Cellular and Molecular Neurobiology, Vol. 12, No. 2, 1992
Characteristics of C6 Glioma Cells Overexpressing a Gap Junction Protein C. C. G. Naus, 1'3 D . Zhu, 2 S. D . L. Todd, z and G. M. Kidder z Received July 20, 1991;accepted October 12, 1991 KEY WORDS: connexin43; transfection; mRNA translation; in situ hybridization; Western blot; immunocytochemistry.
SUMMARY
1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 m R N A and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 m R N A did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling. INTRODUCTION
The gap junction is the site of the intercellular membrane channels which provide for direct cytoplasmic continuity between adjacent cells (reviewed by Bennett et al., 1991; Beyer et al., 1990). The structural unit of the gap junction is the connexon, a proteinaceous cylinder with a hydrophilic channel; connexons spanning the plasma membranes of closely apposed cells align end to end, t Department of Anatomy, The University of Western Ontario, London, Ontario N6A 5C1, Canada. Department of Zoology, The University of Western Ontario, London, Ontario N6A 5B7, Canada. 3 To whom correspondence should be addressed. 163 0272-4340/92/0400-0163506.50/0~ 1992PlenumPublishingCorporation
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forming intercellular channels. Gap junction channels provide for the exchange of small molecules (less than approximately 1000 daltons) between cells to allow metabolic cooperation (Hobbie et al., 1986); they also transmit developmental signals involved in cell patterning (Fraser et al., 1987). Among the metabolites which pass through gap junction channels are second messengers such as cAMP, inositol 1,4,5-trisphosphate, and Ca 2÷, agents involved in cellular regulation (Lawrence et al., 1978; Saez et al., 1989). Due to the recent isolation of gap junction proteins and the cloning of some of their cDNAs, it is now obvious that there is a family of gap junction proteins, called connexins, encoded by multiple genes (Beyer et al., 1990). Beyer et al. (1987) have introduced a nomenclature for the various connexins based on their predicted molecular weights. Thus, the protein initially isolated from liver gap junctions has been termed connexin32 (32 kD), while the protein isolated from heart is connexin43 (43 kD). Other gap junction proteins have been isolated recently as well, for example, connexins 26, 31 and 46 (Beyer et al., 1990; Hoh et al., 1991). In the adult, these connexins are expressed in overlapping but partially tissue-specific fashion. The characterization of connexin expression patterns in the brain has not been completed, but there is evidence for connexin32, connexin43, and connexin26 (Dermietzel et al., 1989; Dupont et al., 1988; E1 Aoumari et al., 1990; Naus et al., 1990, 1991; Paul, 1986; Yamamoto et al., 1989, 1990). From RNA analysis, it is evident that both primary astrocytes and C6 glioma cells express connexin43 (Dermietzel et al., 1991; Giaume et al., 1991; Naus et al., 1991). This is not surprising, since the glioma cell line presumably arose from an astrocytic precursor (Benda, 1968). Of particular significance is the observation that the level of connexin43 mRNA is dramatically decreased in the C6 glioma cells compared to astrocytes. Dye-coupling studies, combined with immunocytochemical localization of gap junction protein, indicate that this decrease in connexin43 mRNA in the C6 cells is reflected in the loss of intercellular coupling via gap junctions (Naus et al., 1991). This lack of intercellular communication may underlie the uncontrolled growth of these cells and their tumorigenic ability in v i v o . To address the role of gap junctions in controlling cell growth, we have initiated a series of transfection experiments aimed at altering gap junction gene expression in glioma cells. By transfecting these C6 glioma cells with appropriate plasmid constructs constitutively expressing connexin43, we have increased the expression of this gene, resulting in dramatic effects on cell coupling, cell growth, and differentiation in culture (Zhu et al., 1991). We have isolated various clones which can be grouped into low, moderate, or high expressers of connexin43 mRNA and protein. The level of expression of this gap junction gene correlates well with both the level of intercellular coupling, as revealed by cell-to-cell spread of low molecular weight dye, and the growth of these transfected clones in culture. The present study examines several of these clones with regard to protein expression and cellular phenotype. These findings support the hypothesis that intercellular communication via gap junctions is involved in controlling cell growth.
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MATERIALS AND METHODS Cell Culture and Transfection
Rat C6 glioma cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 10 ~g/ml streptomycin, and 10 U/ml penicillin. Using methods we have previously described (Zhu et al., 1991), cells were transfected with the plasmid pLTRCX43, an SV40-based vector (pLTR) in which we inserted the connexin43 cDNA clone G2. This clone contains the entire open reading frame for connexin43 and 202 bases of 5'-noncoding sequence. The pLTRCX43 construct also contains a dominant selectable marker, the Escherichia coli xanthineguanine phosphoribosyl transferase (gpt) gene. Transcription of the transfected plasmid resulted in a 2.25-kb connexin43 transcript (1.4kb G 2 c D N A + 0.85 kb SV40 splice and polyadenylation region from pLTR) (Zhu et al., 1991). Glial cultures were prepared as described previously by Hertz et al. (1978), with some modifications. Neopallium of newborn Sprague Dawley rats was disrupted by trituration and vortex-mixing in modified Eagle's minimal essential medium (MEM; GIBCO) with 20% fetal calf serum. Cells were passed twice through sterile nylon sieves (10/tm) prior to seeding in petri dishes or on sterile coverslips and incubated at 37°C in 95% atmospheric air/5% CO2 and 90% relative humidity. Serum concentration was reduced to 10% after 1 week, and cultures reached confluency after 2 weeks. They were then grown with or without 0.25 mM dibutyryl cAMP and harvested 2 weeks later. Previous work from our laboratory has indicated that more than 80% of these cells in primary astrocyte cultures are immunoreactive for GFAP (Naus et al., 1991).
RNA Isolation and Northern Blotting
Total cellular R N A from C6 glioma cells and transfected clones was extracted according to the method of Chirgwin et al. (1979), with some modification. Cellular R N A was isolated by homogenization in guanidine isothiocyanate followed by pelleting through a CsCI gradient at 32,000 rpm for 18 hr in 10oml Beckman conical open tubes. Five micrograms of total cellular RNA was electrophoretically separated in a 1.2% agarose-formaldehyde gel and capillary blotted in 10x SSC onto nitrocellulose. Blots were prehybridized in 1% crystalline grade bovine serum albumin (BSA), 1 m M E D T A , 0.5M sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate (SDS), and 100/tg/ml denatured salmon sperm DNA for 5 hr at 65°C, then hybridized in the same buffer at 65°C overnight with connexin43 cDNA probe radiolabeled with 32p using random primer extension (Feinberg and Vogelstein, 1983). The blots were then washed first with 0.5% BSA (fraction V), i mM sodium EDTA, 40 mM sodium phosphate, pH 7.2, 5% SDS twice for 20 min at room temperature, then five times for
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15min in 40mM sodium phosphate, pH7.2, l mM sodium EDTA, 1% SDS at 65°C before exposure to Kodak X A R film with intensifying screens. To obtain quantitative data on relative levels of connexin43 mRNA, autoradiographs were subjected to densitometric analysis (LKB Ultroscan).
Protein Isolation and Western Blotting Cultures of astrocytes, nontransfected C6 cells, and transfected clones Cx43-8, Cx43-12, Cx43-13, and Cx43-14 were washed three times with phosphate-buffered saline (PBS) and then incubated in 0.3 ml of PBS, 2 mM EDTA/each 60-mm petri dish for 5 min at room temperature. The volume of each sample was brought up to 5 ml with PBS, and the cells were disaggregated with a Pasteur pipette and centrifuged at 500g. For total protein isolation, a pellet of 1 × 107 cells was added to 1 ml of SDS loading buffer (2% SDS, 1 mM phenylmethylsulfonyl fluoride, 5% fl-mercaptoethanol, 20% glycerol, in 80 mM Tris-HC1, pH 6.8). To prepare a plasma membrane-enriched protein fraction, the same number of cells was lysed in 0.5% NP-40 and spun at 10,000 rpm for 3 min. The pellet was added to I ml SDS loading buffer. Samples were resolved on 12% polyacrylamide gels (Laemmli, 1970), followed by electrophoretic transfer to nitrocellulose in a BioRad apparatus at 75 V for 1 hr. The nitrocellulose blot was soaked in PBS with 5% nonfat dry milk (NFDM) for 1 hr at 37°C and then incubated overnight at 4°C with a polyclonal antibody directed against the C-terminal domain of connexin43 (residues 302-319) (gift from Dr. B. J. Nicholson; affinity purified and diluted 1:1000). After five times washing with PBS containing 5% NFDM, this was followed by incubation in 125I-goat anti-rabbit IgG (Amersham) for 1 hr. The blot was then washed five times with PBS containing 5% NFDM and two times in the same buffer with 0.1% Tween-20. The blot was then subjected to autoradiography and densitometric analysis to obtain relative quantitative values of connexin43 protein.
Separation of Polysomes from Subribosomai Ribonucleoproteins Medium was removed from 100-mm culture plates of clone Cx43-13 cells and they were washed three times in cold PBS. They were then lysed by the addition of 400/~1 lysis buffer (40 mM Tris, 150 mM NaCI, 20 mM Mg-acetate, 10 mM EGTA, 5 mM dithiothreitol, 0.035 mM cycloheximide, pH 7.5) with 1% NP-40 500 U/ml RNasin (Sigma), and 20 #g E. coli tRNA. Cells were scraped using a rubber policeman, collected in a Dounce homogenizer, and homogenized with an "A" pestle following the addition of sodium deoxycholate to 0.4%. The homogenate was divided into two portions for centrifugation in a Beckman TLA-100 rotor at 24,000 rpm (20,000 g) for 3 min at 4°C. The postmitochondrial supernatants were removed and each was layered over a 50-#1 cushion of 40% sucrose in TAM buffer (20 mM Tris, 100 mM NH4CI, 5 mM Mg-acetate, 5 mM DTT, pH7.5) and spun in the TLA-100 rotor at 50,000rpm (100,000g) for 40 min, forming a polysomal pellet and a subribosomal supernatant. The pellets were resuspended in 150/tl of lysis buffer and, with the supernatants, loaded onto continuous 15-40% sucrose gradients which were spun at 40,000rpm in a
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Beckman SW40 rotor for 2.5 hr at 4°C. Gradients were then scanned at an absorption wavelength of 254 nm. The UV absorption profiles indicated that polysomes and most of the monosomes were contained in the pellet fractions, leaving ribosomal subunits and the remaining monosomes in the supernatants. To obtain R N A for Northern blot analysis, the supernatant subribosomal fractions from four separate 100-mm plates were made 0.3 M with sodium acetate (pH 5.2), precipitated in 95% ethanol, and centrifuged in a Sorvall HB-4 rotor at 12,000 rpm for 20 min at 4°C. The subribosomal and polysomal pellets were resuspended in 10mM Tris-HC1, p H 8 , 1 mM EDTA, 10mM DTY, and 100 U/ml RNasin. The ribonucleoproteins were denatured by combining 10/zl of solubilized pellet with 100/zl of a solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7, 0.5% Sarkosyl, and 100 mM fl-mercaptoethanol. Protein was extracted using phenol/chloroform/isoamyl alcohol, and the resultant RNA subjected to Northern blotting. Northern blots were hybridized with connexin43 cDNA (Beyer et al., 1987) along with a histone H3 clone, both radiolabeled with 32p using random primer extension. Following hybridization, blots were exposed to X-ray film and the resultant autoradiographs subjected to densitometric analysis (LKB Ultroscan). In Situ
Hybridization
In situ hybridization to localize connexin43 m R N A was carried out using a modification of the method of Whitfield et al. (1990). Briefly, cultures were fixed
for 20 min in 4% paraformaldehyde in PBS, then rinsed several times in PBS and once in 0.1 M triethanolamine-HC1, pH 8.0, followed by 10 min in 0.25% acetic anhydride in the same buffer. After rinsing the slides in 2× SSC, they were dehydrated in graded ethanols and hybridized with the radiolabeled connexin43 probe, suspended in hybridization buffer (60mM NaCI, 10mM Tris-HC1, pH 7.5, 0.02% Denhardt's solution, 1 mM EDTA, pH 8.0, 0.01% herring sperm DNA, 0.05% yeast tRNA, 10% dextran sulfate, 50% formamide, 0.05% SDS, 0.05% sodium thiosulfate, 50 mM DTT) overnight at 45°C. Following hybridization, coverslips were washed in several changes of 2x SSC, then treated with 20/tg/ml RNase A in 500 mM NaC1, 10 mM Tris-HC1, 1 mM E D T A for 30 min at room temperature, followed by a 30-min rinse in the same buffer. The final wash was carried out with 2x SSC at 50°C and 0.2× SSC at 55°C. After drying, coverslips were dipped in Kodak NTB-2 emulsion, exposed for several days, and developed. Antisense 35S-labeled connexin43 probe was generated from a 1.4-kb cDNA (Beyer et al., 1987) subcloned into Bluescript M13+, linearized with SalI, and transcribed with T3 R N A polymerase in the presence of 35S-UTP (New England Nuclear, 1000 Ci/mmol). For control hybridization, radiolabeled sense-strand RNA was transcribed by T7 polymerase from the same plasmid linearized with HindlII.
Immunocytochemistry C6 glioma cells and several of the transfected clones were cultured on glass coverslips coated with poly-L-lysine (30-70 kD; GIBCO). For im-
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munocytochemistry, cells were fixed in 95% ethanol/5% glacial acetic acid for 20min at -20°C, rinsed with PBS, and incubated in rabbit polyclonal anticonnexin43 (diluted 1 : 100) or preimmune serum from the same rabbit for 2 hr at room temperature. After subsequent incubation in fluorescein-conjugated goat anti-rabbit IgG (Vector Laboratories; diluted 1:50), coverslips were rinsed in PBS and mounted on slides in PBS containing 50% glycerol, 0.1% pphenylenediamine. To localize actin, cells were fixed for 10min in 2.5% formaldehyde/l% glutaraldehyde in phosphate buffer, followed by a 1-min treatment with 0.1% Triton X-100 in phosphate buffer. After several washes, cells were then incubated in fluorescein phaUoidin (Molecular Probes, Eugene, OR) (diluted 1:6) for 30 min, washed, and mounted for immunofluorescence microscopy. RESULTS
In our intial study of C6 glioma cells transfected with connexin43 cDNA, we found different levels of connexin43 m R N A in different clones (Zhu et al., 1991). These clones also exhibited different degrees of intercellular coupling, which were correlated with the amount of connexin43 mRNA. In this study, we have examined the amount of connexin43 protein in several of these clones, as well as other aspects of the cellular phenotype. R N A analysis indicated that the level of 3-kb connexin43 m R N A is much lower in the nontransfected C6 cells than in primary astrocytes (Fig. 1). In contrast, different transfected clones contained various levels of this mRNA, as well as the 2.25-kb mRNA transcribed from the transfected pLTRCx43. This 2.25-kb mRNA was detected only in clones which retained the 1.4-kb EcoR1 restriction fragment (Zhu et al., 1991). Clone Cx43-8 contained only the endogenous 3-kb mRNA, while clones Cx43-12, Cx43-13, and Cx43-14 expressed the transfected cDNA to yield the 2.25-kb mRNA (Fig. 1). From densitometric analysis of RNA blots, the connexin43 mRNA levels, relative to the level in the nontransfected C6 cells, were 10-fold greater in primary astrocytes, 2-fold greater in clone Cx43-8, 30-fold greater in clones Cx43-12 and Cx43-14, and 50-fold greater in clone Cx43-13.
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~ Fig. 1. Northern blot analysis of connexin43 in RNA isolated from astrocytes, nontransfected C6 glioma cells, and several ~ transfected clones. All cells express the 3-kb connexin43 mRNA. Astrocytes, C6, and clone Cx43-8 cells express only the 3-kb mRNA (filled arrow). C6 cells exhibit a 10-fold reduction in the level of this mRNA in comparison to astrocytes. The other clones also express the exogeneous 2.25-kb connexin43 mRNA (open arrow).
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Western blot analysis demonstrated a correlation between the amount of detectable steady-state connexin43 mRNA and protein. Comparison was made between the amount of immunodetectable connexin43 in total protein preparations isolated from primary astrocytes, nontransfected C6 cells, and several of the transfected clones (Fig. 2a, lanes 1-6). While connexin43 is readily demonstrable in total protein from cultured astrocytes (Fig. 2a, lane 1), it is barely detectable in the nontransfected C6 cells (Fig. 2a, lane 2). The level of connexin43 is slightly higher in clone Cx43-8 (Fig. 2a, lane 3) and higher still in clones Cx43-12 (Fig. 2a, lane 4) and Cx43-14 (Fig. 2a, lane 5). Finally, clone Cx43-13 displays a dramatic increase in connexin43 protein (Fig. 2a, lane 6). When densitometric values were obtained from Western blots, the level of connexin43 protein, relative to nontransfected C6 cells, was 6-fold greater for primary astrocytes, 1.5-fold greater for clone Cx43-8, 3-fold greater for clones Cx43-12 and Cx43-14, and 8-fold greater for clone Cx43-13. Thus there is a rough correlation between connexin43 mRNA and protein level in the various clones. It is clear that the connexin43 protein detected exists in different forms. At least two, and in some samples three, bands were seen, with different levels of slower- and faster-migrating variants (Fig. 2a). While this is clear in samples of astrocytes (lane 1) and clones expressing the transfected cDNA (lanes 4-6), nontransfected C6 cells and clone Cx43-8 appear to express only the slowermigrating protein species (lanes 2 and 3). To obtain a rough measure of the proportion of Cx43 in plasma membranes, samples were prepared from membrane-enriched fractions (Fig. 2b, lanes 2, 4 and 6) and compared to total protein (Fig. 2b, lanes 1, 3, and 5). In astrocytes, three distinct bands are detectable with approximate Mr's of 40, 43, and 46 kD (Fig. 2b, lane 1). In contrast, a membrane-enriched protein fraction from astrocytes contains only the 43- and 46-kD bands (Fig. 2b, lane 2). Nontransfected C6 cells do not display these multiple bands, and very little of the connexin43 immunoreactivity is present in the membrane-enriched fraction (Fig. 2b, lanes 3 and Fig. 2. (a) Western blot analysis of a membrane-enriched protein fraction prepared from 2 x 105 cells. Primary astrocytes (lane 1) exhibit two sizes of connexin43 protein (Mr, approximately 43 and 46 kD). Nontransfected C6 cells (lane 2) and clone Cx43-8 (lane 3) display predominantly connexin43 with Mr approximately 46 kD. Clones Cx43-12 (lane 4), Cx43-14 (lane 5), and Cx43-13 (lane 6) display three species of connexin43 protein, with M~'s of approximately 40, 43, and 46 kD. (b) Comparison of connexin43 in total vs membrane-enriched protein isolated from 2 x 105 cells. Total 58,.D,.protein from astrocytes (lane 1) clearly displays three bands representing proteins with approximate Mr 40,43, and 46 kD. In contrast, the membrane-enriched fraction (lane 2) shows only the 43- and 46-kD protein. This difference is not apparent in Cx43-13 total (lane 5) vs membrane-enriched 58"~ (lane 6) samples; both contain all three protein species. In 48"~ contrast, nontransfected C6 cells display only the 46-kD protein in both total (lane 3) and membrane-enriched (lane 4) samples. Numbers indicate positions of molecular weight 36.,~ standards in (kD).
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4). In contrast, clone Cx43-13 displays multiple bands of connexin43, the major portion being represented in the faster-migrating species (Fig. 2b, lanes 5 and 6). The possible significance of these findings is considered in the Discussion. In our previous report, we have demonstrated that C6 glioma cells transfected with connexin43 cDNA proliferate more slowly than nontransfected cells and that the rate of cell proliferation is inversely related to the amount of connexin43 mRNA and the level of dye coupling (Zhu et al., 1991). The simplest explanation for this is that increased gap junctional intercellular communication leads to a reduction of cell proliferation. However, it is also possible that overproduction of a particular mRNA can in itself slow the rate of proliferation, perhaps by swamping the translational machinery and thus limiting the production of proteins involved in DNA replication, chromatin assembly, or progression through the cell cycle. We examined this possibility by determining the proportion of connexin43 and histone H3 mRNAs in polysomes during exponential growth of clone Cx43-13 cells, these being the transfected clone with the most severely limited cell proliferation (Zhu et al., 1991). Polysomal and subribosomal fractions were prepared as described under Materials and Methods, and the purified RNA from these fractions was analyzed by Northern blotting (Fig. 3). Simultaneous hybridization with connexin43 and histone H3 DNAs revealed the presence of the endogenous (i.e., 3 kb) and exogenous (i.e., transfected; 2.25 kb) connexin43 mRNAs, as well as the histone H3 mRNA (about 0.7 kb). After 5 hr of exposure, only the exogenous 2.25-kb mRNA was evident in the subribosomal RNA sample. After longer exposures (not shown), histone H3 mRNA could also be detected in the subribosomal sample, but endogenous connexin43 mRNA could not. Densitometric analyses of various exposures of such Northern blots from four separate plates of clone Cx43-13 cells were performed to determine the fraction of mRNA in polysomes. For all three transcripts, the fraction in polysomes was very high: 1.00 for endogenous connexin43 mRNA; 0.94 + 0.01
S
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Fig. 3. Northern blot analysis of RNA isolated from subribosomal (S) and polysomal (P) preparations obtained from clone Cx43-13 cells. Histone H3 mRNA is seen at 0.7kb, exogenous (i.e., transfected) connexin43 mRNA at 2.25kb, and endogenous connexin43 mRNA at 3.0 kb. All three mRNAs are predominantly associated with the polysomal fraction.
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(SD) for exogenous connexin43 mRNA; and 0.94 + 0.03 for histone H3 mRNA. To confirm that the transcripts in the 100,000g pellets were indeed associated with polysomes, postmitochondrial supernatants were treated with 50 mM EDTA for 15 min at room temperature before centrifugation. This caused all of the histone mRNA and 0.87 + 0.09 (n = 3) of the Cx43 m R N A to shift from the polysomal pellet to the subribosomal supernatant (autoradiograms not shown). Expression of connexin43 at the cellular level was examined using in situ hybridization and immunocytochemistry. The level of connexin43 mRNA was dramatically higher in clone Cx43-13 cells than in nontransfected C6 cells (Fig. 4). It is clear that all the Cx43-13 cells contain substantial amounts of this mRNA, as evidenced by the density of autoradiographic grains overlying each cell (Fig. 4b). In contrast, very few autoradiographic grains were localized over nontransfected C6 cells (Fig. 4a). Control hybridization with the sense-strand R N A probe produced only background labeling. Immunocytochemistry revealed a similar abundance of connexin43 protein in Cx43-13 cells, while this immunoreactivity was much lower in nontransfected C6 cells (Figs. 5a and b). While some of the connexin43 immunolabeling was present at membrane areas of intercellular contact, it is clear that a major proportion of connexin43 immunoreactivity in clone Cx43-13 cells is not directly associated with the plasma membrane, but, rather, is located in perinuclear and cytoplasmic regions. To examine cytoskeletal differences between nontransfected C6 cells and the high-expressing transfected clone, Cx43-13, the actin-selective stain, phalloidin, was used. There was no obvious organization of actin filaments in the nontransfected cells (Fig. 5c). In contrast, clone Cx43-13 showed a clear organization of actin into stress fibers (Fig. 5d). This change in cytoskeletal organization was also correlated with a more flattened cell morphology.
Fig. 4. In situ hybridization for connexin43 mRNA in cultures of nontransfected C6 (a) and clone Cx43-13 (b) cells. While C6 cells exhibit very little hybridization with the radiolabeled connexin43 cRNA probe, all clone Cx43-13 cells display strong hybridization. Bar = 50/~m.
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Fig. 5. Immunocytochemical localization of connexin43 in C6 glioma ceils and clone Cx43-13 cells. (a) Some punctate immunofluorescence is detectable in nontransfected C6 glioma cells. (b) In clone Cx43-13 cells, a dramatic increase in immunodetectable connexin43 protein is evident. While some of this is localized to areas of intercellular membrane contract (arrows), abundant immunoreactivity is present in perinuclear and cytoplasmic areas. (c) Fluorescein phalloidin staining in nontransfected C6 cells does not reveal obvious actin organization. (d) In cultures of clone Cx43-13, fluorescein phalloidin dearly demonstrates actin stress fibers. Bar = 50 #m.
DISCUSSION Intercellular c o m m u n i c a t i o n via gap junctions has b e e n implicated as being important in the regulation of cell proliferation and subsequent differentiation (Loewenstein, 1979). In fact, there is g o o d evidence that an interruption of this c o m m u n i c a t i o n p a t h w a y is one of the steps in malignant t r a n s f o r m a t i o n (reviewed by Klaunig and R u c h , 1990): a variety of chemical agents which t r a n s f o r m cells in
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vitro cause a reduction of intercellular communication. Conversely, the growth of transformed cells can be inhibited when they come into contact with normal cells, and this is known to be due to the assembly of gap junction channels between the two cell types (Mehta et al., 1986). Recently, it was demonstrated that a gap junction protein (connexin43) is a direct target for phosphorylation by the product of the v-src oncogene, the result of which is the blockage of intercellular coupling (Swenson et al., 1990). The C6 glioma cell has been extensively used as a model of malignant transformation of glial cells (Benda et al., 1968). While astrocytes are highly coupled via gap junctions, C6 cells exhibit very restricted intercellular coupling (Naus et al., 1991; Tiffany-Castiglioni et al., 1986). We have increased gap junctional coupling in C6 cells by transfection with connexin43 cDNA (Zhu et al., 1991). Several of the selected clones exhibit different degrees of expression of the transfected cDNA, this being evident from the levels of steady-state connexin43 mRNA and protein. There is clear association between the level of steady-state mRNA transcribed from the transfected cDNA and the amount of immunodetectable connexin43 protein. While Western blot analysis of protein isolated from nontransfected C6 cells revealed the presence of only a single band of connexin43, multiple electrophoretic variants of this protein were detected in samples from primary astrocytes and transfected C6 clones. These multiple species have been shown to represent different phosphorylation states of the connexin43 protein, with fully phosphorylated form having the lowest mobility (reviewed by Musil and Goodenough, 1990). Recent studies by Dermietzel et al. (1991) and Giaume et al. (1991) showed the presence of multiple species of connexin43 protein in cultures of astrocytes, similar to our current findings for astrocytes. If these multiple bands of immunoreactivity observed in the present study also represent different phosphorylation states of connexin43, and the single band in nontransfected C6 cells represent the fully phosphorylated form, it is possible that overexpression of this protein in clone Cx43-13 may lead to saturation of the cellular mechanisms involved in phosphorylation. This could explain our finding that much of the connexin43 in clone Cx43-13 cells remains in the cytoplasm; phosphorylation is closely linked with plasma membrane insertion (Musil et al., 1990). We have previously reported a reduction in the proliferation of C6 cells following transfection with connexin43 cDNA (Zhu et al., 1991). It is possible that the observed overproduction of connexin43 mRNA may have affected proliferation indirectly by merely saturating the translational machinery or by increasing translational competition between messages. We chose to monitor the partitioning of histone H3 mRNA between polysomes and subribosomal mRNPs as a test of this possibility because of the tight coupling of histone synthesis to DNA replication (Marzluff and Pandey, 1988). Our results, demonstrating that almost all connexin43 and histone H3 mRNAs are undergoing translation in exponentially growing clone Cx43-13 cells, makes it extremely unlikely that their reduced rate of proliferation is due to interference with translation. In addition to a reduction of proliferation rate, cytoskeletal alterations were noted following transfection of C6 cells with connexin43 cDNA. Phalloidin
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staining revealed an organization of actin filaments into stress fibers in the high-expressing clone, Cx43-13, while no such organization was evident in the nontransfected cells. Several studies have indicated that there is a loss of stress fibers following malignant transformation (reviewed by Burridge, 1986). Taken together, these results establish a relationship among expression of a gap junction protein, enhanced intercellular communication, and phenotypic transformation of glioma cells to resemble more closely their normal progenitors. Although other explanations are still possible, the data strongly indicate that the restoration of gap junctional coupling is the causative element in this process. The C6 cell transfection model provides a unique opportunity to discover the exact role of intercellular communication in maintaining the normal cellular phenotype.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Eric Beyer for providing the connexin43 cDNA, to Dr. A. Raz for providing the plasmid pLTR, to Dr. Gilbert Schultz for the histone H3 clone, to Dr. Bruce Nicholson for supplying the antiserum, and to Sharon Kent, John Bechberger, Daniel Belliveau, and Geralyn Wood for technical assistance. This work was supported by grants from the Medical Research Council of Canada (to C. C. G. Naus), the Natural Sciences and Engineering Research Council of Canada (to G. M. Kidder), and the Cancer Research Society. REFERENCES Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, W. (1986). Differentiated rat glial cell strain in tissue culture. Science 161:370-371. Bennett, M. V. L., Barrio, L. C., Bargiello, T. A., Spray, D. C., Hertzberg, E., and Saez, J. C. (1991). Gap junctions: New tools, new answers, new questions. Neuron 6:305-320. Beyer, E. C., Paul, D. L., and Goodenough, D. A. (1987). Connexin 43: A protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol. 105:2621-2629. Beyer, E. C., Paul, D. L., and Goodenough, D. A. (1990). Connexin family of gap junction proteins. J. Mernbr. Biol. 116:187-194. Burridge, K. (1986). Substrate adhesions in normal and transformed fibroblasts: Organization and regulation of cytoskeletal, membrane and extracellular matrix components at focal contacts. Cancer Rev. 4:18-78. Chirgwin, J. M., Pryzybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. Dermietzel, R., Traub, O., Hwang, T. K., Beyer, E. C., Bennett, M. V. L., Spray, D. C., and Willecke, K. (1989). Differential expression of three gap junction proteins in developing and mature brain tissue. Proc. Natl. Acad. Sci. USA 86:10148-10152. Dermietzel, R., Hertzberg, E. L,, Kessler, J. A., and Spray, D. C. (1991). Gap junctions between cultured astrocytes: Immunocytochemical, molecular, and electrophysiological analysis. J. Neurosci. 11:1421-1432. Dupont, E., E1 Aoumari, A., Roustiau-Severe, S., Briand, J. P., and Gros, D. (1988). Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs. J. Membr. Biol. 104:119-128. El Aoumari, A., Fromaget, C., Dupont, E., Reggio, H., Durbec, P., Briand, J.-P., Boller, K., Kretman, B., and Gros, D. (1990). Conservation of a cytoplasmic carboxy-terminal domain of connexin43, a gap junctional protein in mammalian heart and brain. J. Membr. Biol. 115:229-240.
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Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. Fraser, S. E., Green, C. R., Bode, H. R., and Gilula, N. B. (1987). Selective disruption of gap junctional communication interferes with a patterning process in Hydra. Science 237:49-55. Giaume, C., Fromaget, C., E1 Aoumari, A., Cordier, J., Glowinski, J., and Gros, D. (1991). Gap junctions in cultured astrocytes: Single-channel currents and characteristics of channel-forming protein. Neuron 6:133-143. Graves, R. A., Marzluff, W. F., Gielbelhaus, D. H., and Schultz, G. A. (1985). Quantitative and qualitative changes in histone gene expression during early mouse embryo development. Proc. Natl. Acad. Sci. USA 82:5685-5689. Hertz, L., Bock, E., and Schousboe, A. (1978). GFA content, glutamate uptake and activity of glutamate metabolizing enzymes in differentiating mouse astrocytes in primary cultures. Dev. Neurosci. 1:226-238. Hobbie, L., Kingsley, D. M., Kozarski, K. F., Jackman, R. W., and Krieger, M. (1986). Restoration of LDL receptor activity in mutant cells by intercellular junctional communication. Science 235:69-73. Hoh, J. H., John, S. A., and Revel, J-P. (1991). Molecular cloning and characterization of a new member of the gap junction gene family, connexin31. J. Biol. Chem. 266:6524-6531. Klaunig, J. E., and Ruch, R. J. (1990). Biology of disease: Role of inhibition of intercellular communication in carcinogenesis. Lab. Invest. 62:135-146. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lawrence, T. S., Beers, W. H., and Gilula, N. B. (1978). Transmission of hormonal stimulation by cell-to-cell communication. Nature 272:501-506. Loewenstein, W. R. (1979). Junctional intercellular communication and the control of cell growth. Biochim. Biophys. Acta 560:1-65. Marzluff, W. F., and Pandey, N. (1988). Multiple regulatory steps control histone mRNA concentrations. TIBS 13:49-52. Mehta, P. P., Bertram, J. S., and Loewenstein, W. R. (1986). Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell 44:187-196. Musil, L. S., and Goodenough, D. A. (1990). Gap junctional intercellular communication and the regulation of connexin expression and function. Curr. Opin. Cell. Biol. 2:875-880. Musil, L. S., Cunningham, B. A., Edelman, G. M., and Goodenough, D. A. (1990). Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J. Cell. Biol. 111:2077-2088. Naus, C. C. G., Belliveau, D. J., and Bechberger, J. F. (1990). Regional differences in connexin32 and connexin43 messenger RNAs in rat brain. Neurosci. Lett. 111:297-302. Naus, C. C. G., Bechberger, J. F., Caveney, S., and Wilson, J. X. (1991). Expression of gap junction genes in astrocytes and C6 glioma cells. Neurosci. Lett. 112:33-36. Paul, D. L. (1986). Molecular cloning of cDNA for rat liver gap junction protein. J. Cell Biol. 103:123-134. Saez, J. C., Connor, J. A., Spray, D. C. and Bennett, M. V. L. (1989). Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions. Proc. Natl. Acad. Sci. USA 86:2708-2712. Swenson, K. I., Piwnica-Worms, H., McNamee, H., and Paul, D. L. (1990). Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60..... -induced inhibition of communication. Cell Reg. 1: 989-1002. Tiffany-Castiglioni, E., Neck, K. F., and Caceci, T. (1986). Glial culture on artificial capillaries: Electron microscopic comparisons of C6 rat glioma cells and rat astroglia. J. Neurosci. Res. 16:387-396. Whitfield, H. J., Brady, L. S., Smith, M. A., Mamalaki, E., Fox, R. J., and Herkenham, M. (1990). Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: A detailed protocol. Cell. Mol. Neurobiol. 10:145-156. Yamamoto, T., Shiosaka, S., Whittaker, M. E., Hertzberg, E. L., and Nagy, J. I. (1989). Gap junction protein in rat hippocampus: Light microscope immunohistochemical localization. J. Comp. Neurol. 281:269-281. Yamamoto, T., Ochalski, A., Hertzberg, E. L., and Nagy, J. I. (1990). LM and EM immunolocalization of the gap junctional protein connexin43 in rat brain. Brain Res. 508:313-319. Zhu, D., Caveney, S., Kidder, G. M., and Naus, C. C. G. (1991). Transfection of C6 glioma cells with connexin43 cDNA: Analysis of expression, intercellular coupling and cell proliferation. Proc. Natl. Acad. Sci. USA 88:1883-1887.
Characteristics of C6 Glioma Cells Overexpressing a Gap Junction Protein C. C. G. Naus, 1'3 D . Zhu, 2 S. D . L. Todd, z and G. M. Kidder z Received July 20, 1991;accepted October 12, 1991 KEY WORDS: connexin43; transfection; mRNA translation; in situ hybridization; Western blot; immunocytochemistry.
SUMMARY
1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 m R N A and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 m R N A did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling. INTRODUCTION
The gap junction is the site of the intercellular membrane channels which provide for direct cytoplasmic continuity between adjacent cells (reviewed by Bennett et al., 1991; Beyer et al., 1990). The structural unit of the gap junction is the connexon, a proteinaceous cylinder with a hydrophilic channel; connexons spanning the plasma membranes of closely apposed cells align end to end, t Department of Anatomy, The University of Western Ontario, London, Ontario N6A 5C1, Canada. Department of Zoology, The University of Western Ontario, London, Ontario N6A 5B7, Canada. 3 To whom correspondence should be addressed. 163 0272-4340/92/0400-0163506.50/0~ 1992PlenumPublishingCorporation
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forming intercellular channels. Gap junction channels provide for the exchange of small molecules (less than approximately 1000 daltons) between cells to allow metabolic cooperation (Hobbie et al., 1986); they also transmit developmental signals involved in cell patterning (Fraser et al., 1987). Among the metabolites which pass through gap junction channels are second messengers such as cAMP, inositol 1,4,5-trisphosphate, and Ca 2÷, agents involved in cellular regulation (Lawrence et al., 1978; Saez et al., 1989). Due to the recent isolation of gap junction proteins and the cloning of some of their cDNAs, it is now obvious that there is a family of gap junction proteins, called connexins, encoded by multiple genes (Beyer et al., 1990). Beyer et al. (1987) have introduced a nomenclature for the various connexins based on their predicted molecular weights. Thus, the protein initially isolated from liver gap junctions has been termed connexin32 (32 kD), while the protein isolated from heart is connexin43 (43 kD). Other gap junction proteins have been isolated recently as well, for example, connexins 26, 31 and 46 (Beyer et al., 1990; Hoh et al., 1991). In the adult, these connexins are expressed in overlapping but partially tissue-specific fashion. The characterization of connexin expression patterns in the brain has not been completed, but there is evidence for connexin32, connexin43, and connexin26 (Dermietzel et al., 1989; Dupont et al., 1988; E1 Aoumari et al., 1990; Naus et al., 1990, 1991; Paul, 1986; Yamamoto et al., 1989, 1990). From RNA analysis, it is evident that both primary astrocytes and C6 glioma cells express connexin43 (Dermietzel et al., 1991; Giaume et al., 1991; Naus et al., 1991). This is not surprising, since the glioma cell line presumably arose from an astrocytic precursor (Benda, 1968). Of particular significance is the observation that the level of connexin43 mRNA is dramatically decreased in the C6 glioma cells compared to astrocytes. Dye-coupling studies, combined with immunocytochemical localization of gap junction protein, indicate that this decrease in connexin43 mRNA in the C6 cells is reflected in the loss of intercellular coupling via gap junctions (Naus et al., 1991). This lack of intercellular communication may underlie the uncontrolled growth of these cells and their tumorigenic ability in v i v o . To address the role of gap junctions in controlling cell growth, we have initiated a series of transfection experiments aimed at altering gap junction gene expression in glioma cells. By transfecting these C6 glioma cells with appropriate plasmid constructs constitutively expressing connexin43, we have increased the expression of this gene, resulting in dramatic effects on cell coupling, cell growth, and differentiation in culture (Zhu et al., 1991). We have isolated various clones which can be grouped into low, moderate, or high expressers of connexin43 mRNA and protein. The level of expression of this gap junction gene correlates well with both the level of intercellular coupling, as revealed by cell-to-cell spread of low molecular weight dye, and the growth of these transfected clones in culture. The present study examines several of these clones with regard to protein expression and cellular phenotype. These findings support the hypothesis that intercellular communication via gap junctions is involved in controlling cell growth.
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MATERIALS AND METHODS Cell Culture and Transfection
Rat C6 glioma cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 10 ~g/ml streptomycin, and 10 U/ml penicillin. Using methods we have previously described (Zhu et al., 1991), cells were transfected with the plasmid pLTRCX43, an SV40-based vector (pLTR) in which we inserted the connexin43 cDNA clone G2. This clone contains the entire open reading frame for connexin43 and 202 bases of 5'-noncoding sequence. The pLTRCX43 construct also contains a dominant selectable marker, the Escherichia coli xanthineguanine phosphoribosyl transferase (gpt) gene. Transcription of the transfected plasmid resulted in a 2.25-kb connexin43 transcript (1.4kb G 2 c D N A + 0.85 kb SV40 splice and polyadenylation region from pLTR) (Zhu et al., 1991). Glial cultures were prepared as described previously by Hertz et al. (1978), with some modifications. Neopallium of newborn Sprague Dawley rats was disrupted by trituration and vortex-mixing in modified Eagle's minimal essential medium (MEM; GIBCO) with 20% fetal calf serum. Cells were passed twice through sterile nylon sieves (10/tm) prior to seeding in petri dishes or on sterile coverslips and incubated at 37°C in 95% atmospheric air/5% CO2 and 90% relative humidity. Serum concentration was reduced to 10% after 1 week, and cultures reached confluency after 2 weeks. They were then grown with or without 0.25 mM dibutyryl cAMP and harvested 2 weeks later. Previous work from our laboratory has indicated that more than 80% of these cells in primary astrocyte cultures are immunoreactive for GFAP (Naus et al., 1991).
RNA Isolation and Northern Blotting
Total cellular R N A from C6 glioma cells and transfected clones was extracted according to the method of Chirgwin et al. (1979), with some modification. Cellular R N A was isolated by homogenization in guanidine isothiocyanate followed by pelleting through a CsCI gradient at 32,000 rpm for 18 hr in 10oml Beckman conical open tubes. Five micrograms of total cellular RNA was electrophoretically separated in a 1.2% agarose-formaldehyde gel and capillary blotted in 10x SSC onto nitrocellulose. Blots were prehybridized in 1% crystalline grade bovine serum albumin (BSA), 1 m M E D T A , 0.5M sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate (SDS), and 100/tg/ml denatured salmon sperm DNA for 5 hr at 65°C, then hybridized in the same buffer at 65°C overnight with connexin43 cDNA probe radiolabeled with 32p using random primer extension (Feinberg and Vogelstein, 1983). The blots were then washed first with 0.5% BSA (fraction V), i mM sodium EDTA, 40 mM sodium phosphate, pH 7.2, 5% SDS twice for 20 min at room temperature, then five times for
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15min in 40mM sodium phosphate, pH7.2, l mM sodium EDTA, 1% SDS at 65°C before exposure to Kodak X A R film with intensifying screens. To obtain quantitative data on relative levels of connexin43 mRNA, autoradiographs were subjected to densitometric analysis (LKB Ultroscan).
Protein Isolation and Western Blotting Cultures of astrocytes, nontransfected C6 cells, and transfected clones Cx43-8, Cx43-12, Cx43-13, and Cx43-14 were washed three times with phosphate-buffered saline (PBS) and then incubated in 0.3 ml of PBS, 2 mM EDTA/each 60-mm petri dish for 5 min at room temperature. The volume of each sample was brought up to 5 ml with PBS, and the cells were disaggregated with a Pasteur pipette and centrifuged at 500g. For total protein isolation, a pellet of 1 × 107 cells was added to 1 ml of SDS loading buffer (2% SDS, 1 mM phenylmethylsulfonyl fluoride, 5% fl-mercaptoethanol, 20% glycerol, in 80 mM Tris-HC1, pH 6.8). To prepare a plasma membrane-enriched protein fraction, the same number of cells was lysed in 0.5% NP-40 and spun at 10,000 rpm for 3 min. The pellet was added to I ml SDS loading buffer. Samples were resolved on 12% polyacrylamide gels (Laemmli, 1970), followed by electrophoretic transfer to nitrocellulose in a BioRad apparatus at 75 V for 1 hr. The nitrocellulose blot was soaked in PBS with 5% nonfat dry milk (NFDM) for 1 hr at 37°C and then incubated overnight at 4°C with a polyclonal antibody directed against the C-terminal domain of connexin43 (residues 302-319) (gift from Dr. B. J. Nicholson; affinity purified and diluted 1:1000). After five times washing with PBS containing 5% NFDM, this was followed by incubation in 125I-goat anti-rabbit IgG (Amersham) for 1 hr. The blot was then washed five times with PBS containing 5% NFDM and two times in the same buffer with 0.1% Tween-20. The blot was then subjected to autoradiography and densitometric analysis to obtain relative quantitative values of connexin43 protein.
Separation of Polysomes from Subribosomai Ribonucleoproteins Medium was removed from 100-mm culture plates of clone Cx43-13 cells and they were washed three times in cold PBS. They were then lysed by the addition of 400/~1 lysis buffer (40 mM Tris, 150 mM NaCI, 20 mM Mg-acetate, 10 mM EGTA, 5 mM dithiothreitol, 0.035 mM cycloheximide, pH 7.5) with 1% NP-40 500 U/ml RNasin (Sigma), and 20 #g E. coli tRNA. Cells were scraped using a rubber policeman, collected in a Dounce homogenizer, and homogenized with an "A" pestle following the addition of sodium deoxycholate to 0.4%. The homogenate was divided into two portions for centrifugation in a Beckman TLA-100 rotor at 24,000 rpm (20,000 g) for 3 min at 4°C. The postmitochondrial supernatants were removed and each was layered over a 50-#1 cushion of 40% sucrose in TAM buffer (20 mM Tris, 100 mM NH4CI, 5 mM Mg-acetate, 5 mM DTT, pH7.5) and spun in the TLA-100 rotor at 50,000rpm (100,000g) for 40 min, forming a polysomal pellet and a subribosomal supernatant. The pellets were resuspended in 150/tl of lysis buffer and, with the supernatants, loaded onto continuous 15-40% sucrose gradients which were spun at 40,000rpm in a
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Beckman SW40 rotor for 2.5 hr at 4°C. Gradients were then scanned at an absorption wavelength of 254 nm. The UV absorption profiles indicated that polysomes and most of the monosomes were contained in the pellet fractions, leaving ribosomal subunits and the remaining monosomes in the supernatants. To obtain R N A for Northern blot analysis, the supernatant subribosomal fractions from four separate 100-mm plates were made 0.3 M with sodium acetate (pH 5.2), precipitated in 95% ethanol, and centrifuged in a Sorvall HB-4 rotor at 12,000 rpm for 20 min at 4°C. The subribosomal and polysomal pellets were resuspended in 10mM Tris-HC1, p H 8 , 1 mM EDTA, 10mM DTY, and 100 U/ml RNasin. The ribonucleoproteins were denatured by combining 10/zl of solubilized pellet with 100/zl of a solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7, 0.5% Sarkosyl, and 100 mM fl-mercaptoethanol. Protein was extracted using phenol/chloroform/isoamyl alcohol, and the resultant RNA subjected to Northern blotting. Northern blots were hybridized with connexin43 cDNA (Beyer et al., 1987) along with a histone H3 clone, both radiolabeled with 32p using random primer extension. Following hybridization, blots were exposed to X-ray film and the resultant autoradiographs subjected to densitometric analysis (LKB Ultroscan). In Situ
Hybridization
In situ hybridization to localize connexin43 m R N A was carried out using a modification of the method of Whitfield et al. (1990). Briefly, cultures were fixed
for 20 min in 4% paraformaldehyde in PBS, then rinsed several times in PBS and once in 0.1 M triethanolamine-HC1, pH 8.0, followed by 10 min in 0.25% acetic anhydride in the same buffer. After rinsing the slides in 2× SSC, they were dehydrated in graded ethanols and hybridized with the radiolabeled connexin43 probe, suspended in hybridization buffer (60mM NaCI, 10mM Tris-HC1, pH 7.5, 0.02% Denhardt's solution, 1 mM EDTA, pH 8.0, 0.01% herring sperm DNA, 0.05% yeast tRNA, 10% dextran sulfate, 50% formamide, 0.05% SDS, 0.05% sodium thiosulfate, 50 mM DTT) overnight at 45°C. Following hybridization, coverslips were washed in several changes of 2x SSC, then treated with 20/tg/ml RNase A in 500 mM NaC1, 10 mM Tris-HC1, 1 mM E D T A for 30 min at room temperature, followed by a 30-min rinse in the same buffer. The final wash was carried out with 2x SSC at 50°C and 0.2× SSC at 55°C. After drying, coverslips were dipped in Kodak NTB-2 emulsion, exposed for several days, and developed. Antisense 35S-labeled connexin43 probe was generated from a 1.4-kb cDNA (Beyer et al., 1987) subcloned into Bluescript M13+, linearized with SalI, and transcribed with T3 R N A polymerase in the presence of 35S-UTP (New England Nuclear, 1000 Ci/mmol). For control hybridization, radiolabeled sense-strand RNA was transcribed by T7 polymerase from the same plasmid linearized with HindlII.
Immunocytochemistry C6 glioma cells and several of the transfected clones were cultured on glass coverslips coated with poly-L-lysine (30-70 kD; GIBCO). For im-
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munocytochemistry, cells were fixed in 95% ethanol/5% glacial acetic acid for 20min at -20°C, rinsed with PBS, and incubated in rabbit polyclonal anticonnexin43 (diluted 1 : 100) or preimmune serum from the same rabbit for 2 hr at room temperature. After subsequent incubation in fluorescein-conjugated goat anti-rabbit IgG (Vector Laboratories; diluted 1:50), coverslips were rinsed in PBS and mounted on slides in PBS containing 50% glycerol, 0.1% pphenylenediamine. To localize actin, cells were fixed for 10min in 2.5% formaldehyde/l% glutaraldehyde in phosphate buffer, followed by a 1-min treatment with 0.1% Triton X-100 in phosphate buffer. After several washes, cells were then incubated in fluorescein phaUoidin (Molecular Probes, Eugene, OR) (diluted 1:6) for 30 min, washed, and mounted for immunofluorescence microscopy. RESULTS
In our intial study of C6 glioma cells transfected with connexin43 cDNA, we found different levels of connexin43 m R N A in different clones (Zhu et al., 1991). These clones also exhibited different degrees of intercellular coupling, which were correlated with the amount of connexin43 mRNA. In this study, we have examined the amount of connexin43 protein in several of these clones, as well as other aspects of the cellular phenotype. R N A analysis indicated that the level of 3-kb connexin43 m R N A is much lower in the nontransfected C6 cells than in primary astrocytes (Fig. 1). In contrast, different transfected clones contained various levels of this mRNA, as well as the 2.25-kb mRNA transcribed from the transfected pLTRCx43. This 2.25-kb mRNA was detected only in clones which retained the 1.4-kb EcoR1 restriction fragment (Zhu et al., 1991). Clone Cx43-8 contained only the endogenous 3-kb mRNA, while clones Cx43-12, Cx43-13, and Cx43-14 expressed the transfected cDNA to yield the 2.25-kb mRNA (Fig. 1). From densitometric analysis of RNA blots, the connexin43 mRNA levels, relative to the level in the nontransfected C6 cells, were 10-fold greater in primary astrocytes, 2-fold greater in clone Cx43-8, 30-fold greater in clones Cx43-12 and Cx43-14, and 50-fold greater in clone Cx43-13.
~
,
~
~ Fig. 1. Northern blot analysis of connexin43 in RNA isolated from astrocytes, nontransfected C6 glioma cells, and several ~ transfected clones. All cells express the 3-kb connexin43 mRNA. Astrocytes, C6, and clone Cx43-8 cells express only the 3-kb mRNA (filled arrow). C6 cells exhibit a 10-fold reduction in the level of this mRNA in comparison to astrocytes. The other clones also express the exogeneous 2.25-kb connexin43 mRNA (open arrow).
~]
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Western blot analysis demonstrated a correlation between the amount of detectable steady-state connexin43 mRNA and protein. Comparison was made between the amount of immunodetectable connexin43 in total protein preparations isolated from primary astrocytes, nontransfected C6 cells, and several of the transfected clones (Fig. 2a, lanes 1-6). While connexin43 is readily demonstrable in total protein from cultured astrocytes (Fig. 2a, lane 1), it is barely detectable in the nontransfected C6 cells (Fig. 2a, lane 2). The level of connexin43 is slightly higher in clone Cx43-8 (Fig. 2a, lane 3) and higher still in clones Cx43-12 (Fig. 2a, lane 4) and Cx43-14 (Fig. 2a, lane 5). Finally, clone Cx43-13 displays a dramatic increase in connexin43 protein (Fig. 2a, lane 6). When densitometric values were obtained from Western blots, the level of connexin43 protein, relative to nontransfected C6 cells, was 6-fold greater for primary astrocytes, 1.5-fold greater for clone Cx43-8, 3-fold greater for clones Cx43-12 and Cx43-14, and 8-fold greater for clone Cx43-13. Thus there is a rough correlation between connexin43 mRNA and protein level in the various clones. It is clear that the connexin43 protein detected exists in different forms. At least two, and in some samples three, bands were seen, with different levels of slower- and faster-migrating variants (Fig. 2a). While this is clear in samples of astrocytes (lane 1) and clones expressing the transfected cDNA (lanes 4-6), nontransfected C6 cells and clone Cx43-8 appear to express only the slowermigrating protein species (lanes 2 and 3). To obtain a rough measure of the proportion of Cx43 in plasma membranes, samples were prepared from membrane-enriched fractions (Fig. 2b, lanes 2, 4 and 6) and compared to total protein (Fig. 2b, lanes 1, 3, and 5). In astrocytes, three distinct bands are detectable with approximate Mr's of 40, 43, and 46 kD (Fig. 2b, lane 1). In contrast, a membrane-enriched protein fraction from astrocytes contains only the 43- and 46-kD bands (Fig. 2b, lane 2). Nontransfected C6 cells do not display these multiple bands, and very little of the connexin43 immunoreactivity is present in the membrane-enriched fraction (Fig. 2b, lanes 3 and Fig. 2. (a) Western blot analysis of a membrane-enriched protein fraction prepared from 2 x 105 cells. Primary astrocytes (lane 1) exhibit two sizes of connexin43 protein (Mr, approximately 43 and 46 kD). Nontransfected C6 cells (lane 2) and clone Cx43-8 (lane 3) display predominantly connexin43 with Mr approximately 46 kD. Clones Cx43-12 (lane 4), Cx43-14 (lane 5), and Cx43-13 (lane 6) display three species of connexin43 protein, with M~'s of approximately 40, 43, and 46 kD. (b) Comparison of connexin43 in total vs membrane-enriched protein isolated from 2 x 105 cells. Total 58,.D,.protein from astrocytes (lane 1) clearly displays three bands representing proteins with approximate Mr 40,43, and 46 kD. In contrast, the membrane-enriched fraction (lane 2) shows only the 43- and 46-kD protein. This difference is not apparent in Cx43-13 total (lane 5) vs membrane-enriched 58"~ (lane 6) samples; both contain all three protein species. In 48"~ contrast, nontransfected C6 cells display only the 46-kD protein in both total (lane 3) and membrane-enriched (lane 4) samples. Numbers indicate positions of molecular weight 36.,~ standards in (kD).
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2
3
4
5
6
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4). In contrast, clone Cx43-13 displays multiple bands of connexin43, the major portion being represented in the faster-migrating species (Fig. 2b, lanes 5 and 6). The possible significance of these findings is considered in the Discussion. In our previous report, we have demonstrated that C6 glioma cells transfected with connexin43 cDNA proliferate more slowly than nontransfected cells and that the rate of cell proliferation is inversely related to the amount of connexin43 mRNA and the level of dye coupling (Zhu et al., 1991). The simplest explanation for this is that increased gap junctional intercellular communication leads to a reduction of cell proliferation. However, it is also possible that overproduction of a particular mRNA can in itself slow the rate of proliferation, perhaps by swamping the translational machinery and thus limiting the production of proteins involved in DNA replication, chromatin assembly, or progression through the cell cycle. We examined this possibility by determining the proportion of connexin43 and histone H3 mRNAs in polysomes during exponential growth of clone Cx43-13 cells, these being the transfected clone with the most severely limited cell proliferation (Zhu et al., 1991). Polysomal and subribosomal fractions were prepared as described under Materials and Methods, and the purified RNA from these fractions was analyzed by Northern blotting (Fig. 3). Simultaneous hybridization with connexin43 and histone H3 DNAs revealed the presence of the endogenous (i.e., 3 kb) and exogenous (i.e., transfected; 2.25 kb) connexin43 mRNAs, as well as the histone H3 mRNA (about 0.7 kb). After 5 hr of exposure, only the exogenous 2.25-kb mRNA was evident in the subribosomal RNA sample. After longer exposures (not shown), histone H3 mRNA could also be detected in the subribosomal sample, but endogenous connexin43 mRNA could not. Densitometric analyses of various exposures of such Northern blots from four separate plates of clone Cx43-13 cells were performed to determine the fraction of mRNA in polysomes. For all three transcripts, the fraction in polysomes was very high: 1.00 for endogenous connexin43 mRNA; 0.94 + 0.01
S
P
--3.0 -- 2 . 2 5
--0.7
Fig. 3. Northern blot analysis of RNA isolated from subribosomal (S) and polysomal (P) preparations obtained from clone Cx43-13 cells. Histone H3 mRNA is seen at 0.7kb, exogenous (i.e., transfected) connexin43 mRNA at 2.25kb, and endogenous connexin43 mRNA at 3.0 kb. All three mRNAs are predominantly associated with the polysomal fraction.
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(SD) for exogenous connexin43 mRNA; and 0.94 + 0.03 for histone H3 mRNA. To confirm that the transcripts in the 100,000g pellets were indeed associated with polysomes, postmitochondrial supernatants were treated with 50 mM EDTA for 15 min at room temperature before centrifugation. This caused all of the histone mRNA and 0.87 + 0.09 (n = 3) of the Cx43 m R N A to shift from the polysomal pellet to the subribosomal supernatant (autoradiograms not shown). Expression of connexin43 at the cellular level was examined using in situ hybridization and immunocytochemistry. The level of connexin43 mRNA was dramatically higher in clone Cx43-13 cells than in nontransfected C6 cells (Fig. 4). It is clear that all the Cx43-13 cells contain substantial amounts of this mRNA, as evidenced by the density of autoradiographic grains overlying each cell (Fig. 4b). In contrast, very few autoradiographic grains were localized over nontransfected C6 cells (Fig. 4a). Control hybridization with the sense-strand R N A probe produced only background labeling. Immunocytochemistry revealed a similar abundance of connexin43 protein in Cx43-13 cells, while this immunoreactivity was much lower in nontransfected C6 cells (Figs. 5a and b). While some of the connexin43 immunolabeling was present at membrane areas of intercellular contact, it is clear that a major proportion of connexin43 immunoreactivity in clone Cx43-13 cells is not directly associated with the plasma membrane, but, rather, is located in perinuclear and cytoplasmic regions. To examine cytoskeletal differences between nontransfected C6 cells and the high-expressing transfected clone, Cx43-13, the actin-selective stain, phalloidin, was used. There was no obvious organization of actin filaments in the nontransfected cells (Fig. 5c). In contrast, clone Cx43-13 showed a clear organization of actin into stress fibers (Fig. 5d). This change in cytoskeletal organization was also correlated with a more flattened cell morphology.
Fig. 4. In situ hybridization for connexin43 mRNA in cultures of nontransfected C6 (a) and clone Cx43-13 (b) cells. While C6 cells exhibit very little hybridization with the radiolabeled connexin43 cRNA probe, all clone Cx43-13 cells display strong hybridization. Bar = 50/~m.
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Fig. 5. Immunocytochemical localization of connexin43 in C6 glioma ceils and clone Cx43-13 cells. (a) Some punctate immunofluorescence is detectable in nontransfected C6 glioma cells. (b) In clone Cx43-13 cells, a dramatic increase in immunodetectable connexin43 protein is evident. While some of this is localized to areas of intercellular membrane contract (arrows), abundant immunoreactivity is present in perinuclear and cytoplasmic areas. (c) Fluorescein phalloidin staining in nontransfected C6 cells does not reveal obvious actin organization. (d) In cultures of clone Cx43-13, fluorescein phalloidin dearly demonstrates actin stress fibers. Bar = 50 #m.
DISCUSSION Intercellular c o m m u n i c a t i o n via gap junctions has b e e n implicated as being important in the regulation of cell proliferation and subsequent differentiation (Loewenstein, 1979). In fact, there is g o o d evidence that an interruption of this c o m m u n i c a t i o n p a t h w a y is one of the steps in malignant t r a n s f o r m a t i o n (reviewed by Klaunig and R u c h , 1990): a variety of chemical agents which t r a n s f o r m cells in
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vitro cause a reduction of intercellular communication. Conversely, the growth of transformed cells can be inhibited when they come into contact with normal cells, and this is known to be due to the assembly of gap junction channels between the two cell types (Mehta et al., 1986). Recently, it was demonstrated that a gap junction protein (connexin43) is a direct target for phosphorylation by the product of the v-src oncogene, the result of which is the blockage of intercellular coupling (Swenson et al., 1990). The C6 glioma cell has been extensively used as a model of malignant transformation of glial cells (Benda et al., 1968). While astrocytes are highly coupled via gap junctions, C6 cells exhibit very restricted intercellular coupling (Naus et al., 1991; Tiffany-Castiglioni et al., 1986). We have increased gap junctional coupling in C6 cells by transfection with connexin43 cDNA (Zhu et al., 1991). Several of the selected clones exhibit different degrees of expression of the transfected cDNA, this being evident from the levels of steady-state connexin43 mRNA and protein. There is clear association between the level of steady-state mRNA transcribed from the transfected cDNA and the amount of immunodetectable connexin43 protein. While Western blot analysis of protein isolated from nontransfected C6 cells revealed the presence of only a single band of connexin43, multiple electrophoretic variants of this protein were detected in samples from primary astrocytes and transfected C6 clones. These multiple species have been shown to represent different phosphorylation states of the connexin43 protein, with fully phosphorylated form having the lowest mobility (reviewed by Musil and Goodenough, 1990). Recent studies by Dermietzel et al. (1991) and Giaume et al. (1991) showed the presence of multiple species of connexin43 protein in cultures of astrocytes, similar to our current findings for astrocytes. If these multiple bands of immunoreactivity observed in the present study also represent different phosphorylation states of connexin43, and the single band in nontransfected C6 cells represent the fully phosphorylated form, it is possible that overexpression of this protein in clone Cx43-13 may lead to saturation of the cellular mechanisms involved in phosphorylation. This could explain our finding that much of the connexin43 in clone Cx43-13 cells remains in the cytoplasm; phosphorylation is closely linked with plasma membrane insertion (Musil et al., 1990). We have previously reported a reduction in the proliferation of C6 cells following transfection with connexin43 cDNA (Zhu et al., 1991). It is possible that the observed overproduction of connexin43 mRNA may have affected proliferation indirectly by merely saturating the translational machinery or by increasing translational competition between messages. We chose to monitor the partitioning of histone H3 mRNA between polysomes and subribosomal mRNPs as a test of this possibility because of the tight coupling of histone synthesis to DNA replication (Marzluff and Pandey, 1988). Our results, demonstrating that almost all connexin43 and histone H3 mRNAs are undergoing translation in exponentially growing clone Cx43-13 cells, makes it extremely unlikely that their reduced rate of proliferation is due to interference with translation. In addition to a reduction of proliferation rate, cytoskeletal alterations were noted following transfection of C6 cells with connexin43 cDNA. Phalloidin
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staining revealed an organization of actin filaments into stress fibers in the high-expressing clone, Cx43-13, while no such organization was evident in the nontransfected cells. Several studies have indicated that there is a loss of stress fibers following malignant transformation (reviewed by Burridge, 1986). Taken together, these results establish a relationship among expression of a gap junction protein, enhanced intercellular communication, and phenotypic transformation of glioma cells to resemble more closely their normal progenitors. Although other explanations are still possible, the data strongly indicate that the restoration of gap junctional coupling is the causative element in this process. The C6 cell transfection model provides a unique opportunity to discover the exact role of intercellular communication in maintaining the normal cellular phenotype.
ACKNOWLEDGMENTS
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