Elsevier

Gynecologic Oncology

Volume 94, Issue 2, August 2004, Pages 375-382
Gynecologic Oncology

Down-regulation and promoter methylation of tissue inhibitor of metalloproteinase 3 in choriocarcinoma

https://doi.org/10.1016/j.ygyno.2004.04.019Get rights and content

Abstract

Objective. To assess the differential gene expression in neoplastic and normal trophoblastic cells and evaluate the effect of methylation on tissue inhibitor of metalloproteinase 3 (TIMP3) expression in choriocarcinoma (CCA) cells.

Methods. The Atlas™ Human Cancer 1.2 Array (Clontech) was used to compare differential gene expression in a trophoblastic cell line (B6) established from first term placenta with two choriocarcinoma cell lines (JAR and JEG-3). The differentially expressed candidate genes in the placental and malignant trophoblastic cells in these cell lines were confirmed by real-time polymerase chain reaction (PCR), Western blotting and immunohistochemistry. Differential expression of a specific gene, TIMP3, was confirmed by immunohistochemistry using clinical specimens of choriocarcinoma and placenta. The involvement of promoter methylation in suppression of TIMP3 expression in choriocarcinoma was examined using methylation-specific PCR (MSP) and demethylation treatment.

Results. Differential expression of 23 genes was observed in choriocarcinoma cell lines compared to the placental trophoblastic cells using the cDNA array analysis (Atlas™ Human Cancer Array, Clontech). Among these differentially expressed genes, down-regulation of TIMP3, PLAB, IGFBP3 and up-regulation of CCNB1 were confirmed by real-time PCR determination. Reduced expression of TIMP3 was further confirmed in clinical samples of choriocarcinoma by immunohistochemical staining. Methylation of TIMP3 promoter was detected in choriocarcinoma cell lines and clinical samples of choriocarcinoma. Treatment with a demethylation drug, 5-aza-2′-deoxycytidine, in choriocarcinoma cell lines restored TIMP3 expression.

Conclusion. Down-regulation of TIMP3, PLAB, IGFBP3 and up-regulation of CCNB1 were observed in choriocarcinoma cells compared to placental trophoblasts. Down-regulation of TIMP3 expression was confirmed in clinical specimens of choriocarcinoma and may play a role in its pathogenesis. Promoter methylation of the TIMP3 is involved in suppression of TIMP3 expression. Differentiation expression of TIMP3 in choriocarcinoma may have potential application in clinical diagnosis and patient treatment.

Introduction

Choriocarcinoma (CCA) is a highly malignant tumor characterized by excessive proliferation of trophoblastic cells with high potential to metastasize. Worldwide, CCA is relatively more common in Asia and Africa than in North America and Europe [1], [2], [3], [4]. Trophoblastic cells in normal placenta possess pseudo-malignant property, invade into the uterine epithelium and penetrate the maternal stroma for nutrition of the developing embryo [5]. However, the invasion property of placental trophoblast is under strict regulatory control [6]. The choriocarcinoma cells (malignant trophoblasts), in addition to its abnormal proliferative property, are highly invasive and could metastasize to distinct sites in the body. Despite advancement in the diagnosis and therapy, the curable rate of choriocarcinoma remains poor when the cancer cells have metastasized to lung, vagina, liver and brain [7]. Mortality of choriocarcinoma patients remains significantly high, with cranial metastasis being responsible for up to 50% of death [8].

Very little is known about the molecular mechanisms involved in the metastasis of choriocarcinoma. A few metastasis suppressor genes, which were shown to be involved in the control of metastatic potential in human cancers, have been examined in choriocarcinoma. The NM23, which is a nucleoside diphosphate kinase firstly identified to be down-regulated in metastatic breast carcinoma [9], was reported to have prognostic significance in some gynecologic neoplasms including ovarian cancer [10] and cervical cancer [11]. Down-regulation of NM23 was also reported in gestational trophoblastic tumors [12]. However, another study reported up-regulation of NM23 expression in choriocarcinoma [13]. The KISS1 gene, which encodes metastatin, is a metastasis suppressor gene involved in suppression of human melanoma metastasis. Expression of KISS1, and its receptor (GPR54), have been reported to be suppressed in choriocarcinoma [14]. We have recently reported the reduction of E-cadherin/β-catenin expression in choriocarcinoma [15] and hydatidiform mole [16] and its possible involvement in invasion potential.

Recent advances in molecular methodology have facilitated high-throughput analysis of differential gene expression in choriocarcinoma. A recent study using differential display analysis has identified a few differentially expressed genes in pathological trophoblasts involved in protein synthesis (ribosomal L26 and ribosomal L27), metabolism (ferritin light chain), intercellular communication (decorin) and regulation of gene expression (Krüppel-like zinc-finger protein, EGF-response factor 2 and heterogeneous nuclear ribonucleoprotein A1) [17]. However, the expression pattern of these genes in clinical specimens of choriocarcinoma was not examined. In another study using cDNA array analysis with 588 known genes, comparison of gene expression between choriocarcinoma cells and a SV40 immortalized placental cell line has identified down-regulation of heat shock protein-27 (HSP-27) in choriocarcinoma cells [18]. In this study, we have compared gene expression between choriocarcinoma cells and telomerase-immortalized trophoblastic cells established from first trimester placenta by cDNA array in an attempt to identify differential expressed genes in choriocarcinoma cells for potential clinical applications. Atlas™ Human cancer 1.2 array (Clontech, Palo Alto, CA, USA) with 1176 genes was used in this study. Among the differentially expressed genes identified, we have carried further investigation on TIMP3 expression in both choriocarcinoma cell lines and clinical specimens because of its involvement in invasion and metastasis in other human cancers. The involvement of promoter methylation in the regulation of TIMP3 expression in choriocarcinoma cells and tissues was also examined.

Section snippets

Cell lines and clinical tissue samples

Formalin-fixed and paraffin-embedded tissues of choriocarcinoma before chemotherapy (n = 12) were retrieved from the Department of Pathology, the University of Hong Kong, Queen Mary Hospital and the Harbin Medical University, and used in this study. Fresh tissues of six normal first-trimester placentas (gestational age: 10.0 ± 1.4 weeks) and five choriocarcinomas were also collected and stored frozen at −70°C before use. The histopathology of both frozen specimens and paraffin-embedded tissues

Differential gene expression in choriocarcinoma cells

Radiolabeled cDNA prepared from two choriocarcinoma cell lines (JAR and JEG-3) and one normal trophoblast cell line (B6), were hybridized with the Atlas™ Human Cancer 1.2 Array. This array consists of 1176 cDNA clones known to be associated with cancer. The intensities of hybridization signals from cDNA prepared from choriocarcinoma cells (JAR and JEG-3) were compared with the normal trophoblastic cell line (B6). The hybridization experiment was performed twice for each cell population to

Discussion

In the present study, we have used cDNA array to compare the gene expression profile of human choriocarcinoma cells and normal trophoblastic cells. Among the 1176 genes present on the cDNA array, we found that 23 genes showed significant differential expression (>3-fold) in choriocarcinoma cells compared to normal trophoblastic cells. In general, the genes that are expressed at higher level in choriocarcinoma cell lines are involved in cell cycle regulation [e.g. cdc28 protein kinase regulatory

Acknowledgements

This work was supported by grants from the Hong Kong Research Grant Council Grant (HKU 7281/00M) and the Conference and Research Council Grant (CRCG) from the University of Hong Kong.

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