Pathogenesis Of Gestational Trophoblastic Disease Research Articles

Role of the Insulin-like growth factor axis and the Transforming growth factor-β in the regulation of the placenta and the pathogenesis of Gestational Trophoblastic Diseases

During human pregnancy, the trophoblast develops as the fetal compartment, while in bidirectional communication with the maternal decidua. The trophoblast is responsible for the adequate implantation of the embryo, supply of nutrients and environmental protection of the fetus along the progress of pregnancy. To perform these functions trophoblast cells, undergo a complex and finely tuned differentiation into specialized groups of cells, in a process regulated by several hormones, growth factors and cytokines. Abnormalities in trophoblast function result in several pregnancy complications. In this review, we focus our attention on two growth factors with pivotal roles during human pregnancy. The Insulin-like growth factor (IGF) family and the Transforming growth factor (TGF-β) axis are important regulators of the proliferation, differentiation, apoptosis, migration and invasion of human trophoblasts. We summarize what is described in the literature on how these factors and their receptors are expressed on the different subsets of trophoblasts, the signaling pathways that transduce their corresponding actions and functional biological effects. We describe the associations that have been found between these growth factors and the group of pathologies known as Gestational Trophoblastic Diseases (GTD).

Low molecular weight hyaluronan induces migration of human choriocarcinoma JEG-3 cells mediated by RHAMM as well as by PI3K and MAPK pathways

Hyaluronan (HA) is the major glycosaminoglycan present in the extracellular matrix. It is produced by some tumours and promotes proliferation, differentiation and migration among others cellular processes. Gestational trophoblastic disease (GTD) is composed by non-tumour entities, such as hydatidiform mole (HM), which is the most common type of GTD and also malignant entities such as choriocarcinoma (CC) and placental site trophoblastic tumour (PSTT), being CC the most aggressive tumour. Although there is a growing understanding of GTD biology, the role of HA in the pathogenesis of this group of diseases remains largely unknown. The aim of this work was to study the role of HA in the pathogenesis of GTD by defining the expression pattern of HA and its receptors CD44 and RHAMM, as well as to determine if HA can modulate proliferation, differentiation and migration of CC cells. Receptors and signalling pathways involved were also analyzed. We demonstrated that HA and RHAMM are differently expressed among GTD entities and even among trophoblast subtypes. We also showed that HA is able to enhance the expression of extravillous trophoblast markers and also to induce migration of JEG-3 cells, the latter mediated by RHAMM as well as PI3K and MAPK pathways. These findings indicate a novel regulatory mechanism for CC cell biology and also contribute to the understanding of GTD pathophysiology.

Downregulation of the Gli Transcription Factors Regulator Kif7 Facilitates Cell Survival and Migration of Choriocarcinoma Cells

The kinesin protein Kif7 has been recognized as an integral component of hedgehog signalling. Aberrant activation of hedgehog signalling has been implicated in many human solid tumours. Gestational trophoblastic disease includes frankly malignant choriocarcinoma and potentially malignant hydatidiform mole. Here we investigated the hedgehog signalling components expression profiles in gestational trophoblastic disease. Downregulation of Gli1, Gli2, Gli3 and Kif7 was demonstrated in clinical samples of choriocarcinoma and hydatidiform moles as well as choriocarcinoma cell lines when compared with normal placentas. Ectopic expression of Kif7 in two choriocarcinoma cell lines JAR and JEG-3 led to a decrease in cell growth and increase in apoptosis demonstrated by MTT and TUNEL assays, respectively. Overexpression of Kif7 also led to suppressed cell migration through transwell assay. In contrast, knocking down Kif7 in HTR-8/SVneo, an immortalized trophoblast cell line, increased cell number over time and increased the migratory ability of the cells. Taken together, Kif7 may contribute to pathogenesis of gestational trophoblastic disease through enhancing survival and promoting dissemination of trophoblasts.

P-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases

ObjectivesTo delineate the potential role of p21-activated kinases (PAKs) in the pathogenesis of gestational trophoblastic diseases (GTD) by defining the expression pattern of PAK-1, -4 and -6 and their potential implication in estrogen receptor (ER) regulation of normal placental tissue and GTD. MethodsWe evaluated immunohistochemically 10 normal first-trimester placentas (NP), 10 partial moles (PM), 15 complete moles (CM) and 3 choriocarcinomas (CCA) for PAK-1, PAK-4, PAK-6 and ER expression intensity and localization. Staining outcomes were assessed utilizing non-parametric Kruskal–Wallis one-way analysis of variance test followed by pairwise Wilcoxon Rank Sum tests. Statistical significance was determined by two-sided p-value of <0.05. ResultsIn NP, PAK-6 immunoreactivity was predominantly cytoplasmic. Compared to NP, PM and CM demonstrated significant increase of cytoplasmic PAK-6 in cytotrophoblast (p=0.012, p=0.033 respectively), accompanied by significantly increased nuclear immunoreactivity in cytotrophoblast (p=0.008, p=0.045 respectively) and intermediate trophoblast (p=0.003, p=0.015 respectively). PAK-4 was found significantly upregulated in both cytoplasmic and nuclear compartments of cytotrophoblast and syncytiotrophoblast in PM (p=0.004 and p=0.002 for cytotrophoblast; p=0.018 and p=0.002 for syncytiotrophoblast, respectively) and CM (p=0.001 and p=0.001 for cytotrophoblast; p=0.002 and p=0.001 for syncytiotrophoblast, respectively) when compared to NP, whereas PAK-1 expression was significantly reduced in the syncytiotrophoblast of PM (p=0.025 for cytoplasm and p=0.008 for nucleus). Nuclear expression of ER was undetectable in all stained samples. ConclusionOur results reveal PAK-6 upregulation in GTD compared to NP. The absence of nuclear expression of ER might stem in part from the repressive effect of PAK-6 in trophoblastic tissue.

Application of a NotI subtraction and methylation-specific genome subtractive hybridization technique in the detection of genomic DNA methylation differences between hydatidiform moles and villi

Previous studies indicate that epigenetic modifications play an important role in transcriptional regulation and contribute to the pathogenesis of gestational trophoblastic disease, including complete hydatidiform moles (CHMs). However, the underlying mechanisms and the critical genes have not been clearly identified. In the present study, we developed a novel technique, NotI subtraction and methylation-specific genome subtractive hybridization (MS-G-SH), as a method of screening for methylation changes between hydatidiform moles and villi. Following NotI subtraction and hybridization, three different positive DNA clones were found in 110random clones of DNA samples. Most importantly, two DNA clones having long CpG islands and high homology with exons of insulin-like growth factor 2 (IGF2) and transforming growth factor-β (TGF-β) were identified using bioinformatic tools. After bisulfite treatment and methylation-specific PCR, the specific methylation of certain exons of IGF2 and TGF-β was identified. In addition, the mRNA expression levels of these two genes were markedly different. In conclusion, this novel MS-G-SH technique is an alternative and effective approach for the detection of specific DNA methylation.

Notice of Retraction

Gestational trophoblastic disease (GTD) includes frankly malignant choriocarcinoma (CCA) and placental site trophoblastic tumor and potentially malignant hydatidiform mole. p21-Activated kinase (PAK) 4 promotes cell motility. This study investigated the role of PAK4 in the pathogenesis of GTD. PAK4 messenger RNA and protein expressions in clinical samples and cell lines of normal placentas and GTD were determined by quantitative real-time polymerase chain reaction and western blot, respectively. The effects of human chorionic gonadotropin (hCG) and phosphoinositide 3 kinase (PI3K) on the expression and activation of PAK4 were investigated by treating CCA JEG3 and JAR cells with anti-hCG antibody and PI3K inhibitor, respectively. The effects of PAK4 on CCA cell proliferation, migration and invasion were assessed by corresponding functional assays. We demonstrated overexpression of PAK4 in GTD and CCA cell lines at both RNA and protein level. hCG is one of the upstream regulators of PAK4 expression, whereas activation of PAK4 is PI3K/PKB dependent in JEG3 and JAR cells. Significant correlation was found between PAK4 expression and proliferation index minichromosome maintenance complex component 7 (P = 0.007). In JEG3 and JAR cells, stably transfected PAK4 increased proliferation, migration and invasion, whereas small interfering RNA knockdown of PAK4 decreased proliferation, migration and invasion along with downregulated CDK6 and membrane-type 1 matrix metalloproteinase (MT1-MMP) and upregulated p16. We further found PAK4-mediated transcription of MT1-MMP in CCA cells by luciferase reporter assay. Our results demonstrated for the first time that overexpressed PAK4 was involved in the pathogenesis of GTD, promoting proliferation and enhancing cell migration and invasion in CCA cells.

Morbidity and mortality of patients suffering from gestational trophoblastic diseases at the Clinic of gynecology and obstetrics, Clinical center of Serbia in the period from 2000 to 2007

Gestational trophoblastic disease is a heterogenous group of diseases with malignant potential. The aim of this retrospective study was to evaluate potential risk factors in pathogenesis of gestational trophoblastic disease, its morbidity and mortality as well as treatment results. We investigated 82 patients who were treated at the University Clinic of Gynecology and Obstetrics Clinical Center of Serbia from Jan 1st 2000 to Dec 31st 2007. The data were collected from their hospital charts and referred to gynecological anamnesis, diagnosis, protocols of operated patients, diagnosis, histopathological findings, decisions of expert team for trophoblastic disease and hospital discharge. The incidence was 1.5 per 1000 deliveries. The most frequent finding was hydatiform mole (59.8%). The patients were treated by chemotherapy and surgery. All patients survived. The maternal age and a larger number deliveries and abortions are risk factor for gestational trophoblastic disease. The incidence in our clinic is approximately equal to the incidence in western countries in this period.

P21-Activated Kinase-1 Promotes Aggressive Phenotype, Cell Proliferation, and Invasion in Gestational Trophoblastic Disease

Gestational trophoblastic disease (GTD) includes hydatidiform mole (HM), which can develop persistent gestational trophoblastic neoplasia requiring chemotherapy; choriocarcinoma, which is a frankly malignant tumor; placental site trophoblastic tumor; and epithelioid trophoblastic tumor. p21-Activated kinases (PAKs) promote malignant tumor progression. Therefore, this study investigated PAK1, PAK2, and p-PAK2 Ser(20) in the pathogenesis of GTD. By real-time PCR, PAK1 mRNA was significantly higher in HMs, particularly metastatic HMs (P = 0.046) and HMs that developed persistent disease (P = 0.011), when compared with normal placentas. By immunohistochemistry, significantly increased cytoplasmic PAK1 immunoreactivity in cytotrophoblasts was also detected in HMs (P = 0.042) and choriocarcinomas (P = 0.003). In addition, HMs that developed persistent disease displayed higher PAK1 immunoreactivity than those that regressed (P = 0.016), and elevated PAK1 immunoreactivity was observed in placental site trophoblastic tumors. Indeed, there was significant positive correlation between PAK1 expression and the proliferative indices Ki-67 (P = 0.016) and MCM7 (P = 0.026). Moreover, higher PAK1 mRNA and protein expression was confirmed in the choriocarcinoma cell-lines JEG-3 and JAR; however, PAK2 mRNA and p-PAK2 immunoreactivity showed a similar expression pattern in normal first trimester placentas and GTD. Knockdown of PAK1 in JEG-3 and JAR reduced cell proliferation, migration, and invasion ability, up-regulated p16, and down-regulated vascular endothelial growth factor and MT1-MMP expression. This is the first report revealing the involvement of PAK1 in the pathogenesis and clinical progress of GTD.

Expression of laminin receptor 1 in gestational trophoblastic diseases and normal placenta and its relationship with the development of postmolar tumors

Objectives To investigate the expression of laminin receptor 1 (LR1), a non-integrin-type laminin receptor, in gestational trophoblastic diseases and normal first-trimester placenta, since it may play a role in controlling trophoblast invasion in normal and molar pregnancies. Methods Paraffin sections from 24 gestational age controlled normal first-trimester placentas, 47 partial moles, 56 complete moles, 3 invasive moles, 4 gestational choriocarcinomas, and 1 placental-site trophoblastic tumor were studied immunohistochemically for expression of LR1. Results In complete and partial moles, decidual cells showed significantly stronger LR1 protein staining compared to the normal placenta ( p < 0.01). When compared to the partial moles, weak staining in less than 33% of decidual cells was also more prominent in the normal placenta ( p < 0.05). Complete and partial moles, invasive moles, choriocarcinomas, and placental-site tumors did not differ from each other with respect to staining intensity. Strong immunostaining for LR1 in decidual cells, cytotrophoblasts, syncytiotrophoblasts, and extracellular matrix cells of partial and complete moles was not significantly correlated with the development of persistent postmolar gestational trophoblastic tumors. Conclusions LR1 may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of LR1 in decidual cells of partial and complete moles may not influence the development of persistent gestational trophoblastic tumor. Since they are seen rarely, multicentric studies should be planned to study LR1 expression in invasive moles and gestational trophoblastic neoplasms.

P63 Expression in Gestational Trophoblastic Disease: Correlation With Proliferation and Apoptotic Dynamics

p63 regulates cell growth and differentiation and contributes to tumorigenesis through its complex isoforms. Gestational trophoblastic disease encompasses a heterogeneous family of lesions with different malignant potential that arise from various trophoblast subpopulations. This study investigated the expression of p63 isoforms in various trophoblastic diseases and correlated with clinical progress, proliferation, and apoptotic activities. 4A4 and anti-p40 antibodies were applied to assess expressions of total and DeltaNp63 isoforms in 20 placentas, 62 hydatidiform moles, 9 choriocarcinomas, 5 placenta site trophoblastic tumors, and 2 epithelioid trophoblastic tumors immunohistochemically. The immunoreactivity of p63 was localized to the nuclei of cytotrophoblast, villous, and chorionic-type intermediate trophoblasts with significant correlation between 2 p63 indices (P<0.001). p63 indices were significantly lower in placentas of advanced gestational age (P<0.001). Hydatidiform moles demonstrated significantly higher p63 indices than normal placentas (P<0.001). Epithelioid trophoblastic tumors displayed the highest p63 indices (45%-80%) whereas immunoreactivity was only focal in choriocarcinoma (0%-5.62%) and was essentially absent in placenta site trophoblastic tumors. There was no significant correlation between p63 indices and subsequent development of trophoblastic neoplasia in hydatidiform moles (P>0.05). Both p63 indices positively correlated with the proliferative index (Ki67) (P<0.05), apoptotic index (M30) (P<0.005), p53 (P<0.005), and p21(WAF1/CIP1) expression (P<0.005). Our results indicate that DeltaNp63, the dominant isoforms expressed in trophoblasts, display heterogeneous expression patterns in relation to trophoblast subtypes. We also demonstrate for the first time the possible role of p63 in the pathogenesis of gestational trophoblastic disease (GTD) through its interaction with p53-dependent proliferation and apoptotic activities.

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.

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.

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.

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.

Differential expression profiling of genes in a complete hydatidiform mole using cDNA microarray analysis

Objectives. To gain a better understanding of the genes involved in the pathogenesis of gestational trophoblastic diseases, we evaluated the genome-wide expression levels of genes in complete hydatidiform mole (H-mole) as compared to normal placenta using cDNA microarray technique. Methods. The expression profiles of complete H-mole tissues were compared with those of normal placenta using cDNA microarray technique. The data obtained from 10,305 human genes were normalized by the print-tip-based LOWESS method. Significance analysis of microarray (SAM) was used to identify genes with statistically significant changes in expression. The expression levels of genes which showed significant differences between normal early placenta and complete H-mole tissues were further confirmed by RT-PCR. Results. A cDNA microarray analysis consisting of 10,305 human genes revealed significant changes in the expression of 213 genes, with 91 genes being upregulated and 122 being downregulated. SAM revealed significant changes in gene expression, including those associated with signal transduction, cell structure, transcription, and apoptosis. Further RT-PCR analysis of altered gene expression in mole tissues supported the microarray analysis results. We confirmed the upregulation of TLE4, CAPZA1, PRSS25, RNF130, and USP1 in complete H-mole tissues. Moreover, our study provides the first evidence that ELK3, LAMA3, LNK, STAT2, and TNFRSF25 are downregulated in complete H-mole compared to normal early placenta tissues. Conclusions. These findings provide a large body of information regarding gene expression profiles associated with complete H-mole tumorigenesis and allow the identification of potential targets for tumor prevention or therapy.

Caspase activity is downregulated in choriocarcinoma: a cDNA array differential expression study

Background: Placental trophoblast can be considered to be pseudomalignant tissue and the pathogenesis of gestational trophoblastic diseases remains to be clarified. Aims: To examine the role of caspases 8 and...

Expression of the High-Mobility Group Protein HMGI(Y) in Gestational Trophoblastic Diseases

The high-mobility group protein HMGI(Y) is a member of a family of nonhistone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes, and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. Recently we could show that HMGI(Y) is expressed in the normal human placenta, where it is localized in the nuclei of villous cytotrophoblast, in the anchoring villi at the implantation site and in extravillous (intermediate) trophoblast invading the maternal decidua. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. The purpose of this study was to investigate the expression pattern of HMGI(Y) in gestational trophoblastic diseases (GTD), which has not been studied so far. To analyze the expression of HMGI(Y), we performed immunohistochemistry on a total of 29 cases of GTD, including 21 hydatidiform moles and 8 choriocarcinomas. Hydatidiform moles showed a positivity for HMGI(Y) in villous cytotrophoblast and in areas of the trophoblast proliferations on the villous surface; villous syncytiotrophoblast was negative. The choriocarcinomas showed strong immunoreactivity in all cases. The expression pattern of HMGI(Y) in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD and might be potentially useful as an additional diagnostic marker for such lesions.

Expression Pattern of the Activating Protein-1 Family of Transcription Factors in Gestational Trophoblastic Lesions

The human placenta is an endocrine tissue with a unique capacity for rapid, but tightly controlled, proliferation and invasion. Gestational trophoblastic diseases (GTDs) are placental pathologies with endocrine activity and partially malignant potential and include hydatidiform moles, placental site nodules, and tumors such as placental site trophoblastic tumor and choriocarcinomas. The activating protein-1 (AP-1) family of transcription factors is composed of the cellular homologs of the Jun and Fos oncoproteins, which are immediately involved in cellular proliferation, differentiation, and invasion processes and in the regulation of endocrine genes. The expression pattern of the AP-1 family in the normal human placenta has been recently described, where most of the factors were found in the extravillous (invasive) trophoblast. Their systematic expression in GTD has not been studied thus far. For this reason in this study, we investigated the expression pattern of the AP-1 family in GTDs and compared it with the expression in normal placenta using immunohistochemistry with specific polyclonal antibodies against all members of the AP-1 family (JunB, JunD, c-Jun, c-Fos, FosB, Fra-1, Fra-2). Immunohistochemistry was performed on normal human placentas (positive control) and on 28 cases of GTD including 7 choriocarcinomas and 21 hydatidiform moles. In the normal placenta and in hydatidiform molar samples, most AP-1 factors (especially c-Jun, JunD, and Fra2) were expressed in the intermediate (extravillous) trophoblast. In addition, in molar lesions, strong expression was found in trophoblasts proliferating from the surface of villi. There was only a weak expression of JunB and Fra2 in small fractions of villous cyto- and syncytiotrophoblast nuclei. In choriocarcinomas, there was a strong expression for c-Jun, JunD, Fra1, and Fra2. The specific localization to extravillous trophoblasts and their expression pattern in GTDs indicate that the AP-1 family of transcription factors might be implicated in regulating proliferation and invasion of trophoblasts and play a role in the pathogenesis of GTDs.

Osteopontin Expression in Gestational Trophoblastic Diseases: Correlation With Expression of the Adhesion Molecule, CEACAM1

The human placenta is a complex tissue with multiple endocrine and nutritional functions and a unique capacity for rapid proliferation but tightly controlled invasion, differentiating it from malignant tumors. Osteopontin (OPN) is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. OPN also could be implicated in regulating implantation and placentation by promoting cellular migration and invasion in a placenta-specific fashion. We could demonstrate the expression pattern of OPN in the normal human placenta in which it is localized in the extravillous (intermediate) trophoblast and the villous cytotrophoblast. CEACAM1 is an adhesion molecule, which we have recently found to be expressed at the maternal-fetal interface of the normal placenta with a localization to the extravillous (invasive) trophoblast and in gestational trophoblastic disease (GTD) and also to be potentially implicated in trophoblast invasion and tumorigenesis. Both OPN and CEACAM1 have been shown to interact with integrin beta3. The purpose of this study was to investigate the expression pattern of OPN in GTD and to correlate it with the expression of CEACAM1. To analyze the expression of OPN, we performed immunohistochemistry on a total of 27 cases of GTD, including 21 hydatidiform moles and 6 choriocarcinomas, which had previously been characterized with respect to their CEACAM1 expression. Hydatidiform moles showed a positivity for OPN in villous cytotrophoblast and in the trophoblast proliferations on the villous surface. The strongest OPN expression could be observed in the choriocarcinomas with a heterogenous OPN expression pattern. CEACAM1 had shown similar results and was found to be expressed in choriocarcinoma. The expression pattern of osteopontin in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD (possibly as a functional complex with CEACAM1 and integrin beta3) and might be useful as an additional diagnostic marker for such lesions.

Protein profiling of complete mole and normal placenta using ProteinChip analysis on laser capture microdissected cells

Introduction Surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) is a novel method for biomarker discovery that can provide a rapid protein expression profile from a variety of biological samples. Since SELDI-MS requires a small amount of biological material, this technique is ideal for analyzing proteins isolated from microdissected tissue samples. The current study was undertaken to investigate potential differences in protein expression between normal and molar trophoblast procured by laser capture microdissection (LCM) utilizing SELDI ProteinChip array technology. Further knowledge of protein expression in complete mole may advance our understanding of the pathogenesis of gestational trophoblastic diseases. Materials and methods Laser capture microdissected trophoblast cells from nine fresh complete moles were analyzed and compared to the trophoblast cells from 10 fresh normal placentas of comparable gestational age, using SELDI ProteinChip to identify potential differences in protein expression. Results Three metal binding polypeptides were identified with the estimated molecular weights of 11.3, 13.8, and 14.0 kDa, which appeared in significantly lower levels in complete mole as compared to normal trophoblast cells ( P < 0.001, P < 0.03, and P < 0.01). Discussion While further characterization of these protein peaks is important and necessary, our current work clearly demonstrates that the combined technology of SELDI and LCM is effective in distinguishing protein expression between normal placenta and complete mole. Further knowledge of protein expression in complete mole may advance our understanding of molecular mechanisms and improve management in gestational trophoblastic diseases.