Identification of genes differentially expressed in C6 glioma cells transfected with connexin43

doi:10.1016/S0165-0173(99)00087-9

Brain Research Reviews

Volume 32, Issue 1, 24 March 2000, Pages 259-266

https://doi.org/10.1016/S0165-0173(99)00087-9 Get rights and content

Abstract

Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated primarily by channels composed of connexin43. In contrast, C6 glioma cells are deficient in connexin expression and gap junctional communication. Transfection of these glioma cells with connexin cDNAs results in changes in cellular phenotype following increased GJIC. Specifically, connexin expression correlates with reduced cellular proliferation and tumorigenicity. To characterize the role of gap junctions in this growth control, we have screened for changes in gene expression by differential display. We have observed that these changes in GJIC are associated with changes in expression of several genes, including those coding for a number of secreted factors which may play a role in modulating the tumor phenotype of these cells. These include the immediate early gene cyr61, ostoepontin and the KC gene (murine homologue of the human gro gene).

Introduction

Rat C6 glioma cells are a well-characterized neural cell line, originally isolated from N-nitrosomethylurea-induced glial tumors [1]. C6 cells show both decreased levels of the gap junction protein connexin43 and limited intercellular communication [42]. It has been hypothesized that the reduced cell-to-cell communication found in these and other cancer cells may result in both increased cell growth and other tumour characteristics [58]. Several studies have linked aberrant gap junctional intercellular communication (GJIC) in tumors, down-regulation of GJIC by cancer-causing agents or genes, and up-regulation of GJIC by inhibitors of carcinogenesis [58]. Several approaches have been used to upregulate intercellular communication to restore growth control [reviewed in Refs. 26, 48, 53, 55, 56. For example, many reports describe effects of retinoids on increasing GJIC and decreasing cell growth and transformation 2, 38, 57.

With the recent cloning of many of the connexin cDNAs, a direct approach to increasing connexin expression and subsequent GJIC has allowed a more thorough examination of the role of intercellular communication on tumor suppression. The introduction and overexpression of connexin cDNAs in tumor cells by transfection has shown that the presence of functional gap junctions can suppress growth and/or tumorigenicity of most transformed cells. For example, transfection and expression of connexin43 9, 17, 19, 27, 37, 39, 43, 47, 61, 62, connexin32 3, 17, 18and connexin26 40, 52in various cells resulted in increased communication and, in most cases, reduced growth and tumor formation. Goldberg et al. [22]transfected Rat-1 fibroblasts with a Cx43 antisense vector, significantly reducing the expression of functional gap junction channels. While there was no effect on cell growth or saturation density, these cells had reduced ability to inhibit formation of foci of pp60^v-src transformed fibroblasts in a coculture assay.

Chen et al. [9]reported changes in the expression of genes involved in regulating the cell cycle, namely decrease in cyclins A, D1 and D2 and cyclin-dependent kinases CDK5 and CDK6, in transformed cells transfected with connexin43. These changes were accompanied by density-dependent inhibition of proliferation and prolongation of the G₁ and S phases of the cell cycle. These findings support the notion that GJIC can affect gene expression.

It was the objective of this study to determine if the expression of other genes could be altered by gap junctional coupling. Changes in gene expression between control C6 cells and C6 cells transfected with connexin43 were detected using differential display [36]. Genes expressed in one cell type and not the other were isolated and identified by sequencing. Differential expression was confirmed by Northern blot analysis. Using this approach, we have identified several candidate genes which appear to be differentially expressed between C6 cells and those expressing connexin43.

Section snippets

RNA isolation and poly A+selection

Rat C6 cells were transfected with connexin43 in a transfection vector (pLTR) or with the transfection vector alone as a control, as described previously [61]. Total RNA was isolated from confluent cultures of various C6 glioma cell subclones.

Differential display and sequencing

We have used the Genomyx Hieroglyph mRNA Profile System (Genomyx) and the GenomyxLR™ DNA Sequencer. This system has been designed to provide greater sensitivity and eliminates many of the problems associated with earlier differential display systems. With

Results

We have previously described the C6 glioma cell clones used in this study [61]. Following transfection, we obtained several clones which exhibited different levels of connexin43 expression (Fig. 1). The growth suppression observed in these clones was more pronounced in the higher expression clones [61]. We subsequently used RNA from these clones to verify the candidate genes which appeared to be differentially expressed when comparing the parental C6 cells with the high expressing clone Cx13.

Discussion

Differential cloning strategies have been used to identify genes associated with tumorigenic phenotypes 12, 13, 51. This approach has been recently improved through the use of differential display, a novel approach to identifying differentially expressed genes 34, 36, 50. This new strategy allowed for significant increase in screening the number of genes with differential expression. After candidate genes have been detected, they are isolated, re-amplified, identified by DNA sequencing, and

Acknowledgements

The authors are grateful to L. Bechberger for technical assistance. This work was supported by a grant from the Medical Research Council of Canada.

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Citation Excerpt :
Among the most exciting current topics in translational research is that of susceptibility factors by which gene expression alteration, environmental changes, and other stimuli bias in favor or against acquisition of a phenotype. In the gap junction field, it was initially surprising that expression of numerous other genes was affected in Cx43 null brain, heart and astrocytes (Iacobas et al., 2002, 2003; Naus et al., 2000). Moreover, such pervasive transcriptomic changes provide the expectation that phenotype resulting from knockdown of Cx43 might be highly variable with regard to strain, age or sex or other environmental conditions.
Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression level change of numerous other genes located on all chromosomes and involved in a wide diversity of functional pathways. The significant overlap between alterations in gene expression level, control and coordination in Cx43 knockout and knockdown astrocytes raised the possibility that Gja1 represents a transcriptomic node of gene regulatory networks. However, conditional deletion of Gja1 in astrocytes of two mouse strains resulted in remarkably different phenotypes. In order to evaluate the influence of the genetic background on the transcriptome, we performed microarray studies on brains of GFAP-Cre:Cx43^f/f C57Bl/6 and 129/SvEv mice. The surprisingly low number of Cx43 core genes (regulated in all Cx43 nulls regardless of strain) and the high number of differently regulated genes in the two Cx43 conditional knockouts indicate high influence of mouse strain on brain transcriptome.
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Opposing roles of connexin43 in glioma progression
2012, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
In addition to changes in cell cycle regulatory proteins, Cx43 may also affect the expression of other growth suppressors (Table 2). For instance, Cx43 transfection of C6 rat glioma cells also modifies the expression of secreted proteins, which either resulted in increasing (osteopontin, CCN3, IGFBP4) or decreasing (MPG-E8, IGF-1, IGFBP3) protein levels [46–50]. Similarly, a diffusible factor such as MCP-1 was found to be downregulated in human glioblastoma cells transfected by Cx43 [51], which opens up the possibility of which changes in the expression pattern of secreted proteins may modulate growth via paracrine effect [52,53].
Despite the tremendous amount of data over the last 40 years, lack of gap junctional intercellular communication (GJIC) or altered expression of gap junction proteins is still a lesser known ‘hallmark’ of cancer. Expression of astrocytic gap junction protein, connexin43 (Cx43), is often reduced in astrocytomas, the most common neoplasia of the central nervous system (CNS) in adults. Supported by a number of evidences, the global decrease of Cx43 expression appears to be advantageous for the growth of glioma cells. Although the mechanisms by which Cx43 regulates the expression levels of proteins involved in cell growth is unclear, there are evidences to suggest that it might be independent of their channel forming properties. In this regard, the carboxyl tail of Cx43 may have the ability to control the translocation of transcription factor regulators into the nucleus. However, this putative tumor suppressor effect of Cx43 is counterbalanced by its capacity to enhance the migration of glioma cells out of the tumor core through mechanisms that seems to implicate its carboxyl tail, possibly by interacting with the actin cytoskeleton. This ambivalence between the tumor suppressor effect and promotion of cell migration may partly be explained by the heterogeneous expression of Cx43 in the glioma core especially at the malignant glioblastoma stage; some tumor cells would be expected to migrate (Cx43 expressing cells) and others to proliferate (non-expressing Cx43 cells). Moreover, the involvement of Cx43 in glioma progression seems to be more complex since, in addition, GJIC may increase their resistance to apoptosis and Cx43 may also affect cell homeostasis in a paracrine fashion via hemichannel action. In conclusion, Cx43 appears to be involved at different levels of the glioma progression by acting on cell growth regulation, promotion of cell migration and resistance to apoptosis. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Non-channel functions of connexins in cell growth and cell death
2012, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Ectopic expression of Cx43 in glioma cells increases GJIC and decreases cell growth in vitro and in vivo, coinciding with elevated expression of the cysteine rich 61 protein and the nephroblastoma overexpressed (NOV) protein. The latter colocalizes with Cx43 gap junction plaques at the plasma membrane surface [118–121]. It has indeed been shown that NOV interacts with Cx43, but not with other connexins, including Cx32 and Cx40, in glioma cells and choriocarcinoma cells [122–124].
Cellular communication mediated by gap junction channels and hemichannels, both composed of connexin proteins, constitutes two acknowledged regulatory platforms in the accomplishment of tissue homeostasis. In recent years, an abundance of reports has been published indicating functions for connexin proteins in the control of the cellular life cycle that occur independently of their channel activities. This has yet been most exemplified in the context of cell growth and cell death, and is therefore as such addressed in the current paper. Specific attention is hereby paid to the molecular mechanisms that underpin the cellular non-channel roles of connexin proteins, namely the alteration of the expression of tissue homeostasis determinants and the physical interaction with cell growth and cell death regulators. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Connexins: Sensors and regulators of cell cycling
2011, Biochimica et Biophysica Acta - Reviews on Cancer
Citation Excerpt :
Ectopic expression of Cx43 in glioma cells increases GJIC and decreases cell growth in vitro and in vivo, coinciding with elevated expression of the cysteine rich 61 (Cyr61) protein and the nephroblastoma overexpressed (NOV) protein. The latter hereby colocalizes with Cx43 gap junction plaques at the cell plasma membrane surface [259–262]. It has indeed been shown that NOV interacts with Cx43, but not with other connexins, including Cx32 and Cx40, in glioma cells and choriocarcinoma cells [263–265].
It is nowadays well established that gap junctions are critical gatekeepers of cell proliferation, by controlling the intercellular exchange of essential growth regulators. In recent years, however, it has become clear that the picture is not as simple as originally anticipated, as structural precursors of gap junctions can affect cell cycling by performing actions not related to gap junctional intercellular communication. Indeed, connexin hemichannels also foresee a pathway for cell growth communication, albeit between the intracellular compartment and the extracellular environment, while connexin proteins as such can directly or indirectly influence the production of cell cycle regulators independently of their channel activities. Furthermore, a novel set of connexin-like proteins, the pannexins, have lately joined in as regulators of the cell proliferation process, which they can affect as either single units or as channel entities. In the current paper, these multifaceted aspects of connexin-related signalling in cell cycling are reviewed.
The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis
2010, Toxicology and Applied Pharmacology
Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules < 1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.
Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium
2009, International Journal of Biochemistry and Cell Biology
The gap junction proteins, connexins (Cx), are present in the testis and among them Cx43 play an essential role in spermatogenesis. By using an in vitro proliferation model of germ cells and Sertoli cells, we tempted here to clarify the role of Cx43 in the control of Sertoli and germ cell proliferation and apoptosis. Cx43 was detected in purified preparations of Sertoli cells and spermatogonia and immunolocalized in both cell types identified by vimentin and c-kit, respectively. Inhibition of gap junction coupling by the gap junction inhibitor α-GA significantly enhanced BrdU incorporation in Sertoli cells and reduced the number of activated caspase-3 positive germ cells. Similarly, inhibitory Cx43 and pan-Cx mimetic inhibitory peptides increased proliferation of Sertoli cells and stimulated survival of germ cells. Cx32 mimetic inhibitory peptide also stimulated Sertoli cell proliferation without altering germ cell proliferation and apoptosis. The present results reveal that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation.

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Short reviewIdentification of genes differentially expressed in C6 glioma cells transfected with connexin43

Abstract

Introduction

Section snippets

RNA isolation and poly A+selection

Differential display and sequencing

Results

Discussion

Acknowledgements

FEBS Lett.

Reg. Peptides

J. Biol. Chem.

Neurosci. Lett.

Mol. Cell. Endocrinol.

Trends Neurosci.

Gene

Dev. Biol.

Neurosci. Lett.

Cell Biol. Int.

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Trends Biochem. Sci.

Differentiated rat glial cell strain in tissue culture

Science

Inhibition of chemically induced neoplastic transformation by carotenoids. Mechanistic studies

Ann. N. Y. Acad. Sci.

Transfection of C6 glioma cells with connexin32: the effects of expression of a nonendogenous gap junction protein

Cell Growth Differ.

Insulin-like growth factor binding protein-4 gene expression is induced by transfection of gap junction connexin43 gene in a C6 glioma cell line

Growth Regul.

Identification of a gene family regulated by transforming growth factor-b

DNA Cell Biol.

Connexin43 reverses the phenotype of transformed cells and alters their expression of cyclin/cyclin-dependent kinases

Cell Growth Differ.

Cloning and identification of genes differentially expressed in metastatic and non-metastatic rat rhabdomyosarcoma cell lines

Clin. Exp. Metastasis

Differential expression of a novel gene, WDNM1, in nonmetastatic rat mammary adenocarcinoma cells

Cancer Res.

Osteopontin: between a rock and a hard plaque

Circ. Res.

Osteopontin: a protein with diverse functions

FASEB J.

Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo

Proc. Natl. Acad. Sci. U. S. A.

Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin32

Proc. Natl. Acad. Sci. U. S. A.

Molecular characterization and functional expression of the human cardiac gap junction channel

J. Cell Biol.

Gap junctions in the developing nervous system

Perspect. Dev. Neurobiol.

A mammalian homologue of a transcript from the Drosophila pecanex locus

J. Neurogenet.

A connexin 43 antisense vector reduces the ability of normal cells to inhibit the foci formation of transformed cells

Mol. Carcinogen.

Expression of the Elm1 gene, a novel gene of the CCN (connective tissue growth factor, Cyr61/Cef10, and neuroblastoma overexpressed gene) family, suppresses in vivo tumor growth and metastasis of K-1735 murine melanoma cells

J. Exp. Med.

Short review
Identification of genes differentially expressed in C6 glioma cells transfected with connexin43