Gap junctional intercellular communication (GJIC) has been reported to have a close relationship to cell differentiation and carcinogenesis1). The human colon cancer cell line, Caco-2, undergoes spontaneous enterocytic differentiation characterized by the expression of alkaline phosphatase (ALP) and dome formation in the media containing fetal calf serum (FCS). We have reported that FCS in culture media induces these differentiation events dose-dependently2). In order to elucidate the correlation between enterocytic differentiation and GJIC, we examined GJIC in Caco-2 cells cultured with various concentrations of FCS. GJIC was analyzed quantitatively using the scrape-loading/dye transfer assay3).

Caco-2 cells were cultured in Eagle's minimun essential medium (EMEM), suppplemented with 1% non-essential amono acid and 10%FCS in a 35 mm dish. Twenty four hours after the culture, the cells were washed with PBS, then transferred to EMEM containing different concentrations of FCS (0%, 0.1%, 1%, 5%, 10%). On the 12th day of culture, GJIC was demonstrated by scrape-loading/dye transfer assay3). In order to evaluate GJIC quantitatively, number of rows labeled with lucifer yellow CH was counted at ten sites of the scraped cells as shown in Figure A. Dome formation was seen in over 5% FCS in EMEM, and induction of ALP was seen in over 1% FCS in EMEM2). GJIC in Caco-2 cells cultured with various concentrations of FCS was 3.0+0.3 (mean+SE, 10%FCS containing EMEM, Figure B), 2.3+0.6 (5%FCS), 2.5+0.4 (1%FCS), 1.1+0.1 (FCS free) respectively.

These data suggest that homologous communication ability appears to be correlated with the degree of cellular differentiation, and that our analytical method of the results by scrape-loading/dye transfer assay is useful to evaluate the GJIC ability, quantitatively.