Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines Pamela MJ McLaughlin, 1,2 Monika Trzpis, 1,2 Bart-Jan Kroesen, 1,2 Wijnand Helfrich, 1,2 Peter Terpstra, 1,2 Wim HA Dokter, 1,2 Marcel HJ Ruiters, 1,2 Lou FMH de Leij, 1,2 and Martin C Harmsen 1,2 1 Department of Pathology and Laboratory Medicine, Section of Medical Biology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands; and 2 GUIDE, Groningen University Institute for Drug Exploration, Oostersingel 59, 9713 EZ Groningen, The Netherlands. EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated and assayed for their ability to control the expression of the EGP-2 cDNA, the green fluorescent protein, the luciferase reporter gene and the thymidine kinase and cytosine deaminase suicide genes. Expression of these chimeric constructs as assessed in a range of different cell lines was restricted to cell lines expressing EGP-2. In addition, only cells expressing EGP-2 were sensitive for gancyclovir after being transiently transfected with EGP-2 promoter-driven thymidine kinase. Deletion analyses defined 687 bp upstream as the basic proximal promoter region, which could confer epithelial-specific expression to the GFP reporter gene in vitro. As these EGP-2 sequences can confer promoter activity to reporter and suicide genes in an EGP-2 restricted manner, they may be useful for gene therapy of EGP-2 expressing carcinomas. Cancer Gene Therapy (2004) 11, 603–612. doi:10.1038/sj.cgt.7700725 Published online 9 July 2004 Keywords: transcriptional targeting; GA733-2; suicide gene therapy D espite numerous improvements in radiological, chemotherapeutical and surgical techniques, current treatments for metastatic malignant disease are often ineffective. Therefore, new treatment strategies, which can enhance the selectivity of systemic therapy so that tumor response is increased without toxicity to normal tissue, have gained interest. 1 In this respect, gene therapy provides an attractive option, in combination with promising suicide gene/prodrug systems as effector mechanism. 2 However, a major impediment to the development of gene therapy treatments is the lack of suitable expression cassettes for directing selective trans- gene expression. The epithelium specific but highly abundant expression of the human epithelial glycopro- tein-2 (EGP-2) makes it a useful target for carcinoma directed treatment modalities, such as EGP-2-restricted gene therapy. The 38 kb EGP-2 protein, also referred to as Ep-CAM or 17-1A, is encoded by the GA733-2 gene. 3 Although it has been described as a homotypic adhesion molecule and as ligand of the leukocyte-associated immunoglobulin-like receptor (LAIR-1) the physiological function of EGP-2 is still unclear. 4–6 Since its discovery in 1979, numerous immunotherapeutical strategies using EGP-2 as a target have been developed and are at present used in clinical settings. 7,8 Use of the EGP-2 protein’s carcinoma specificity for the development of gene therapy strategies, however, has been held up by the fact that the regulatory sequences directing this specificity had not yet been characterized. Here, we describe the isolation of the 5 0 sequences from the GA733-2 gene and the identification of cis-acting sequences needed for selective expression of heterologous genes in EGP-2-positive cells. By deletion analysis, the basic proximal promoter region capable of directing expression in an EGP-2-restricted manner was defined. Subsequently, the EGP-2 transcriptional regulatory se- quences were successfully used to direct transient, carcinoma-specific expression of the cytosine deaminase (CD) and thymidine kinase (TK) suicide genes. The use of these constructs, especially in combination with an EGP- 2-specific gene delivery system as has been developed recently, 9 should enhance the safety and efficacy of vector-based carcinoma-specific gene therapy approaches. Materials and methods Cell culture The GLC-1 and GLC-45 SCLC cell lines were generated at our laboratory, previously. 10 The fetal lung fibroblasts Received April 2, 2003. Address correspondence and reprint requests to: Dr MC Harmsen, PhD, Department of Pathology and Laboratory Medicine, Section Medical Biology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail: m.c.harmsen@med.rug.nl Cancer Gene Therapy (2004) 11, 603–612 r 2004 Nature Publishing Group All rights reserved 0929-1903/04 $30.00 www.nature.com/cgt