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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell Commun Adhes. Author manuscript; available in PMC 2010 May 27.
Published in final edited form as:
Cell Commun Adhes. 2003 Jul–Dec; 10(4-6): 299–303.
PMCID: PMC2877511
NIHMSID: NIHMS203125

Maintaining Connexin43 Gap Junctional Communication in v-Src Cells Does Not Alter Growth Properties Associated with the Transformed Phenotype

Abstract

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine “normal” cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.

Keywords: Connexin43, gap junctional communication, oncogene, transformation, tumor suppressor, v-Src

INTRODUCTION

Connexins have long been considered to have a tumor-suppressor function due to a large body of data that supports a correlation between the presence of connexins and functional gap junctions with reduced rates of cell proliferation in “normal” cells as compared to communication-deficient tumor cells (6, 11). Up-regulation of gap junctional communication (GJC) in tumor cells by the expression of exogenous connexins has been shown to reduce the growth rates of the tumor cells, whereas the down-regulation of GJC in normal cells following treatment with tumor promoting agents or growth factors or by the induced expression of oncogenes has been associated with an increase in the rates of cell proliferation. Furthermore, fibroblast cells isolated from the Cx43 knockout mouse were found to grow faster in cell culture and to higher saturation densities than fibroblasts that were isolated from wild type mice (7). These Cx43 knockout mouse cells were not transformed and were not able to grow in an anchorage-independent manner. Re-expression of the rat wild type Cx43 protein (wt Cx43) in these fibroblasts made them communication-competent and significantly reduced their rates of growth in culture. Taken together, these and many other studies have provided support for a growth-suppressive function of the connexins and/or GJC.

One of the ways by which GJC is regulated is through the post-translational phosphorylation of the connexin protein on serine, threonine and/or tyrosine sites (4, 10). In cells that express the v-Src protein kinase, wt Cx43 is phosphorylated on tyrosine residues and GJC is dramatically disrupted (2, 5, 8, 12). This loss of GJC does not occur in mammalian cells that co-express v-Src together with a mutant form of Cx43 with phenylalanine mutations at the Tyr247 and Tyr265 sites and tyrosine phosphorylation on Cx43 is dramatically reduced in these cells (1, 5). Since the cells that express this Cx43 mutant maintain GJC in the presence of the co-expressed v-Src kinase, the question has been raised of whether the disruption of normal growth control that is characteristic of v-Src transformed cells is also observed in the cells that retain the ability to communicate through Cx43 gap junctions in the presence of v-Src.

In the present studies, we have utilized Cx43 knockout mouse cells that stably express either wt Cx43 or a mutant form of Cx43 that lacks the tyrosine sites targeted by v-Src, Y247F/Y265F Cx43, to examine how the expression of v-Src in these cells alters their growth properties. Although we have previously shown that the expression of wt Cx43 in Cx43 knockout mouse fibroblasts is sufficient to reduce their growth rates in culture, we found that maintaining Cx43-mediated GJC in cells that express the v-Src oncoprotein was not sufficient to alter growth properties that have been associated with the transformed cell phenotype. The expression of v-Src in cells that expressed wt Cx43 or the double tyrosine mutant Y247F/Y265F Cx43 resulted in similar growth properties for these two cell types, despite their differences in the ability to communicate through Cx43 gap junctions. The v-Src cells expressing either of these forms of Cx43 were able to grow in an anchorage-independent manner as opposed to the non-transformed cells that did not express the v-Src kinase. Thus, our studies do not support the hypothesis that maintaining Cx43-mediated GJC in v-Src cells is sufficient to prevent the loss of normal cell growth controls.

METHODS

Generation of Cell Lines

Cx43 knockout cell clones expressing rat wt Cx43, or a double tyrosine mutant form of rat Cx43, Y24F/Y265F Cx43, were generated by retroviral infection with a pBABE/puro/Cx43 virus as described previously (9). Selected stable cell clones expressing Cx43 were then infected with a pLxSH (vector control) or a pLv-SrcSH retrovirus. Cells that stably expressed the v-Src protein were selected with hygromycin and then subcloned (5). To address concerns due to clonal differences, we also prepared stable cell pools of hygromycin-resistant cells by retro-viral infection of cells that expressed either wt Cx43 or the Y247F/Y265F tyrosine mutant Cx43.

Characterization of the Cell Clones and Cell Pools

The expression of the Cx43 and v-Src proteins in the cell clones and cell pools was confirmed by Western blotting analysis of whole cell lysates using antibodies to Cx43, to Src or to phosphotyrosine, as described previously (5). GJC was measured in selected cell clones and cell pools by the microinjection of single cells in a monolayer with Lucifer Yellow dye (10%) and then counting the number of neighboring cells that became fluorescent due to the transfer of dye through functional gap junctions.

Cell Proliferation Studies

Changes in the growth properties of the cells were assessed in assays that measured the ability of the cell clones or the cell pools to form foci and to grow in soft agar as described previously (3). Rat-1 v-Src cells were used as a positive control in these studies and Cx43 knockout cells that expressed wt Cx43 and the empty pLxSH vector were used as a negative control. In addition, the rates of cell proliferation were monitored in culture for cells plated onto 60 mm plates and fed every two days. Cell growth was determined by counting the numbers of cells on the plates on different days after plating in replicate experiments. The rates of cell growth were examined both in normal cell media containing 10% fetal calf serum (FCS) and in media supplemented with 0.5% FCS.

RESULTS AND DISCUSSION

Western blotting analysis confirmed that the cell clones and cell pools used in these studies expressed the Cx43 protein and that the v-Src protein was expressed in the pLv-SrcSH infected cells. Dye transfer studies carried out to confirm that the cells expressing wt Cx43 and the pLxSH vector and the cells expressing the Y247F/Y265F Cx43 tyrosine mutant and v-Src were communication competent showed that, in all clones, Lucifer Yellow dye diffused to an average of 10–13 neighboring cells. Whereas GJC was severely reduced in the cell clones and cell pools that expressed the wt Cx43 protein together with v-Src, average dye transfer to ≤ 1 neighboring cell. Subsequent studies to characterize the growth of the v-Src expressing Cx43 knockout cells were carried out with two individual cell clones and a hygromycin-selected cell pool for each cell type.

Ability of the Cells to Form Foci and to Grow in Soft Agar

To assess the ability of the cell clones or the cell pools to escape from normal growth restraints and grow in three-dimensional clumps or foci rather than in monolayers, the cells were plated at a low density and maintained in culture for two weeks. The growth medium was then removed and the cells were stained to detect the presence of high-density cell foci. Rat-1 v-Src cells formed a significant number of foci under these conditions (145–312 foci/plate). As shown in Table 1, the individual cell clones and cell pool that expressed the wt Cx43 protein and the pLxSH vector did not form foci (≤ 0.3% of the number formed by the Rat-1 v-Src cells). The Cx43 knockout cells that expressed either the wt Cx43 protein or the Y247F/Y265F Cx43 mutant and v-Src exhibited a low ability to form foci compared to the Rat-1 v-Src cells (0.2–9% ± 0–5% of the number of foci). The cell pools that expressed v-Src demonstrated less ability to form foci than the individual cell clones that expressed v-Src. This may have been due, in part, to the clonal selection process in which the more aggressively growing cells were the first clones to grow up under hygromycin selection. Importantly, there was no significant difference in the levels of foci formation between the communicating cells that expressed the Y247F/Y265F Cx43 with v-Src (GJC to 10–13 neighboring cells) and the non-communicating cells that expressed wt Cx43 with v-Src (GJC ≤ 1 neighboring cell).

TABLE 1
Foci formation*

The ability of the cells to escape anchorage-dependent growth was assessed by seeding the cells in soft agar and allowing them to grow in suspension for two weeks. Rat-1 v-Src cells were used as a positive control in these studies. As shown in Table 2, the Cx43 knockout cells that expressed wt Cx43 and the pLxSH vector did not grow in soft agar. The cells that expressed v-Src with wt Cx43 or with the Y247F/Y265F Cx43 tyrosine mutant did grow in soft agar and formed colonies at ~36–78% (±6–24%) of the number that was formed by the Rat-1 v-Src cells. The cell pools expressing v-Src appeared to grow a little better in soft agar than the individual cell clones that expressed v-Src. Importantly, we found no significant difference in the ability of the cells to grow in soft agar for the v-Src cells that maintained GJC (expressing the Y247F/Y265F Cx43 mutant) and the cells that had lost the ability to communicate through Cx43 gap junctions (expressing wt Cx43).

TABLE 2
Growth in soft agar*

Rates of Cell Proliferation in 10% Serum and in 0.5% Serum

We also examined the rates of cell proliferation for the cell clones that expressed v-Src. We measured growth in normal growth media (10% FCS) and under conditions of serum-deprivation (0.5% FCS) for each cell type. The cells were plated, then fed every two days and counted on different days following plating. As shown in Figure 1, the v-Src expressing cell clones grew much faster in 10% FCS than the non v-Src cells that expressed wt Cx43 with the vector alone (wt Vector). Maintaining GJC in the cells that expressed the Y247F/Y265F Cx43 mutant and v-Src was not sufficient to slow their growth in media that contained normal levels of serum and these cells grew at a similar rate to the non-communicating cells that expressed wt Cx43 and v-Src. Under the conditions of serum-deprivation (0.5% FCS), the growth advantage of the v-Src expressing cells was lost and all of the cell types grew slowly.

Figure 1
Effects of maintaining GJC on cell proliferation in cells that express wt Cx43 or a Y247F/Y265F Cx43 mutant and the v-Src kinase

In these studies we have assessed the ability of cells that express the v-Src protein to form foci and to grow in soft agar and measured their rates of proliferation in cell culture under both normal and serum-deprived conditions. We examined whether maintaining Cx43 GJC in cells in the presence of v-Src had an affect on these aspects of cell growth. Taken together, the data that is reported here do not support the hypothesis that maintaining the ability of cells to communicate through functional Cx43 gap junctions is sufficient to prevent the loss of normal growth restraints in cells that are transformed by the v-Src oncoprotein. These studies would suggest that v-Src might effect changes to the regulation of cell growth through its actions on other cellular substrates.

Acknowledgments

This research was supported by grants from the NIH (RR16453, Charles Boyd, P.I.; Bonnie Warn-Cramer, Project P.I.) and the NCI (CA52098, Alan Lau, P.I.).

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