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Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1., (Copyright © 2011 Wiley Periodicals, Inc.)

Gap junctions are cellular structures which permit direct exchanges of small molecules from cytoplasm to cytoplasm in most of the cells of metazoan organisms. For four decades, it has been observed that the inhibition of this type of intercellular communication is often associated with tumorigenesis. The assumption that loss of homeostasis which characterizes tumor growth could be a consequence of a lack of gap junctional intercellular communication (GJIC) has been reinforced by strategies able to reinduce both GJIC and normalization of the phenotype. So far, no molecular data may explain clearly how gap junctions can regulate cell proliferation. It has been argued that the gap-junction tumor suppressive effect may depend specifically on the connexin type which is expressed. For instance, the transfection of connexin30 (Cx30), a gap junction protein, has been previously associated with a slower growth of rat glioma cells (9L cells). Here, we show that these cells do communicate less compared to the Cx43-expressing parental cells even if the Cx30-transfected cells do express more Cx43. This result was related to the cytoplasmic distribution of Cx43 and a nuclear localization of both the Cx30 and a 20-kDa fragment corresponding to a Cx43 signal. According to these data, it seems that cell growth regulation may depend more on the behavior of connexins than the simple establishment of GJIC., ((c) 2007 Wiley-Liss, Inc.)

Gap junctional intercellular communication is thought to play an important role in cell differentiation and tissue homeostasis. Gap junctional intercellular communication is mediated by intercellular channels connecting adjacent cells and composed of connexin (Cx) proteins. Until now, approximately 20 different Cx have been characterized in mammals, and they are expressed in a tissue-specific manner. The downregulation of Cx expression is often observed in tumors and transformed cell lines and is believed to contribute to the loss of proliferating control. Connexin 26 (Cx26) is a Cx constitutively expressed in the normal epithelial esophageal tissue. In the majority of esophageal tumors, Cx26 expression is low or totally absent. CpG island hypermethylation is known to be associated with gene silencing in cancer. Because the promoter and exon 1 region of Cx26 are rich in CpG dinucleotides, we examined whether the loss of Cx26 expression in human esophageal TE cell lines was related to the hypermethylation of this region. We analyzed several TE cell lines derived from different human esophageal carcinomas and exhibiting different levels of Cx26 expression by using methylation-sensitive restriction digestion and Southern blot analysis. We did not find any correlation between the Cx26 expression and the methylation level of the promoter region of the Cx26 gene. Our results suggest that methylation was probably not involved as a primary mechanism of Cx26 regulation in human esophageal cancer cell lines., (Copyright 2003 Wiley-Liss, Inc.)

We previously showed that gap junction intercellular communication mediates the bystander effect in anticancer gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir. Because most cancer cell lines have lost their ability to communicate through gap junctions, we investigated whether we could induce such a communication by transferring a gene for a gap junction. We transfected a vector carrying the HSV-tk (tk) and gap junction (connexin (Cx) 32) genes (Cx32(+)tk(+)) into noncommunicating HeLa cells. We compared the cytotoxicity of ganciclovir with mixtures of these cells and HeLa cells that expressed (Cx32(+)) or did not express (Cx32(-)) the Cx32 gene. The bystander effect was strong when the two mixed cell types expressed Cx32 (i.e., Cx32(+)tk(+) cells and Cx32(+)tk(-) cells). Only 25% of cells survived in this communicating mixture, even when only 10% of the cells were Cx32(+)tk(+). There was also a moderate bystander effect when the Cx32(+)tk(+) cells were mixed with noncommunicating HeLa cells in a 50% ratio. These results demonstrated that the bystander effect is enhanced by Cx32 and suggested that expression of Cx in only one cell type in a mixture can cause a bystander effect. Mol. Carcinog. 30:176--180, 2001., (Copyright 2001 Wiley-Liss, Inc.)

The expression of connexin 32 (the major liver gap junction protein) and connexin 43 (the major cardiac gap junction protein) was examined in six surgically removed human hepatocellular carcinoma tissues and the surrounding nontumorous livers using specific rat connexin probes. No decrease in connexin 32 mRNA expression was found in carcinomas compared with the surrounding nontumorous tissue. Morphometrical analysis also showed that in most of the carcinomas the number of gap junction spots stained with connexin 32 antibody was not less than that in the surrounding livers. These results are in striking contrast to the significant reductions in connexin 32 mRNA and protein expression observed in rat primary liver tumors induced by chemicals. On the other hand, all of the six human hepatocellular carcinomas exhibited elevated levels of connexin 43 mRNA, which was expressed at a very low level in the surrounding nontumorous livers. These carcinomas exhibited no detectable amplification of the connexin 43 gene. The present study suggests that gap junctional intercellular communication is altered in human hepatocellular carcinomas by molecular mechanisms different from those in rat hepatocarcinogenesis.

The gene expression of liver major gap junction (GJ) protein was studied in rats systemically administered phenobarbital, a rat liver tumor promoter. Using a GJ protein cDNA and northern blot analysis, the level of GJ protein mRNA in liver was observed to be markedly reduced at 4 and 11 wk of phenobarbital exposure (0.1% in drinking water). However, the level of GJ protein mRNA was not altered in kidney at 11 wk of exposure. In liver, phenobarbital did not induce expression of the neoplasm-associated marker genes glutathione S-transferase (placental form) and gamma-glutamyltranspeptidase, while in kidney the observed expression of these genes was not changed. These in vivo results indicate that phenobarbital reduces GJ protein gene expression specifically in rat liver without altering expression of genes often altered during liver carcinogenesis, and they support assigning a role for the impairment of gap junctional intercellular communication in phenobarbital-mediated liver tumor promotion.