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

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. © 2004 Elsevier Inc. All rights reserved.