A connexin 43 mutant lacking the carboxyl cytoplasmic domain inhibits both growth and motility of mouse 3T3 fibroblasts

Connexins have been shown to inhibit the growth of a wide number of communication-deficient cells both in vivo and in vitro, but the molecular mechanism remains largely unknown. In previous work we have shown that stable transfectants 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 tag, exhibit a decreased basal growth rate and weakened mitogenic response to platelet derived growth factor compared with either the parent cell line or cells transfected with an expression vector that did not encode a functional protein. Here we have investigated further the growth characteristics of these cells in order to establish the mechanism by which this protein suppresses cell growth. Analysis of DNA synthesis in individual cells by immunofluorescence staining of bromodeoxyuridine incorporation demonstrated that 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 G2/M phases of the cell cycle suggesting growth may be arrested during G2 or metaphase. In addition to effects on cell growth, Cx43-256M expression inhibited cell motility as assayed both in a wounding assay and in a Boyden chamber assay. These results now raise the question as to whether the primary effect of the Cx43-256M protein is on cell growth or cell motility.