Differential Display RT-PCR Analysis of Human Choriocarcinoma Cell Lines and Normal Term Trophoblast Cells: Identification of New Genes Expressed in Placenta J. Garcı´a and J. L. Castrillo* Centro de Biologı´a Molecular ‘‘Severo Ochoa’’, Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Paper accepted 8 October 2003 In this study, we performed the differential display technique to identify genes specifically expressed in human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and normal placental term cells. Few differences were found among the expression profiles of the three choriocarcinoma cell lines and most of the differentially expressed genes were detected in normal term placenta. A total of 36 cDNA fragments were isolated and analysed. Of these, 19 sequences corresponded to regions in the human genome coding for potential novel genes. We confirmed by RT-PCR, the placental mRNA expression of three selected new human genes, on chromosomes 16q12, 9q32 and 6q22. The other 17 sequences showed high similarity to known human genes (like PSG3, FN1, PAI-2). Interestingly, the functions of five known proteins (from genes IK, TRA-1, HERPUD1, UBA-2, and TRAP240) have not yet been well characterized in placenta tissue. In addition, new alternative spliced mRNAs were detected for IK, TRAP240 and PLAC3 genes. The differential expression of the PAI-2 gene among the choriocarcinoma cell lines was also confirmed. The genes identified in this analysis will be of interest for future studies regarding both a better understanding of the biology of the trophoblast cell and the formation of placental tumors. Placenta (2004), 25, 684–693 Ó 2004 Elsevier Ltd. All rights reserved. INTRODUCTION Human placenta is a unique organ that is dedicated to the establishment, maintenance and regulation of pregnancy. The main components of the placenta are the trophoblast cells. These cells, derived from the blastocyst, differentiate from mononuclear cytotrophoblast into a layer of multinucleated syncytiotrophoblast [1]. Trophoblast functions are essential for pregnancy: they mediate implantation, produce pregnancy hormones, protect the fetus from rejection and infections, separate maternal from fetal blood, mediate the gas, nutrient and waste exchanges and finally, participate in the delivery at term [2]. The precise mechanisms of human placental develop- ment and maintenance are still poorly understood. Many hor- mone receptors, peptides and enzyme intermediates involved in the various signalling pathways in the placenta remain to be identified. Abnormally extensive invasion during pregnancy may lead to pre-malignant or malignant conditions like choriocarcinoma, a relatively infrequent tumor which originates from the tropho- blast. This kind of tumor belongs to the group of disorders known as gestational trophoblastic disease (GTD), which includes benign hydatidiform mole (complete and partial), malignant invasive mole and the rare placental site/epitheloid trophoblastic tumor [3]. Although important advances in the diagnosis and therapy of GTD have been done, the molecular mechanisms leading to these diseases remain unknown. A fundamental question that remains unanswered is, what are the genetic alterations that allow an uncontrolled trophoblast invasiveness in choriocarcinoma? It is suspected that molecules like cell–cell adhesion proteins, matrix metalloproteinases and their inhibitors, which regulate the invasion process, may play a role in the pathogenesis of GTD [4]. Technologies developed in the 1990s for the study of dif- ferences in gene-expression patterns have contributed enor- mously to our understanding of basic biological processes such as differentiation and development. They have led to the identification of new therapeutic targets in many different cell systems [5,6]. These methods include the differential display RT-PCR technique (DDRT-PCR), expressed sequence tag (EST) methodology, representational difference analysis (RDA), serial analysis of gene expression (SAGE) and DNA arrays technologies. In particular, DDRT-PCR has proved to be a useful tool for the identification and isolation of novel genes, as well as different alternative spliced mRNAs from a gene. Ten years after the original description of the * Corresponding author. Centro de Biologı ´a Molecular ‘‘Severo Ochoa’’, C-106, Universidad Auto ´noma de Madrid, Cantoblanco, E-28049, Madrid, Spain. Tel.: +34-913978074; fax: +34-913978087. E-mail address: jlcastrillo@cbm.uam.es ( J.L. Castrillo). 0143–4004/$–see front matter Ó 2004 Elsevier Ltd. All rights reserved. Placenta (2004), 25, 684–693 doi:10.1016/j.placenta.2003.10.020