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Carcinogenesis, Vol. 22, No. 3, 507-513, March 2001
© 2001 Oxford University Press

CARCINOGENESIS

Rat gap junction connexin-30 inhibits proliferation of glioma cell lines

Frédéric Princen1, Pierre Robe1,,2,,3, Daniel Gros4, Thérèse Jarry-Guichard4, Jacques Gielen1, Marie-Paule Merville1 and Vincent Bours1,,5

1 Laboratory of Medical Chemistry and Medical Oncology,
2 Department of Human Physiology and
3 Department of Neurosurgery, University of Liège, Sart-Tilman, 4000 Liège, Belgium,
4 Laboratoire de Génétique et Physiologie du Développement (UMR 6545), and Institut de Biologie du Développement de Marseille (IBDM), Université de la Méditerranée, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Connexins, the structural components of gap junctions, control cell growth and differentiation and are believed to belong to a family of tumour suppressor genes. Studies on connexin localization in brain showed that several of these proteins were expressed in distinct compartments of the brain in a cell-type specific manner, indicating that different gap junctions play specific roles in the physiology of the mammalian brain. In this report, we first cloned rat connexin-30 cDNA from brain and showed that it was expressed in long-term primary culture of rat astrocytes. In order to examine the potential role of connexin-30 in tumour cell proliferation, we transfected the connexin-30 cDNA into two rat glioma cell lines (9L and C6) which have lost its expression. Transfected clones adequately expressed membrane-bound connexin-30 protein. Connexin-30-expressing clones showed slower growth, lower DNA synthesis and reduced proliferation in soft agar as compared with the parental and control cells. We concluded that connexin-30 may also probably be considered as a tumour suppressor in rat gliomas.

Abbreviations: FITC, fluorescein isothiocyanate; GJIC, gap junctional intercellular communication; MTN, multiple tissue northern; TBS-T, Tris-buffered saline–Tween 20; TRITC, tetramethylrhodamine isothiocyanate.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Adjacent cells can exchange low molecular weight molecules, such as ions or second messengers, through intracellular channels called gap junctions (1–3). These structures arise from the intercellular docking of two transmembrane hemichannels (connexons) that are each made up of six proteins called connexins (4,5). Fourteen different genes encoding for connexins have been characterized thus far in the murine genome, and are expressed in a cell-type specific manner (6–8).

Gap junctions are thought to contribute to the maintenance of tissue homeostasis by allowing rapid ion and water exchange, and to participate to the control of cell proliferation and differentiation (9–11). Interestingly, individual cells frequently express several types of connexins, and since different connexins form gap junctions that display distinct molecular permeabilities, gap junctional intercellular communication (GJIC) may allow specific types of signalling between cells (12,13). In the central nervous system for instance, different gap junctions appear to mediate the GJIC between astrocytes and between astrocytes and oligodendrocytes (14,15), and to participate in the complex regulation of pH, ionic concentrations or calcium wave propagation (16,17).

Numerous reports have also suggested an important role for gap junctions in carcinogenesis. Tumour tissues are often characterized by a reduced level of connexin mRNA or aberrant localization of connexins, which suggests that both transcriptional and post-translational regulation of connexins can be altered during carcinogenesis (18). Moreover, carcinogens can directly inhibit the function of gap junctions (19), whereas several anti-carcinogenetic agents up-regulate GJIC (20). Furthermore, the restoration of GJIC by transfection of connexin genes can reverse the transformed phenotype of tumour cells, as demonstrated on rat glioma and human glioblastoma cells with connexin-43 (21, 22) or for human mammary and cervix carcinoma cells with connexin-26 (23, 24). However, the mechanisms responsible for this connexin-mediated growth suppression remain elusive thus far.

We report here the characterization of rat connexin-30 (Cx30) cDNA, and show that this connexin is mainly expressed in the brain of adult rats. It is also expressed in astrocytes in vitro, but is absent from two rat glioma cell lines. Re-introduction of the Cx30 gene in these cancer cells leads to a decreased proliferation, suggesting a tumour-suppressor role in these cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
PCR cloning
Total RNA from rat brain was reverse transcribed using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) and then amplified according to Haefliger et al. (25) with a sense degenerate oligonucleotide corresponding with the first extracellular domain of the connexin consensus sequence and an anti-sense degenerate oligonucleotide complementary to the second extracellular domain. PCR products were separated on a 2% agarose gel and bands between 300 and 500 bp were excised by the agarose gel DNA extraction method (Roche, Germany) and subcloned in the pCR® topo vector using the TOPO TA cloning method (Invitrogen, Gröningen, The Netherlands). The cloned PCR products were sequenced using the Sequenase 2.0 Sequencing kit (USB, Cleveland, OH). The Marathon® cDNA amplification kit for amplification of 5' and 3' cDNA ends (Clontech, Palo Alto, CA) was used to obtain a complete coding sequence for Cx30.

Cell culture
Rat gliosarcoma 9L cells were grown in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovin serum (FBS), 1% non-essential amino acids, 1% 100 mM sodium pyruvate and 1% 5 mg/ml penicillin–streptomycin. Rat glioma C6 cells were grown in DMEM (Life Technologies) supplemented with 3% FBS, 1% 5 mg/ml penicillin–streptomycin. Cells were grown at 37°C in 5% CO2.

Plasmid construction and stable transfections
The expression vector encoding the Cx30 protein was made by insertion of the Cx30 cDNA coding sequence into EcoRI–XbaI sites of the mammalian expression vector pcDNA3 (Invitrogen). The final construction contained the neomycin resistance gene driven by the SV40 early promoter and the Cx30 gene driven by the cytomegalovirus promoter. 9L and C6 cells were transfected by the DOTAP liposomal transfection reagent (Roche, Germany) with the Cx30 expression vector or with a control empty vector. Transfected cells were then selected in culture medium containing 500 µg/ml G418 (Life Technologies). The selected clones were characterized for expression of Cx30 mRNA and protein levels.

Long-term glial and oligodendrocyte cultured cells
Long-term primary culture of glial cells was obtained as described previously (26). Briefly, newborn rat cortices were dissected and freed from vessels and meninges, dissociated by sieving on 225 and 25 µM nylon meshes and collected in MEM (Life Technologies) supplemented with 10% FBS. The medium was renewed after 24 h and then every 48 h for at least 10 weeks. Rat oligodendrocytes were prepared as described previously (27). Briefly, rat pup cortices were dissected and freed from vessels and meninges, dissociated by sieving on 225 and 25 µM nylon meshes. The cell suspension was then layered on the top of a pre-centrifuged Percoll density gradient and centrifuged. The interface lying between cell debris and red blood vessels was resuspended in PBS–HEPES buffer (Life Technologies) and centrifuged. The pellet was resuspended in DMEM supplemented with N1, biotin and 30% neuroblastoma B 104-conditioned DMEM-N1 medium (Life Technologies), seeded on a tissue culture plate and incubated for 48 h. The plates were then gently shaken and non-adherent cells were transferred to a new plate and grown. These cultures were then switched to DMEM-N1 medium and transferred to polyornithine-coated plates, allowing their transformation into oligodendrocytes.

Northern blot analysis
Total RNA was extracted by the tripure isolation reagent method (Roche). Total RNA (10 µg) from each sample was migrated by electrophoresis in denaturing formaldehyde–agarose gels (1%). Gels were capillary blotted onto Hybon-N+ nylon membranes (Pharmacia Biotech, Roosendaal, The Netherlands). Blots were pre-hybridized for 30 min at 65°C in express hybridization solution (Clontech). Hybridization was carried out for 1 h at 65°C in the express hybridization solution with an [{alpha}-32P]dCTP-radiolabelled cDNA probe prepared by random-primed DNA labelling (Roche). Blots were then washed according to the express hybridization solution protocol and exposed at –70°C to Fuji medical X-ray film with an intensifying screen. Rat Cx43 full-length cDNA, rat Cx30 full-length cDNA and human GAPDH cDNA were used as probes. Analysis of Cx30 mRNA expression was performed using a rat multiple tissue northern blot (Clontech) containing 2 µg poly-A+ RNA from eight different rat tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis).

Western blot analysis
Whole-cell protein extracts (50 µg) obtained by SDS lysis were separated on a 12.5% SDS–PAGE gel. After transfer to a nylon membrane (Immobilon-P; Millipore, Bedford, MA) and blocking with Tris-buffered saline–Tween 20 (TBS–T) (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 0.2% Tween 20) plus 5% dry milk powder, the membranes were incubated for 1 h with an anti-Cx30 antibody (1/300), washed and finally incubated for 1 h with a secondary anti-rabbit peroxidase-conjugated antibody (1/5000). The reaction was revealed by the enhanced chemiluminescence detection method (ECL kit; Amersham, UK).

Immunofluorescence staining of Cx30 and Cx43 proteins
Cells were seeded on coverslips and fixed when in 95% ethanol containing 5% (v/v) acetic acid for 20 min at –20°C and then rinsed with DMEM (Life Technologies). The cells were permeabilized with DMEM containing 10% FBS and 0.1% saponin for 60 min at room temperature. Primary rabbit anti-Cx30 (10 µg/ml) and mouse anti-Cx43 antibodies (dilution 1/250; Chemicon, Harrow, UK) were added and incubated overnight at 4°C in saturated medium. After washing, the secondary tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit (Sigma–Aldrich, Lyon, France) and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse antibodies (Jackson, Immunoresearch Laboratories, Baltimore, MD) were added at a final dilution of 1/40 and 1/250, respectively, for 1 h at room temperature. After washing, the staining was visualized with a Zeiss Axiovert microscope.

Communication assay
GJIC was assessed as described (28). Briefly, cells were harvested in culture medium and seeded at low density on polyornithine-coated glass coverslips. Twenty-four hours later, the coverslips were flooded with media and the cells allowed to grow to confluence for 5–6 days. Media were changed every other day. The coverslips were then placed in the perfusion chamber of a Zeiss fluorescence microscope and perfused with EA01 buffer (137 mM NaCl, 5.7 mM KCl, 1.8 mM CaCl2, 22.2 mM D-glucose and 10 mM HEPES). Cells in confluent areas were injected with lucifer yellow dye [5% (w/v) in 0.1 M LiCl] by passing 0.5 Hz 500 ms 1 mA hyperpolarizing current pulses for 30 s through the electrode. Impalement was considered effective when the membrane potential remained negative throughout the injection period and if a single cell was stained by passive dye diffusion before current application. The total number of neighbouring cells marked with lucifer yellow was then counted 60 s after the end of the injection and served as a measure of GJIC.

Cell growth rate (generation time t-test)
Cells (104/well) were plated in 24-well tissue culture plates. Cells were trypsinized and counted by the trypan blue exclusion method every 2 days for 8 days. Cell growth rate was represented as generation time (Tn = t/ln (Nf /Ni)ln 2, where Ni represents plated cell number, Nf, counted cell number at time t (48, 96, 144, 192 h) and ln, natural logarithm). Generation time T is the average of Tn at 8 days (29).

[3H]Thymidine incorporation
Cells were seeded in flat-bottom 96-well tissue culture plates at a density of 5x103 cells/well in 200 µl culture medium. After 3 days, 0.4 µCi [3H]TdR (Amersham, UK) was added and cells were incubated for 24 h. Cells were then washed, lyzed with NaOH and harvested on glass fibre filters with an automatic cell harvester. The radioactivity retained on the filters was measured in a liquid scintillation counter. Each experiment was reproduced six times and mean c.p.m. values were calculated.

Anchorage-independent growth assay
This test was performed by seeding 5x103 cells from each clone in growth medium containing 0.34% soft agar (Noble agar; Difco Laboratories, Detroit, MI). Cell suspension (1.5 ml) was added to 6-well plates pre-coated with 2 ml solidified (0.9% agar) basal layer. Three weeks after seeding, colonies containing at least 30 cells were stained and counted.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cloning and expression analysis of rat connexin-30
Total RNA isolated from rat brain was reverse transcribed and amplified by RT–PCR using degenerate primers designed on the basis of sequence similarity between previously characterized connexin genes (25). The fragments obtained were subcloned and sequenced. One amplified clone showed a sequence that had not been previously described in rats. The cDNA of this connexin was incomplete and the full-length cDNA was obtained by amplifying 5' and 3' cDNA ends.

The nucleotide sequence of the full-length cDNA encoded a protein of 261 amino acids with a theoretical molecular mass of 30 000 Da, which was named Cx30. An alignment of this sequence with the human and mouse homologues showed a close identity with the two species, 94 and 99%, respectively (Figure 1Go). The rat Cx30 amino acid sequence shared 75% identity with Cx26, showing a late divergence in evolution with Cx26 as previously described in mice (30).



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  		Fig. 1. Amino acid sequence comparison of rat, mouse and human Cx30 proteins. The four putative transmembrane regions are underlined. A dash indicates identical amino acid residues. Connexin-specific conserved cysteine residues in the extracellular domain are shown in bold. The nucleotide sequence is available from Genbank under accession number AF 170184. The asterisk indicates the stop codon.

 
Analysis of Cx30 mRNA expression in different tissues was performed using a rat multiple tissue northern (MTN) blot hybridized with Cx30 full-length cDNA as a probe (Figure 2Go). Cx30 mRNA was most abundant in brain and a weak expression was also detected in lung, confirming the results obtained with mouse Cx30 (30). The slower migrating band detected on the blot in liver, kidney and lung samples was due to cross-hybridization of the probe with Cx26 mRNA (Figure 2Go). Cx26 expression was also detected in brain on shorter expositions (data not shown). Expression of Cx26 mRNA in these tissues was confirmed by hybridization of the same MTN blot with full-length Cx26 cDNA as a probe (data not shown).



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  		Fig. 2. Northern blot analysis on multiple tissue northern (MTN) filter containing poly-A+ RNA isolated from eight different adult rat organs (heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis). The filter was hybridized with full-length Cx30 cDNA as a probe. The higher transcript detected in brain, liver, kidney and lung was generated by the cross-hybridization of the probe with Cx26 mRNA.

 
Furthermore, Cx30 mRNA expression was detected in rat primary cultures of astrocytes and not of oligodendrocytes. Interestingly, Cx30 mRNA expression was not detected in two glioma cell lines (9L and C6) (Figure 3Go, upper panel) while Cx43 mRNA expression was found in all the tested samples, but was less abundant in the C6 glioma cell line (Figure 3Go, lower panel). In this experiment, total RNA from brain of adult rat was used as a positive control for expression of Cx30 and Cx43 mRNAs. Cx30 and Cx43 expression patterns in brain and glial cells confirmed the results obtained in another report (31). Indeed, we did not observe any Cx30 expression in freshly removed glial cells from newborn rats (data not shown), but this expression was induced in long-term culture (12 weeks) which is reminiscent of Cx30 expression pattern in mouse glial cells (30).



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  		Fig. 3. Northern blot analysis of total RNA isolated from rat brain, oligodendrocytes, long-term culture glial cells, 9L and C6 cells. Full-length Cx30 cDNA and full-length Cx43 cDNA were used as probes (upper and lower panels, respectively). Total RNA from adult rat brain was used as positive control for the detection of Cx30 and Cx43 mRNA.

 
Stable transfection of connexin-30 in 9L and C6 cells
The glioma cell lines 9L and C6 were characterized by the absence of detectable Cx30 expression (Figure 3Go). In order to determine the possible role of Cx30 in tumour suppression, we transfected a Cx30 expression vector or a control empty vector into the 9L and C6 cell lines. After G418 selection, several clones of Cx30 stably transfected cells were selected by Northern blot (Figure 4Go). Northern blots showed that Cx30-transfected 9L and C6 cells (9Lcx30 and C6cx30) expressed a 1.6-kb transcript whose size coresponded to that of the Cx30 cDNA plus the bovine growth hormone (bGH) polyadenylation signal and transcription termination sequences from the expression vector. However, the expression level of Cx30 mRNA was lower in C6 than in 9L clones. Neither the parental cell lines (9Lwt and C6wt) nor the control cell lines (9Lneo and C6neo) expressed detectable levels of Cx30 transcript mRNA (Figure 4Go, upper panel). The neomycin resistance gene transcript was not detected in the parental cell lines (9Lwt and C6wt), but was observed in all transfected cells (Figure 4Go, middle panel). The Northern blot was normalized using a probe for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene (Figure 4Go, lower panel).



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  		Fig. 4. Northern blot analysis on total RNA from Cx30-transfected 9L and C6 cells (9Lcx30 and C6cx30), control 9Lneo and C6neo cell lines and parental 9Lwt and C6wt cell lines. In the upper panel, the filter was hybridized with full-length Cx30 cDNA. In the middle panel, the filter was re-probed with neomycin cDNA as a probe. In the lower panel, the Northern blot was normalized by re-probing the filter with a GAPDH probe.

 
Western blot analysis revealed an immunoreactive band at 30 kDa, corresponding to the Cx30 protein in all the Cx30 transfectants with the exception of the C6cx30-14 clone (the 9Lcx30-14 clone showed only a faint Cx30 expression). As expected, the control (9Lneo and C6neo) and parental cell lines (9Lwt and C6wt) did not show an immunoreactive band at 30 kDa (Figure 5Go).



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  		Fig. 5. Cx30 protein expression in Cx30-transfected 9L and C6 clones. (A) Cx-30-transfected 9L cells (9Lcx30), control 9Lneo cells and parental 9Lwt cells and (B) Cx30-transfected C6 cells (C6cx30), control C6neo cells and parental C6wt cells.

 
Finally, double immunofluorescence labelling of 9L cells with Cx30 and Cx43 antibodies revealed a partial membrane co-localization for both connexins in the 9Lcx30-26 clone as already described in astrocytes (32), but the signal intensity was higher for Cx43. As expected, control 9L cells did not shown a Cx30 signal in the membrane (Figure 6Go). A partial membrane co-localization of both connexins was also observed for the 9Lcx30-27 clone, but the signal intensity for Cx30 was lower than in the 9Lcx30-26 clone (data not shown).



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  		Fig. 6. Double immunofluorescence labelling with antibodies to Cx30 and Cx43 (A, B, C) control 9Lneo cells. Cx30-transfected 9L cells (9Lcx30-26) (D, E, F). (A) and (D) show phase-contrast micrographs done on the same cells. (B) and (E) represent FITC labelling with anti-Cx43 antibodies and (C) and (F) correspond to TRITC-labelling with anti-Cx30 antibody. Arrows indicate common staining of Cx43 and Cx30. The bar shown in (A) equals 20 µm.

 
Moreover, in the Cx30-transfected cells, we also analysed the Cx43 mRNA and protein levels and did not observe any modification of Cx43 expression in Cx30-transfected cells as compared with the parental (9Lwt or C6wt) or transfected control cells (9Lneo or C6neo) (data not shown).

GJICs were measured in transfected clones to determine whether Cx30 expression led to modifications in cellular coupling. After micro-injection of a single cell with lucifer yellow, the number of communicating cells was decreased in the C6Cx30.14 and C6Cx30.20 clones while it was slightly increased in clone C6Cx30.26, as compared with a control C6neo clone (Figure 7Go). All these values were very close to those observed with unmodified C6 cells (28) and were confirmed by analysis of calcein transfer (data not shown). The same experiment could not be performed in 9L cells, as these cells communicate very efficiently through Cx43, thus impeaching the recording of any GJIC enhancement.



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  		Fig. 7. GJIC in C6 clones. Iontophoresis of lucifer yellow dye was used to assess the GJIC of confluent C6 cells. The figure indicates means (± SEM) of the number of stained collateral cells per injected cell for the different transfected C6 clones (C6 Neo control clone and C6Cx30.14, C6Cx30.20 and C6Cx30.26 for Cx30-expressing clones).

 
In vitro cell proliferation of connexin-30-transfected clones
Cells (104/well) were seeded in 24-well plates and counted every 2 days for 8 days. Cx30 transfectants were analysed in comparison with the parental cell lines (9Lwt or C6wt) and neomycin-resistant control cells (9Lneo or C6neo). For 9L and C6 cells, two Cx30-transfected clones from each cell line grew much slower than control and parental cells (Figure 8A and BGo). Similarly, the average generation time, T, based on cell counting data was much longer for these Cx30-transfected clones than for the parental 9Lwt and C6wt and control cell lines 9Lneo and C6neo (Table IGo). Generation time, T, was 49.9 and 45.3 h for 9Lwt and 9Lneo, respectively, and 68.7 and 64.3 h for 9Lcx30-26 and 9Lcx30-27, respectively. In C6 cells, the Cx30-transfected C6cx30-20 and C6cx30-26 cells had a growth rate of 125 and 105.5 h, respectively, whereas the parental cell line C6wt and the control cells C6neo showed a T value of 73.2 and 69.2 h, respectively. Interestingly, a difference in the proliferation curves was observed early after cell seeding, i.e. when the cells were growing at low density and before they had reached the saturation density.



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  		Fig. 8. Effect of Cx30 expression on cell growth. Growth curves were established for 9Lcx30and C6cx30-tranfected cells, 9Lneo and C6neo control transfectant cells and parental 9Lwt and C6wt cells. The test was assessed for (A) 9L cells and (B) C6 cells by seeding 104 cells/well in 24-well tissue culture plates. Subsequently, cells were harvested in triplicate and counted every 2 days for 8 days.

 

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  		Table I. Cell growth rate represented as generation time
 
In order to confirm these results, [3H]thymidine incorporation was measured in Cx30-transfected, parental and control cells (Figure 9A and BGo). Cells (5x103/well) were seeded in a 96-well tissue culture plate. After three days, [3H]thymidine was added to the medium for an additional 24 h. The DNA synthesis levels of C6cx30-20 and C6cx30-26 cells were lower than in parental C6wt and control C6neo cells. The same observations were made for 9Lcx30-26 and 9Lcx30-27 cells as compared with parental 9Lwt and control 9Lneo cells. The same results were obtained when thymidine was added 6 days after cell plating (data not shown).



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  		Fig. 9. Effect of Cx30 expression on thymidine incorporation. [3H]Thymidine incorporation was measured in 9Lcx30and C6cx30-tranfected cells, 9Lneo and C6neo control cells and parental 9Lwt and C6wt cells. The test was assessed for (A) 9L cells and (B) C6 cells by seeding 5x103 cells/well in 96-well tissue culture plates. Three days later, 0.4 µCi [3H]TdR was added and the [3H]thymidine incorporation was measured after another 24 h. Each experiment was done in triplicate and mean c.p.m. values (± SD) are represented.

 
Anchorage-independent growth of connexin-30-expressing 9L cells
In order to examine whether the Cx30 gene can alter anchorage-independent growth of 9L cells, soft agar assays were performed by seeding 5x103 cells/well in 6-well plates (Figure 10A and BGo). Control 9Lneo and parental 9Lwt cells grew well in anchorage-independent cultures, whereas the Cx30 transfectants 9Lcx30-26 and 9Lcx30-27 grew poorly with very few small-size colonies. The same experiment was not performed with C6 cells because, in our hands, the control C6 clones did not form colonies in soft agar.



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  		Figure 10. Anchorage-independent growth capacity of Cx30-transfected 9L cells, control 9Lneo cells and parental 9Lwt cells. The test was assessed by seeding 5x103 cells in growth medium containing 0.34% agar on pre-coated 6-well plates. (A) Colonies containing at least 30 cells were counted. Each experiment was done in triplicate and the values represent the means of at least six counts (± SD). (B) The appearance of large clones was readily detected in control 9Lneo and parental 9Lwt cells. Very few small-size colonies were observed in the Cx30 transfectants 9Lcx30-26 and 9Lcx30-27 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Connexins are the structural components of gap junctions responsible for communication between cells. These proteins control cell growth and some have a tumour suppressor function. Studies on connexin expression in brain showed that these proteins were expressed differently in a cell-type-specific manner (33). In the present work, we first cloned Cx30 cDNA from rat brain and investigated the role of Cx30 in rat glioma cells. The Cx30 protein shared a close identity with its murine counterpart and, as expected, Cx30 RNA was mainly expressed in brain (30). A recent report demonstrated that Cx30 expression appeared during the post-natal development of the rat brain (32), thus confirming our finding of Cx30 expression in rat brain and long-term primary culture of glial cells (31). Conversely, Cx43 expression was detected early in brain and astrocytes.

Interestingly, in the two glioma cell lines analysed (9L and C6), Cx43 mRNA, but not Cx30 mRNA, was detected. Cx30 gene transfection of these glioma cell lines showed that restoration of Cx30 expression did not modify intercellular communications. It is possible that exogenously expressed Cx30 is not properly processed and cannot therefore form functional GJICs, as assessed by dye transfer.

In the two cell lines, Cx30-transfection reduced the proliferation rate. It is now well established that connexins are specifically expressed in different tissues and/or cell types, suggesting a cell-type-specific role. In agreement with these findings, astrocytes co-expressed Cx43 and Cx30, and glioma cell lines (C6 and 9L), which expressed only Cx43, showed a reduced growth rate after Cx30 cDNA transfection.

In the present study, all transfectants did not show a reduced growth rate. One Cx30 transfectant from each tested cell line exhibited the same behaviour as the control and parental cell lines. This could be explained by a lower Cx30 protein expression in these transfectants. Mesnil and colleagues analysed several independent Cx26-transfected HeLa clones and observed different patterns of tumorigenicity: the clone exhibiting the lowest level of Cx26 transcript was still tumorigenic, whereas the clones expressing high amounts of Cx26 were non-tumorigenic (23). This observation was confirmed in different experimental systems, suggesting that the tumour suppressor effect was correlated with the level of Cx expression (34,35).

In our clones, inhibition of cellular proliferation was not correlated with increased intercellular communications and the inhibition of cell proliferation was even observed at low cellular density, when intercellular contacts through gap junctions were likely to be very limited. Several reports had indeed previously suggested that the tumour suppressor effect mediated by connexins was not related to the restoration and/or up-regulation of GJIC. For instance, Cx43 stable expression suppresses human glioblastoma cell proliferation despite a level of GJIC similar to that of the control cells (36). Similarly, Cx32 expression in C6 cells induces a reduced growth in vitro that is not correlated with Cx32 expression levels or intercellular coupling (37). Moreover, transfection of Cx26 mutants in Cx26-expressing HeLa cells enhanced the tumorigenicity of the cells without affecting GJIC. Conversely, transfection of another mutant reduced the GJIC of Cx26-expressing HeLa cells without affecting their in vivo growth (38). Thus, our results showed that Cx30 exerted a tumour suppressor effect independently of the restoration of GJIC, as it had been previously described for Cx43, Cx32 and Cx26.

Finally, as in all studies that have investigated the tumour suppressor effect of connexins, we observed a partial growth reduction of the two glioma cell lines after Cx30 transfection. Astrocytes co-express Cx30 and Cx43, whereas the C6 and 9L glioma cell lines express only Cx43. Moreover, the expression of Cx43 is rather weak in C6 cells (Figure 3Go) and it has been reported that transfection of Cx43 cDNA into these cells produced a significant but partial decrease in cell proliferation (34). Co-transfection of Cx30 and Cx43 could, therefore, lead to a better tumour suppressor effect and provide evidence for the combined role of the two connexins in growth control. The mechanisms of tumour suppression mediated by connexins are still unknown and further investigation is required to explore the signalling pathways originating from the connexins and leading to the inhibition of cell proliferation.


   Notes
 
5 To whom correspondence should be addressed Email: vbours{at}ulg.ac.be Back


   Acknowledgments
 
We thank Dr S.Belachew (University of Liège, Liège, Belgium) for rat oligodendrocytes. F.P. is supported by an F.R.I.A. fellowship. P.R. is a Research Assistant, V.B. is a Senior Research Associate and M.-P.M. a Research Associate at the National Fund for Scientific Research (FNRS, Belgium). This research was supported by grants from FNRS-Télévie, `Centre Anti-Cancéreux' (Liège, Belgium), `Concerted Action Program, convention 97/02-214', Communauté Franciaise de Belgique and `Oeuvre Belge du Cancer' (Brussels, Belgium).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Simpson,I., Rose,B. and Loewenstein,W.R. (1977) Size limit of molecules permeating the junctional membrane channels. Science, 195, 294–296.[Abstract/Free Full Text]
  2. 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-triphosphate and calcium ions. Proc. Natl Acad. Sci. USA, 86, 2708–2712.[Abstract/Free Full Text]
  3. Kumar,N.M. and Gilula,N.B. (1996) The gap junction communication channel. Cell, 84, 381–388.[ISI][Medline]
  4. Makowski,L., Caspar,D.L., Phillips,W.C. and Goodenough,D.A. (1977) Gap junction structures. II. Analysis of the X-ray diffraction data. J. Cell Biol., 74, 629–645.[Abstract/Free Full Text]
  5. Yeager,M. and Gilula,N.B. (1992) Membrane topology and quaternary structure of cardiac gap junction ion channels. J. Mol. Biol., 223, 929–948.[ISI][Medline]
  6. Bruzzone,R., White,T.W. and Paul,D.L. (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem., 238, 1–27.[ISI][Medline]
  7. Söhl,G., Degen,J., Teubner,B. and Willecke,K. (1998) The murine gap junction gene connexin 36 is highly expressed in mouse retina and regulated during brain development. FEBS Lett., 428, 27–31.[ISI][Medline]
  8. Manthey,D., Bukauskas,F., Lee,C.G., Kozak,C.A. and Willecke,K. (1999) Molecular cloning and functional expression of the mouse gap junction gene connexin-57 in human HeLa cells. J. Biol. Chem., 274, 14716–14723.[Abstract/Free Full Text]
  9. Loewenstein,W.R. (1979) Junctional intercellular communication and the control of cell growth. Biochem. Biophys. Acta, 560, 1–65.[Medline]
  10. Pitts,J.D. and Finbow,M.E. (1986) The gap junction. J. Cell Sci., 4 (suppl.), 239–266.
  11. Yamasaki,H., Krutovskikh,V., Mesnil,M. and Omori,Y. (1996) Connexin genes and cell growth control. Arch. Toxicol., 18 (suppl.), 105–114.
  12. Bruzzone,R., Haefliger,J.A., Gimlich,R.L. and Paul,D.L. (1993) Connexin 40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol. Biol. Cell, 4, 7–20.[Abstract]
  13. Elfgang,C., Eckert,R., Lichtenberg-Fraté,H., Butterweck,A., Traub,O., Klein,R.A., Hülser,D.F. and Willecke,K. (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J. Cell Biol., 129, 805–817.[Abstract/Free Full Text]
  14. Massa,P.T. and Mugnaini,E. (1982) Cell junctions and intramembrane particles of astrocytes and oligodendrocytes: a freeze-fracture study. Neuroscience, 7, 523–538.[ISI][Medline]
  15. Nagy,J.I., Ochalski,P.A.Y. and Hertzberg,E.L. (1997) Evidence for co-localization of another connexin with connexin-43 at astrocytic gap junctions in rat brain. Neuroscience, 78, 533–548.[ISI][Medline]
  16. Giaume,C. (1996) Communicating junctions of glial cells in the central nervous system. Ann. Endocrinol., 57, 487–491.[Medline]
  17. Naus,C.C., Bechberger,J.F., Zhang,Y., Venance,L., Yamasaki,H., Juneja,S.C., Kidder,G.M. and Giaume,C. (1997) Altered gap junctional communication, intercellular signaling and growth in cultured astrocytes deficient in connexin 43. J. Neurosci. Res., 49, 528–540.[ISI][Medline]
  18. Yamasaki,H. (1990) Gap junctional intercellular communication and carcinogenesis. Carcinogenesis, 11, 1051–1058.[Free Full Text]
  19. Trosko,J.E., Madhukar,B.V. and Chang,C.C. (1993) Endogenous and exogenous modulation of gap junctional intercellular communication: toxicological and pharmacological implications. Life Sci., 53, 1–19.[ISI][Medline]
  20. Yamasaki,H. (1996) Role of disrupted gap junctional intercellular communication in detection and characterization of carcinogens. Mutat. Res., 365, 91–105.[ISI][Medline]
  21. Huang,R.-P., Fan,Y., Hossain,M.Z., Peng,A., Zeng,Z.-L. and Boynton,A.L. (1998) Reversion of the neoplastic phenotype of human glioblastoma cells by connexin 43 (cx43). Cancer Res., 58, 5089–5096.[Abstract/Free Full Text]
  22. Naus,C.C., Elisevich,K., Zhou,D., Beliveau,D.J. and Del Maestro,R.F. (1992) In vivo growth of C6 glioma cells transfected with connexin 43 cDNA. Cancer Res., 52, 4208–4213.[Abstract/Free Full Text]
  23. Mesnil,M., Krutovskikh,V., Picolli,C., Elfgang,C., Traub,O., Willecke,K. and Yamasaki,H. (1995) Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res., 55, 629–639.
  24. Hirschi,K.K., Xu,C.-E., Tsukamoto,T. and Sager,R. (1996) Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential. Cell Growth Differ., 7, 861–870.[Abstract]
  25. Haefliger,J.-A., Bruzzone,R., Jenkins,N.A., Gilbert,D.J., Copeland,N.G. and Paul,D.L. (1992) Four novel members of the connexin family of gap junction proteins. J. Biol. Chem., 267, 2057–2064.[Abstract/Free Full Text]
  26. Rogister,B., Leprince,P., Bonhomme,V., Rigo,J.-M., Delree,P., Colige,A. and Moonen,G. (1990) Cultured neurons release an inhibitor of astroglia proliferation (astrostatine). J. Neurosci. Res., 25, 58–70.[ISI][Medline]
  27. Belachew,S., Rogister,B., Rigo,J.-M., Malgrange,B., Mazy-Servais,C., Xhauflaire,G., Coucke,P. and Moonen,G. (1998) Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform. Eur. J. Neurosci., 10, 3556–3564.[ISI][Medline]
  28. Robe,P.A., Princen,F., Martin,D., Malgrange,B., Stevenaert,A., Moonen,G., Gielen,J., Merville,M. and Bours,V. (2000) Pharmacological modulation of the bystander effect in the herpes simplex virus thymidine kinase/ganciclovir gene therapy system: effects of dibutyryl adenosine 3',5'-cyclic monophosphate, {alpha}-glycyrrhetinic acid and cytosine arabinoside. Biochem. Pharmacol., 60, 241–249.[ISI][Medline]
  29. Proulx,A., Lin,Z.X. and Naus,C.C.G. (1997) Transfection of rhabdomyosarcoma cells with connexin 43 induces myogenic differentiation. Cell Growth Differ., 8, 533–540.[Abstract]
  30. Dahl,E., Manthey,D., Chen,Y., Schwarz,H.-J., Chang,Y.S., Lalley,P.A., Nicholson,B.J. and Willecke,K. (1996) Molecular cloning and functional expression of mouse connexin-30, a gap junction gene highly expressed in adult brain and skin. J. Biol. Chem., 271, 17903–17910.[Abstract/Free Full Text]
  31. Kunzelmann,P., Schröder,W., Traub,O., Steinhäuser,C., Dermietzel,R. and Willecke,K. (1999) Late onset and increasing expression of the gap junction protein connexin 30 in adult murine brain and long-term cultured astrocytes. Glia, 25, 111–119.[ISI][Medline]
  32. Nagy,J.I., Patel,D., Ochalski,P.A.Y. and Stelmack,G.L. (1999) Connexin 30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin 43 at gap junctions and late developmental appearance. Neuroscience, 88, 447–468.[ISI][Medline]
  33. Dermietzel,R. and Spray,D.C. (1993) Gap junctions in the brain: where, what type, how many and why? Trends Neurosci., 16, 186–192.[ISI][Medline]
  34. Zhu,D., Caveney,S., Kidder,G.M. and Naus,C.C.G. (1991) Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling and cell proliferation. Proc. Natl Acad. Sci. USA, 88, 1883–1887.[Abstract/Free Full Text]
  35. Zhang,Z.Q., Zhang,W., Wang,N.Q., Bani-Yaghoub,M., Lin,Z.X. and Naus,C.C.G. (1998) Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin 43. Carcinogenesis, 19, 1889–1894.[Abstract/Free Full Text]
  36. Huang,R.-P., Fan,Y., Hossain,A.P., Zeng,Z.-L. and Boynton,A.L. (1998) Reversion of the neoplastic phenotype of human glioblastoma cells by connexin 43 (cx43). Cancer Res., 58, 5089–5096.
  37. Bond,S.L., Bechberger,J.F., Khoo,N.K. and Naus,C.C. (1994) Transfection of C6 glioma cells with connexin 32: the effects of expression of a nonendogenous gap junction protein. Cell Growth Differ., 5, 179–186.[Abstract]
  38. Duflot-Dancer,A., Mesnil,M. and Yamasaki,H. (1997) Dominant-negative abrogation of connexin-mediated cell growth control by mutant connexin gene. Oncogene, 15, 2151–2158.[ISI][Medline]
Received July 5, 2000; revised September 18, 2000; accepted December 8, 2000.


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