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.
Several reports show that cancerous cells are linked to early decrease in gap-junction number and functionality diminution. This precancerous phenomenon may be accessed by different fluorescence techniques and particularly by gap-FRAP technique (gap-junction fluorescence recovery after photobleaching). Measurements at cell or tissue scale allowed by this method lead to consider its potential interest in endoscopic technics applied to early cancer detection. Experiments were performed on HT-29 (human colon adenocarcinoma), MCF-7 (human breast cancer cell) and CCD-1137Sk (human Fibroblasts) in the presence of 5.6CFDA. Dye was bleached by laser light (488nm) during few seconds depending on the region of interest (one or fewer cells). Fluorescence recovery kinetic after photobleaching was measured by imaging and spectral analysis with a confocal laser scanning microscope as reference technique. Then, a microspectrofluorimeter was used in order to evaluate the faisability on a fiber optics based system offering measurement condition close to the tissue clinical endoscopy conditions.Preliminary results obtained on the various cells lines show significant differences in kinetics for normal and cancerous cells. We have shown that CCD-1137Sk line cells possess functional communicating junctions, contrary to the carcinogenic HT-29 and MCF-7 cells. Results obtained by microspectrofluorimetry are related to confocal microscopes ones confirming the feasibility in endoscopy.