SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. II. Changes in gap junctional intercellular communication

Poor gap junctional intercellular communication (GJIC) has been associated with uncontrolled cell growth and neoplasia. We have successfully propagated normal first trimester invasive extravillous trophoblast (EVT) cells, and have produced premalignant EVT lines after SV40 Tagtransformation: RSVT-2 is an uncloned line that is long-lived; RSVT2/C is a clonal line that is immortal. Both are hyperproliferative, hyperinvasive and variably refractory to the anti-proliferative and anti-invasive effects of transforming growth factor β (TGFβ). Possible changes in gap junctions during the transition of normal invasive EVT cells to the premalignant stage were examined by comparing expression of connexin proteins (by immunolabeling for Cx26, Cx32, Cx40, Cx43), and mRNA (by Northern blot with cDNA probes for Cx26, Cx32, Cx43), and functional GJIC (by dye transfer using the preloading method) in normal parental EVT cells and their SV40 Tag transformants. Results from immunofluorescence and Northern blot analysis revealed that, of the panel of connexins examined, only Cx43 was variably expressed in these cell lines in vitro. Expression of Cx43 protein and mRNA was abundant in normal EVT cell line HTR8, reduced in long-lived RSVT-2 cells and undetectable in immortalized RSVT2/C cells. GJIC, as measured by dye transfer between donor and recipient cells, was also similarly reduced in recipient RSVT-2 cells, and drastically reduced in RSVT2/C cells, irrespective of whether the dye donor was of the same cell type (homocellular coupling) or HTR8 cells (heterocellular coupling). Treatment with TGFβ reduced Cx43 mRNA expression as well as GJIC in normal EVT cells, but not in the SV40 Tag transformants. Our findings suggest that downregulation of connexins with the resultant impairment in GJIC is an early event in tumor progression, as observed in the premalignant SV40 Tag transformants. Int. J. Cancer77:440–448, 1998. © 1998 Wiley-Liss, Inc.     

Altered gap junctional intercellular communication in neoplastic rat esophageal epithelial cells

Gap junctional intercellular communication (GJIC) is reduced in many neoplastic cells, but few data exist for esophageal neoplasms. GJIC was examined by fluorescent dye microinjection in two nontumorigenic and two highly tumorigenic rat esophageal epithelial cell lines. All lines expressed high levels of dye coupling in homologous cell culture. In cocultures of nontumorigenic and tumorigenic cells, however, only one of six cell combinations displayed significant heterologous GJIC. Northern, Western, and immunohistochemical analyses indicated that all four cell lines expressed comparable levels of connexin43 (Cx43), but not connexin32 or connexin26, and formed Cx43-containing gap junction plaques at cell-cell interfaces. Immunostaining of rat esophageal frozen sections demonstrated that esophageal epithelial cells expressed Cx43 in vivo. In normal epithelium, the highest expression was seen in the basal cells and little suprabasal staining was evident. In preneoplastic and neoplastic lesions of the esophageal epithelium which were induced by treating rats with N-nitrosomethylbenzylamine, Cx43 staining of the basal layer was also seen but appeared to be more diffuse compared to normal epithelium. In addition, suprabasal Cx43 staining was apparent in dysplastic and papillomatous lesions. These results indicate that Cx43 is expressed in normal and neoplastic rat esophageal cells and that the cells exhibit extensive homologous GJIC, but little heterologous GJIC. This lack of heterologous GJIC may be due to differences in cell adhesion proteins or other factors.      

Expression of cell adhesion molecules and connexins in gap junctional intercellular communication deficient human mesothelioma tumour cell lines and communication competent primary mesothelial cells

Gap junctional intercellular communication (GJIC) has been reportedto be markedly reduced in human mesothelloma tumour cell linescompared with primary mesothelial cells. Iminunofluorescencestainings have shown that the gap junction protein connexin43(Cx43) is expressed In both malignant and normal mesothelialcells. In this study the mRNA expression of Cx43 and three differentconnexlns—Cx37, Cx40 and Cx45, which are highly expressedin lung tissue—was investigated in eight human mesotheliomacell lines, and in human primary mesothellal cells from severaldonors. The expression of the intercellular adhesion moleculesA-CAM (N-cadherln) and L-CAM (E-cadherin) was studied at theprotein level. No mRNA expression of Cx37, Cx40 or Cx45 in eithermesothelioma tumour cells or the primary mesothelial cells wasdetected. Cx43 was expressed at both the mRNA and the proteinlevel, in seven out of eight mesothelloma cell lines, as wellas in all the primary mesothellal cell cultures. The intercellularadhesion molecule A-CAM was expressed at the cell—cellborders In six out of seven mesothelioma cell lines, as wellas in normal mesothellal cells. No expression of L-CAM was observedin these cells. The results suggest that Cx43 and A-CAM arethe major proteins in gap and adherens Junctions respectivelyin human mesothellal cells. Most mesothelioma tumour cell lineswith markedly reduced GJIC still express both Cx43 and A-CAM.Only one of our mesothelloma tumour cell lines severely deficientin GJIC lacks both the gap junction protein Cx43 and the celladhesion molecule A-CAM.     

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap-junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myclras all displayed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src- and neu-transformed cells, but not in the ras- and myclras-transformed cells. Nuclei isolated from WB-neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB-neo control cells, supporting the idea that the nuclear-localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC-deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane-localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane-associated Cx43 immunostaining. These data suggest that altered localization of the gap-junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells. © 1996 Wiley-Liss, Inc.     

Connexin 43 expression promotes malignancy of HuH7 hepatocellular carcinoma cells via the inhibition of cell-cell communication

In normal liver, Connexin (Cx) 43 is not detected, but up-regulated in some liver cancers. We herein investigated the role of Cx43 in hepatoma cell carcinogenesis. Cx43-silenced HuH7 cells using shRNA showed lower growth and higher differentiation, and Cx43-overexpressing cells exhibited rapid growth and low differentiation. Unexpectedly, gap junctional intercellular communication (GJIC) was inversely correlated with Cx43 levels. Furthermore, the expression level and promoter activity of Cx32 was negatively regulated by the expression of Cx43. From these data, Cx43 expression may be in part responsible for the malignancy of hepatoma cells through a decrease in GJIC composed of Cx32.     

细胞连接蛋白基因在胃癌中的表达研究

目的： 为了系统研究人胃癌基因组中Ｃｘ 基因的表达状况。方法： 本文采用Ｎｏｒｔｈｅｒｎ 印迹杂交和ＲＴＰＣＲ 方法, 对１５ 例人胃癌组织、配对癌旁组织、正常组织及人胃腺癌ＭＧＣ８０３ 细胞株的Ｃｘ２６ 、Ｃｘ３１－１ 、Ｃｘ３２ 、Ｃｘ３３、Ｃｘ３７ 、Ｃｘ４０、Ｃｘ４３ 、Ｃｘ４６ 八种Ｃｘ 基因表达进行了一系列检测。结果：发现在人正常胃粘膜上皮中高水平表达而在胃癌中表达极其微弱或根本无表达的细胞连接蛋白基因的表达规律,首次明确了Ｃｘ 基因在人胃癌基因组中的表达谱。其中人正常胃粘膜上皮细胞中Ｃｘ３２ 、Ｃｘ３７ 、Ｃｘ４３ 在ｍ ＲＮＡ 水平上有高水平表达; 与正常相反, 人胃癌细胞除Ｃｘ４３ 在ｍ ＲＮＡ 水平上有低水平表达外, 其他连接蛋白基因均无转录活性; 而人胃腺癌ＭＧＣ８０３ 细胞株中, Ｃｘ３７ 、Ｃｘ４６ 在ｍ ＲＮＡ 水平上有一定程度的表达。结论：本研究表明Ｃｘ３２ 可能是人胃上皮细胞基因组中维持细胞间隙连接通讯功能的特异表达的Ｃｘ 基因,Ｃｘ４６ 可能是胃癌Ｃｘ 基因表达的一种变异。Ｃｘ３７、Ｃｘ４３ 不是人胃上皮细胞的特异表达基因。本研究证实了Ｃｘ 基因在肿瘤细胞中表达下调,Ｃｘ 基因具有潜在的抑瘤基因的生物学活性。     

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C

In this study, we investigated whether the tumor promoters, 12-O- tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1- bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB- F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin- specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.      

Restoration of gap-junctional intercellular communication in a communication-deficient rat liver cell mutant by transfection with connexin 43 cDNA

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC. © 1993 Wiley-Liss, Inc.     

Inhibition of intrinsic gap-junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant-negative connexin 43 mutant

The tumor-suppressive property of the connexin gap-junction proteins was postulated from the fact that their function of cell coupling is impaired in most cancer cells. However, in conflict with this notion, certain cancer cells are able to communicate through gap junctions despite their malignancy. To explain this phenomenon, we studied by using a dominant-negative strategy the effect on tumorigenicity of loss of intrinsic gap-junction intercellular communication (GJIC) in the rat bladder carcinoma cell line BC31, which shows both expression of connexin 43 (Cx43) and intercellular communication. In cells transfected with a mutant Cx43 with seven residues deleted from the internal loop at positions 130–136 (Cx43Δ), transport of the resulting connexin protein to the plasma membrane occurred normally, but the GJIC of the cells was effectively abolished at the level of permeability of established gap junctions. Dominant-negative inhibition of GJIC by Cx43Δ accelerated growth of BC31 cells in nude mice. In contrast, when GJIC in BC31 cells was artificially enforced by transfection of wild-type Cx43, the cells lost the capacity to grow in vivo. Decreased phosphorylation of Cx43Δ suggested close interaction of the internal loop of connexin with its commonly phosphorylated domains in the C-terminal tail and involvement of this interaction in gap-junction permeability. Therefore, we conclude that the intrinsic GJIC observed in cancer cells should be considered a tumor-suppressor factor and that its level may influence malignant growth capacity. Mol. Carcinog. 23:254–261, 1998. © 1998 Wiley-Liss, Inc.     

Expression and function of connexin in normal and transformed human keratinocytes in culture

We have studied the gap junctional intercellular communication(GJIC) of immortalized and tumourigenic human keratinocyte celllines and of a spontaneously immortalized non-tumourigenic anda highly differentiating keratinocyte cell line (HaCaT) as thecontrol. In homologous cultures, the GJIC capacity of five squamouscell carcinoma-derived cell lines was 1–27% that of theHaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenicpotential and an SV40 DNA-immortalized cell line had markedlyreduced GJIC capacities. Northern analysis and immunohisto-chemistryshowed that connexin (Cx) 43 is the major gap junction proteinexpressed in the communicating cells. They do not express Cx26 or 32. The low or absent communication observed in certaincell lines was due in some to a lack of Cx 43 gene expression,but in others to aberrant localization of the gap junction protein.GJIC of these cell lines, as well as that of primary normalhuman epidermal keratinocytes, was susceptible to 12-O-tetra-decanoylphorbol-13-acetate-mediatedinhibition. Moreover, GJIC of HaCaT cells and their tumourigenicderivatives is Ca²⁺-dependent. These results, when comparedwith those previously obtained for mouse keratinocyte cell lines,reveal that GJIC of human keratinocytes was correlated to thedegree of differentiation and is controlled in a similar wayto that of murine keratinocytes. Aberrant GJIC seems to be acommon feature of human and murine skin carcinogenesis.     

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.      

Reduction of malignant phenotype of HEPG2 cell is associated with the expression of connexin 26 but not connexin 32

Connexin (Cx) genes have a negative growth effect on tumour cells with certain specificity. However, it is not clear whether each Cx gene can act similarly in growth control. Hepatocytes normally express Cx26 and Cx32 as their major gap junction genes, but HepG2 cells, a hepatoma cell line, are deficient in gap junctional intercellular communication (GJIC) based on the down-regulation of Cx26 and aberrant localization of Cx32. In this study, we showed that some of the expressed Cx26 protein in HepG2 cells localized in the plasma membrane and contributed to recovery of GJIC, while the Cx32 protein remained localized in the cytoplasm. The Cx26-transfected clones showed a significantly slower growth in vivo as well as in vitro and reduced anchorage-independent growth ability compared with a mock-transfected clone. Cx26-transfected cells had more regular cell layers due to the re-establishment of the E-cadherin cell adhesion complex. E-cadherin expression following Cx26 transfection was induced. Cx26 expression simultaneously brought E-cadherin and beta-catenin proteins into the plasma membrane without any change in the expression level of beta-catenin protein. These results suggest that the expression of Cx26 contributes to negative growth control of HepG2 cells and the morphological change through the induction of E-cadherin and subsequent formation of cell adhesion complex.     

Growth control of 3T3 fibroblast cell lines established from connexin 43–deficient mice

Connexins are considered to be involved in cell growth control, on the basis of studies mainly with tumorigenic cells. To study the role of connexin genes in normal cell growth control, we established fibroblast cell lines from connexin 43 (Cx43)–deficient mice and characterized their growth. Embryonic fibroblasts from wild-type mice (Cx43^+/+) and those with heterozygous (Cx43^+/–) and homozygous (Cx43^–/–) deficiencies of the Cx43 gene were cultured and passaged by a 3T3 protocol (every 3 d, 3 × 10⁵ cells/60-mm dish). All cell lines showed a growth crisis during passages 6–15 and then started to grow well. All cell lines grew at similar rates under the 3T3 protocol, but Cx43-deficient (Cx43^–/–) cell lines tended to grow faster when they were plated at 10⁵ cells per dish. Cx43^–/– cells did not express Cx43 and showed little gap-junctional intercellular communication (GJIC), confirming that Cx43 is the major connexin responsible for GJIC of these fibroblasts. While all Cx43^+/+ and Cx43^+/– cell lines expressed Cx43 protein, some of them showed very little GJIC. Those cell lines with high GJIC showed higher levels of the P2 form of Cx43 protein, and more Cx43 was localized in the plasma membrane than in cell lines with lower GJIC levels. We investigated effects of serum concentration on cell growth in these cell lines. Although different cell lines responded differentially to these agents, there was no clear relationship between Cx43 expression and cell growth stimulation by them. This suggests that Cx43 expression alone is not a strong regulator of mouse fibroblast growth. Mol. Carcinog. 23:121–128, 1998. © 1998 Wiley-Liss, Inc.     

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner

Many lines of evidence support the hypothesis that connexins form a family of tumor-suppressor genes. Transfection of connexin43 (Cx43) into rat C6 glioma cells have revealed that Cx43 functions as a growth- and tumor-suppressor in C6 cells. In previous studies, we and others have reported that several mutant connexins can inhibit gap junctional intercellular communication (GJIC) realized by the wild type in a dominant-negative manner. We have now examined dominant-negative effects of Cx43 mutants on cell growth control exerted by wild-type Cx43 in C6 cells. When 2 Cx43 mutants (L160M and A253V) were transfected into Cx43-transfected C6 cells, they restored anchorage-independent growth capacity and reinforced the tumorigenicity of these cells, meaning that these 2 mutants can inhibit growth-suppressive function of wild-type Cx43 in a dominant-negative manner. Neither of the mutants appeared to affect phosphorylation states and subcellular localization of Cx43 proteins. Intriguingly, the mutant A253V did not suppress GJIC capacity, implying a growth-suppressive pathway mediated by Cx43 may not be related to GJIC.Int. J. Cancer 78:446–453, 1998. © 1998 Wiley-Liss, Inc.     

v-Src requires Ras signaling for the suppression of gap junctional intercellular communication

Cell transformation by v-Src causes suppression of gap junctional intercellular communication (GJIC). Although tyrosine phosphorylation of connexin43 (Cx43), a gap junctional component, appears to be necessary for the suppression, involvement of other signaling remains unclear. We investigated the role of Ras signaling in the suppression of GJIC by v-Src. Conditional expression of either S17N Ras or mtGap1m dramatically recovered GJIC in v-Src-transformed cells. Although expression of S17N Ras or mtGap1m substantially decreased the levels of active Ras, tyrosine phosphorylation of cellular proteins including Cx43 remained unchanged. Similarly, treatment of v-Src-transfomed cells with a Ras farnesyltransferase inhibitor, manumycin A, restored GJIC, whereas tyrosine phosphorylation of Cx43 remained unchanged. Thus, these results strongly suggest that, in addition to Cx43 phosphorylation, constitutive activation of Ras signaling is required for the suppression of GJIC by v-Src.     

连接蛋白基因Cx43抑制胶质瘤细胞增殖及其机理的初步探讨

目的 探讨连接蛋白基因Cx43对胶质瘤细胞增殖的抑制及其可能的机理。方法 将含Cx43cDNA的质粒以脂质体介导转染Cx43表达缺失的人和鼠的恶性胶质瘤细胞,通过Northem杂交、原位杂交及免疫组化染色检测Cx43mRNA及蛋白表达;MTT法测定细胞增殖率;核仁组成区嗜银蛋白染色检测细胞增殖活性;TUNEL法检测细胞凋亡;划痕标记荧光染料示踪技术检测细胞间隙连接通讯(GJIC);Western杂交及免疫组化染色检测bFGF、PDGF、EGFR、IGF-I和IGFBP3的表达。结果 转染Cx43基因的胶质瘤细胞增殖下降,GJIC恢复,同时伴有bFGF、PDGF、IGF-I和IGFBP3表达下降,而EGFR表达和细胞凋亡则无改变。结论 Cx43基因可能通过恢复GJIC功能及抑制某些重要生长因子的自分泌,实现对胶质瘤细胞增殖的抑制。     

Gap junction intercellular communication propagates cell death in cancerous cells

Gap junction intercellular communication (GJIC) or cell coupling has an important function in maintaining tissue homeostasis and is thus a critical factor in the life and death balance of cells. While the role of GJIC in cell growth regulation has been much studied, its involvement in apoptosis remains unclear. In this study we elucidated the possibility that cell death is propagated via gap junctions, employing the rat bladder carcinoma cell line BC31. BC31 cells proliferate quickly, are tumorigenic, and are well-coupled via gap junctions that contain the gap junction protein Connexin43 (Cx43). In addition, these cells are predisposed to spontaneous death by apoptosis, particularly upon achieving confluency. We found that many dying BC31 cells express Cx43 just as their non-apoptotic counterparts do. Furthermore, Cx43 in apoptotic cells could be functionally competent, supporting coupling of these cells with their non-apoptotic neighbors, and as a result, clusters of coordinately dying cells were observed. The role of Cx43 and GJIC in propagating cell death was shown by analysing clones of BC31 cells expressing a mutant of Cx43 that is a dominant negative inhibitor of GJIC, and by using beta-glycyrrhetinic acid to inhibit intrinsic cell coupling in BC31 cells: in both cases the formation of clusters of dying cells was abrogated, and the intensity of cell death was considerably decreased. These results suggest that GJIC spreads cell-killing signals initially generated by a single cell that spontaneously initiates apoptosis, into healthy surrounding cells, thus increasing the level of cell death. Treatment of BC31 cells with the sleep-inducing lipid Oleamide, which selectively restricts gap junction permeability to Ca(2+) ions, did not abrogate coordinated cell death by clusters, indicating that Ca(2+) ions are the most probable cell-killing signals spread through gap junctions.     

Comparison of glucocorticoid-mediated changes in the expression and function of rat hepatocyte gap junctional proteins

Gap junctional intercellular communication (GJIC) is often modulatedby chemical carcinogens and during carcinogenesis, in part,through changes in gap junction mRNA levels. However, the mechanismsby which gap junction mRNA levels are altered in either normalor cancer cells are largely unknown. Since glucocorticoids arepotent modulators of gene expression and stability, we haveinvestigated the effects of these hormones on GJIC and gap junctionmRNA expression in rat hepatocytes cultured in three differentmedia. Addition of dexamethasone to cultures of rat hepatocytesresulted in a maintenance of GJIC and both major liver gap junctionalmRNAs, connexin (Cx)26 and Cx32, at levels above those in hepatocytescultured in glucocorticoid-free media. In addition, hepatocytescultured without dexamethasone for 24 h could be induced tocommunicate and increase Cx mRNA levels by the addition of dexamethasoneto their medium. These media-independent changes in GJIC andgap junction mRNA levels by dexamethasone warrant further investigationsinto their mechanisms of action and the potential therapeuticvalue of glucocorticoids in the treatment of cancer.