In vitro characterization of gap junctional intercellular communication by gap-FRAP technique M. Abbaci 1 , J-R. Stines 1 , M. Barberi-Heyob 1 , W. Blondel 2 , D. Dumas 3 , F. Guillemin 1 and J. Didelon 1 1 Centre Alexis Vautrin, CRAN UMR 7039 CNRS-UHP-INPL, Vandoeuvre-les-Nancy, France; 2 UHP- Nancy 1, CRAN UMR 7039 CNRS-UHP-INPL, Vandoeuvre-les-Nancy; 3 UHP- Nancy 1, LEMTA - UMR 7563 CNRS-INPL-UHP et IFR 111, Faculté de Médecine, Vandoeuvre-les-Nancy. ABSTRACT Gap junctional intercellular communication (GJIC) has been shown to be involved in the carcinogenesis process. Gap- FRAP (Fluorescence Recovery After Photobleaching) technique could be used to estimate gap junctions functionality and their potential involvement for distinguish normal and cancer cells. In this study, the gap-FRAP technique was used to analyse functional gap-junction-mediated communication for cell lines with different GJIC status. Gap-FRAP data and connexin 43 protein expression decreased for FaDu cancer cell line, in contrast to fibroblast and KB positives cell lines. To check the involvement and functionality of gap junctions in the restitution of the fluorescence after photobleaching, we used a gap junction channel inhibition assay with 18 α-glycyrrhetinic acid. Our results indicate that the degree of gap junctional intercellular communication could be estimated by this technique in vitro. Keywords: gap junction, gap-FRAP, connexin 43 1. INTRODUCTION Gap junction channels provide a direct intercellular communication. The juxtaposition of two half channels, called connexons, constitutes this junction. Each connexon is inserted into the cytoplasmic membrane of two neighbouring cells. It is made of six protein sub-units called connexins (Cx) [1]. The family of the Cx is composed of 21 types of proteins classified according to their molecular weight [2]. The Cx43 is the protein sub-unit the most extensively expressed in human tissues. A link between an impairment of intercellular communication and the process of carcinogenesis seems to be defined. In fact, the majority of malignant cells are characterized by a decrease of the number and functionality of gap junctions, compared to their healthy counterparts [3, 4]. The distinction between cancerous and healthy cell populations appears possible in studying functionality of gap junction channels. We used the gap-FRAP (Fluorescence Recovery After Photobleaching) technique which enlarges the FRAP technique to study the functionality of gap junctional intercellular communication channels [5]. After exposure with a specific fluorescent tracer in vitro, a gradient of concentration is generated, followed by a light irradiation leading to dye photobleaching in the targeted cell. If the tracer is hydrophilic and its molecular weight lower than 1.2 kDa, the relaxation of concentration gradient means that the dye intercellular flux was made by gap junctions [6]. In this study, we have used FRAP approach, which has been validated as a reliable technique to analyse functional gap-junction-mediated communication, because of several advantages that it offers over alternative techniques. To investigate the relationship between intercellular communication of fibroblast cells and two head and neck carcinoma cell lines, FRAP was used to detect function of gap junctional intercellular communication (GJIC). 2. MATERIALS AND METHODS 2.1. Materials Cell culture materials were purchased from Costar (Dutscher, Brumath, France). Culture media and additives were obtained from life technologies (Gibco BRL, Cergy-Pontoise, France) except for foetal calf serum (FCS), which was