A Connexin 43 mutant lacking the carboxyl cytoplasmic domain inhibits bothgrowth and motility of mouse 3T3 fibroblasts

Connexins have been shown to inhibit the growth of a wide number of communi cation-deficient cells both in vivo and in vitro, but the molecular mechani sm remains largely unknown. In previous work we have shown that stable tran sfectants of 3T3 A31 fibroblasts, which express a Connexin 43 (Cx43) mutant (Cx43-256M) consisting of amino acids 1-256 of rat Cx43 fused to a c-myc t ag, exhibit a decreased basal growth rate and weakened mitogenic response t o platelet derived growth factor compared with either the parent cell line or cells transfected with an expression vector that did not encode a functi onal protein. Here we have investigated further the growth characteristics of these cells in order to establish the mechanism by which this protein su ppresses cell growth. Analysis of DNA synthesis in individual cells by immu nofluorescence staining of bromodeoxyuridine incorporation demonstrated tha t the slow growth of Cx43-256M cells was due to a decrease in the number of cells that undergo DNA synthesis following growth factor stimulation. This was associated with an increased proportion of the cell population in the G(2)/M phases of the cell cycle suggesting growth may be arrested during G( 2) or metaphase. In addition to effects on cell growth, Cx43-256M expressio n inhibited cell motility as assayed both in a wounding assay and in a Boyd en chamber assay. These results now raise the question as to whether the pr imary effect of the Cx43-256M protein is on cell growth or cell motility. M el. Carcinog. 28:23-30, 2000. (C) 2000 Wiley-Liss. Inc.