEP in syncytiotrophoblastic cells, as opposed to weak or negative staining in cytotrophoblastic cells. These data demonstrate that induction of choriocarcinoma cell differentiation is associated with an increase of NEP/CD10 expression at the cell surface, suggesting a role of this enzyme in regulating differentiated trophoblast functions such as hCG secretion. NEP/CD10 may also be a new cellular differentiation marker of both the normal and neoplastic trophoblast.

The Expression and Localization of Neutral Endopeptidase 24.11/CD10 in Human Gestational Trophoblastic Diseases

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.

Expression of Epidermal Growth Factor Receptor-Related Family Products in Gestational Trophoblastic Diseases and Normal Placenta and Its Relationship with Development of Postmolar Tumor

Objective.The goal of this work was to study the expression of epidermal growth factor receptor (EGFR) and c-erbB-3 and c-erbB-4 oncogenes in gestational trophoblastic diseases and normal first-trimester placenta.Study design. Paraffin sections of 16 cases of partial mole, 25 cases of complete mole, 10 cases of gestational choriocarcinoma, and 11 cases of therapeutic abortion were studied immunohistochemically for EGFR, c-erbB-3, and c-erbB-4 proteins. The presence of EGFR mRNA was studied using in situ hybridization.Results. Staining for EGFR was detected immunohistochemically in all cell types in gestational trophoblastic diseases and normal placenta. In situ hybridization for EGFR mRNA correlated with immunostaining for EGFR in all tissues studied. All 10 cases of choriocarcinoma exhibited strong immunoreactivity for EGFR. The levels of expression of EGFR in choriocarcinoma and syncytiotrophoblasts and cytotrophoblasts in complete mole were significantly greater than those in syncytiotrophoblasts and cytotrophoblasts in both normal placenta and partial mole (P < 0.01, P < 0.01). Expression of c-erbB-3 did not significantly differ among placental and gestational trophoblastic disease tissues and trophoblastic cell types except for significantly increased expression in choriocarcinoma as compared with cytotrophoblasts of partial mole (P = 0.02). The placenta, complete and partial mole, and choriocarcinoma tissues demonstrated similar immunoreactivity for c-erbB-4. Strong immunostaining for EGFR (P = 0.02) and c-erbB-3 (P < 0.01) in extravillous trophoblasts of complete mole was found to be significantly correlated with the development of persistent postmolar gestational trophoblastic tumor.Conclusion. The EGFR-related family of oncogenes may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of EGFR and c-erbB-3 in complete mole may also influence the development of persistent gestational trophoblastic tumor.

Expression of Aminopeptidase A in Human Gestational Choriocarcinoma Cell Lines and Tissues

Aminopeptidase A (AP-A), a cell-surface metallopeptidase hydrolyzing peptide with N-terminal acidic residues, has been proved to be identical to the B cell differentiation antigen BP-1 and to the kidney differentiation antigen gp160, suggesting recognition of AP-A as a differentiation-related marker on certain normal and transformed cells. AP-A has also been purified from human placenta and been shown to be localized in the trophoblasts. In the present study, we examined the expression and enzymatic activity of AP-A in human gestational choriocarcinoma, a neoplastic transformant from trophoblasts which comprises a heterogenous population of trophoblastic cells in different stages of differentiation. Flow cytometry and immunoblot analysis demonstrated that AP-A was expressed in five choriocarcinoma cell lines which were secreting low or moderate levels of human chorionic gonadotropin (hCG), while two high hCG-secreting cell lines lacked AP-A expression. The AP-A enzymatic activity correlated with cell-surface levels of AP-A and was abrogated by amastatin, an inhibitor of AP-A. Immunohistochemical analysis revealed that AP-A was present in seven of eight choriocarcinoma tissues and was localized on the cell membrane of cytotrophoblastic choriocarcinoma cells, but not on cells with syncytiotrophoblast-like features. These results demonstrate that AP-A is expressed on most choriocarcinomas and its expression is restricted to low hCG-secreting, cytotrophoblastic cells and down-regulated as a function of cell differentiation, suggesting an involvement of AP-A in the differentiation/maturation process of neoplastic trophoblasts.

Telomerase activity in gestational trophoblastic disease.

AIMS: To investigate the pattern of telomerase activity in hydatidiform mole as compared with normal placenta and choriocarcinoma, and to determine the prognostic significance of telomerase activity in hydatidiform mole....

Telomerase activity in complete hydatidiform mole

Objective: The purpose of this study was to evaluate the association of telomerase activity in complete hydatidiform moles with subsequent development of persistent gestational trophoblastic tumor. Study Design: By means of the standard telomerase repeat assay, we examined telomerase activity in 4 normal placentas, 31 complete hydatidiform moles (16 cases of uneventful regression, according to serum levels of β-human chorionic gonadotropin, after evacuation and 15 cases in which persistent gestational trophoblastic disease developed after evacuation), 7 invasive moles, and 5 choriocarcinoma tissue samples. Results: Telomerase activity was detected in 13 of 15 (86.7%) complete hydatidiform moles in patients who eventually underwent chemotherapy for the treatment of persistent gestational trophoblastic tumors. All 9 patients with metastatic disease (International Federation of Gynecology and Obstetrics stage III) had telomerase activity in the initial molar tissue sample. In contrast, telomerase activity was evident in only 3 of 16 (12.5%) complete hydatidiform moles from patients with spontaneous remission after evacuation ( P < .05). Telomerase activity was detected in all 7 invasive moles and all 5 choriocarcinoma tissue samples but was not detected in normal placentas. Conclusion: The presence of telomerase activity in complete hydatidiform moles is associated with the development of persistent gestational trophoblastic tumors, such as invasive moles and choriocarcinoma. (Am J Obstet Gynecol 1999;180:328-33.)

Integrin αvβ3, expression on the endometrium in infertile women

Choriocarcinoma is a highly malignant trophoblastic neoplasm. Most of them are gestational in origin, while non-gestational ones are exceedingly rare. The genetic origin, immunogenicity, sensitivity to chemotherapy and prognosis of these two kinds of conditions are quite different, so identification of these two kinds of choriocarcinoma is of great importance. The objective of this study is to distinguish choriocarcinoma as gestational or non-gestational and identify the causative pregnancy of gestational choriocarcinoma through molecular genetic analysis.Twelve patients with choriocarcinoma, who had experienced surgery prior to chemotherapy, were enrolled in this study. DNA was prepared from blood samples from the patient and her partner using standard techniques. In order to prepare DNA from choriocarcinoma tissue, areas of choriocarcinoma were firstly microdissected from haematoxylin and eosin-stained sections. PCR amplification and fluorescent microsatellite genotyping were performed using DNA from the couples and captured tissue. The genetic contributions to the choriocarcinoma were determined by comparing the genotypes of the choriocarcinoma and that of the couples.Four of twelve cases had only a maternal contribution, indicating a non-gestational origin. The remaining eight cases were all gestational in origin and the causative pregnancies were identified as AnCHM (androgenetic complete hydatidiform mole) in six and normal pregnancies in two respectively.Microsatellite polymorphism analysis is a molecular approach for distinguishing the non-gestational choriocarcinoma from the gestational one, and can also be used to identify the causative pregnancy of gestational choriocarcinoma. Antecedent pregnancy prior to choriocarcinoma is not always its causative pregnancy. Therefore, it is reasonable to identify the causative pregnancy by its genetic origin, instead of clinical impression.

Endoglin, a component of the TGF-beta receptor system, is a differentiation marker of human choriocarcinoma cells.

Endoglin is an integral membrane glycoprotein that binds transforming growth factor-beta1 (TGF-beta1) with high affinity and it is strongly expressed on syncytiotrophoblasts throughout pregnancy. Here, we describe the expression of endoglin by the choriocarcinoma cell line JAR as evidenced by flow cytometry, immunoprecipitation, Western blot and reverse transcriptase polymerase chain reaction analyses. Cross-linking experiments of [125I]-labeled TGF-beta1 to JAR cells indicated that endoglin expressed at the surface of these cells binds TGF-beta. Furthermore, staining of human choriocarcinoma tissue sections with a polyclonal antibody to endoglin demonstrated a high expression of endoglin in syncytiotrophoblast-like areas, as opposed to a negative staining of cytotrophoblast-like cells. This pattern of endoglin expression was confirmed by experiments with methotrexate, an inducer of giant, multinucleated, non-proliferative cells, morphologically indistinguishable from the naturally occurring syncytiotrophoblasts. Thus, treatment of the JAR and JEG-3 choriocarcinoma cell lines with methotrexate led to an increase in endoglin expression, as demonstrated by Western and Northern blot analyses. Taken together, our results suggest that endoglin, in addition to being involved in placental development, may also be a cellular differentiation marker.

Human chromosome 7 carries a putative tumor suppressor gene(s) involved in choriocarcinoma.

Choriocarcinoma developed from a complete hydatidiform mole has an unique genetic feature that involves monoallelic contribution from the paternal genome. To determine the chromosome carrying putative tumor suppressor gene(s), microcell-hybrids were isolated following fusion of choriocarcinoma cells with microcells from mouse A9 cells containing a single human chromosome (1, 2, 6, 7, 9 or 11). Microcell-hybrids with the introduction of chromosome 7 were suppressed or modulated for tumorigenicity and exhibited altered in vitro growth properties. Introduction of chromosomes 1, 2, 6, 9 or 11 had no effect. Tumorigenic revertants isolated from microcell-hybrids with the introduced chromosome 7 contains reduced numbers of chromosome 7. These findings suggest that chromosome 7 contains a putative tumor suppressor gene(s) for choriocarcinoma. Alterations in tumorigenic phenotypes seen in microcell-hybrids were not associated with the presence of either ERV3 or H-plk locus located on the introduced chromosome 7, indicating the putative tumor suppressor gene(s) is outside of ERV3 and H-plk gene loci. Furthermore, we obtained evidence to define a critical region on chromosome 7 (7p12-7q11.23) that was frequently lost in surgically removed choriocarcinoma tissues and cell lines. Using a panel of microsatellite markers, biallelic deletions were observed, which strongly suggests the presence of a tumor suppressor gene(s) within this critical region.