Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than nontransfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.Intercellular communication by gap junctions has been implicated in the control of cell growth and differentiation (1, 2). These intramembranous channels, or connexons, are composed of hexameric assemblies of the gap junction proteins connexins. Isolation ofconnexins and cloning oftheir cDNAs indicate that they are encoded by a family of genes that exhibit some degree of cellular and tissue specificity (3, 4).C6 glioma cells exhibit a low level of intercellular communication by gap junctions, as shown by morphological and functional studies (5-7). Consistent with these observations, these cells contain low levels of the same gapjunction mRNA and protein that is expressed in astrocytes, namely connexin 43 (6, 8). Since tumorigenicity has been correlated with a low level or absence of intercellular communication by gap junctions (1, 9), the low level of connexin 43 mRNA and protein in C6 glioma cells may relate to the invasive growth of these cells in vivo (10).As an initial step to understand the role of gap junctions in normal and abberant cellular growth, we sought to alter the expression of connexin 43 in C6 glioma cells. To this end, we transfected these cells with a full-length cDNA for connexin 43 (11). We have obtained several clones that transcribe the transfected cDNA at moderate to high levels. Whereas connexin 43-like immunoreactivity is not readily detectable in nontransfected cells, such reactivity is prevalent in these transfected cells. The level of intercellular dye coupling correlates with the relative level of connexin 43 mRNA. Furthermore, the growth rate of the glioma cells expressing the transfected connexin 43 cDNA was significantly lower than that of nontransfected C6 cells. MATERIALS AND METHODSExpression Vector. The plasmid used for expression of the connexin 43 cDNA in rat C6 glioma cells was derived from pLTR ( Fig. 1), a simian virus 40 (SV40)-based vector, which includes the enhancer portion of the Harvey sarcoma virus long terminal re...

Abstract. Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. E Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36

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The endogenous Mr 34,000 galactoside-binding lectin (L-34) is found at elevated levels in a wide variety of neoplastic cells and correlative evidence suggests that it is involved in tumor metastasis in vivo and in transformation in vitro. We demonstrate here that introduction of recombinant L-34 into tumorigenic, weakly metastatic UV-2237-cl-15 fibrosarcoma cells results in an increased incidence of experimental lung metastases in syngeneic and nude mice. Transfection of normal BALB/c-A31 cloned fibroblasts with functional L-34 results in acquisition of anchorage-independent growth and in morphological transformation in vitro but not in tumorigenicity in vivo. These results provide direct evidence that the cellular expression of L-34 is associated with some aspects of transformation and with metastasis, but not with tumorigenicity per se.