Genetic manipulation of gap junctions in cultured cells.

Intercellular signalling is one system whose imbalance could trigger lost cell growth control. Among the mechanisms involved in intercellular singalling, gap junctional communication (GJC) has emerged as a possible regulator of cell growth. This dissertation used molecular genetic approaches to study the role of GJC in cell growth regulation. Two antisense systems were designed to inhibit GJC in WB$\sp{\rm r}$ cells, a non-tumorigenic, rat liver cell line which expresses connexin43 gap junction protein and has a high endogenous GJC capacity. The first method, using an expression vector containing a connexin43 cDNA in the antisense orientation, was not useful for blocking GJC in WB$\sp{\rm r}$ cells, with antisense expression apparently unstable. Vector sequences were lost from clones expressing connexin43 antisense RNA, and these cells grew more slowly than did control cells. These data suggested a selective disadvantage to antisense RNA expression. The second antisense system was more successful. A phosphorothioate DNA oligomer (oligo), targeted against the connexin43 gene, qualitatively decreased connexin43 protein as evidenced by decreased immunofluorescence staining of contacting cell membranes with an anti-connexin43 antibody. Oligo-treated cells assayed by dye transfer microinjection showed a slight reduction in dye transfer capacity among antisense oligo-treated cells. However, both immunofluorescence and dye transfer assays suggested that cytotoxicity was associated with the antisense oligo treatment. These data do not directly address a role for GJC in cell growth regulation, however, a possible requirement for gap junctions in cell survival is supported by these results. Because decreased GJC is associated with oncogene expression and with specific malignancies, it was of interest to map human gap junction genes and correlate their chromosomal location to the loci of proto-oncogenes. Somatic cell mapping panels were used with rat cDNA probes to map two human connexin gene loci, connexin32 (GJB1) to Xq13.1, and connexin43 (GJA1) to 6q21-23.1. Although the role of gap junctions in cell growth regulation is still ambiguous, the work contained in this dissertation extends the current genetic analysis of gap junctions in cultured cells.PhDBiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103059/1/9303722.pdfDescription of 9303722.pdf : Restricted to UM users only