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ABSTRACT: Connexins (Cxs), the gap junction proteins, have been found to be downregulated in many types of cancers including gliomas. By restoring gap junctional communication in cancer cell models, the neoplastic phenotype can be reversed, suggesting Cxs are tumor suppressors. Pannexin2 (Panx2) is a member of the novel gap junction protein family, Panxs, and it has been proposed as a brain-specific protein. Recently, gene array analysis showed an overall reduction of Panx2 in gliomas, and a direct correlation was observed between Panx2 expression and post-diagnosis survival in patients. In this study, we explored the potential inverse correlation between Panx2 and glioma oncogenicity. A decrease or absence of Panx2 expression in a panel of human glioma cell lines was found, whereas an appreciable amount of Panx2 was detected in both human brain and astrocytes. Stable Panx2 expression revealed a flattened morphology and increased cell-cell contacts in rat C6 glioma cells similar to Panx1. However, in contrast to Panx1 and Panx3, Panx2 was predominately detected in the cytoplasm in vesicle-like patterns but not at the plasma membrane. Coexpression of Panx2 and Panx1 did not show colocalization of both Panxs. Strikingly, restoration of Panx2 expression significantly reduced in vitro oncogenicity parameters, including monolayer saturation density and anchorage-independent growth, as well as in vivo tumor growth. This study suggests a role of aberrant Panx2 expression during gliomagenesis, and that Panx2 independently functions as a negative growth regulator without Panx1.
Oncogene 09/2009; 28(49):4402-8. · 6.37 Impact Factor
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ABSTRACT: Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein connexin43 (Cx43) and provide a substrate for formation of a functional syncytium implicated in the spatial buffering capacity of astrocytes. To study the function of gap junctions in the brain, we used heterozygous Cx43 null mice, which exhibit reduced Cx43 expression. Western blot analysis showed a reduction in the level of Cx43 protein and GJIC in astrocytes cultured from heterozygote mice. The level of Cx43 is reduced in the adult heterozygote cerebrum to 40% of that present in the wild-type. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild-type and heterozygote mice after focal ischemia. In our model of focal stroke, the middle cerebral artery was occluded at two points, above and below the rhinal fissure. Four days after surgery, mice were killed, the brains were sectioned and analyzed. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared with wild-type (14.4 +/- 1.4 mm(3) vs. 7.7 +/- 0.82 mm(3), P < 0.002). These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection after ischemic injury.
The Journal of Comparative Neurology 12/2001; 440(4):387-94. · 3.81 Impact Factor
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ABSTRACT: Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.
Cell Communication & Adhesion 02/2001; 8(4-6):325-8. · 1.18 Impact Factor
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ABSTRACT: Tumour cells often exhibit erratic cell growth, as well as decreased gap junctional intercellular communication (GJIC). C6 glioma cells are characterized by low levels of gap junction mRNA and protein, and decreased amounts of GJIC when compared with astrocytes. Previous work has shown that C6 glioma cells transfected with connexin43 (C6-Cx43) exhibit decreased proliferation in vivo and in vitro, as well as genes that are differentially expressed between these cells. In this study, RNA levels of two CCN (connective tissue growth factor [CTGF], Cyr61/Cef-10, nephroblastoma overexpressed [NOV]) gene family members are shown to be upregulated in C6-Cx43 cells: Cyr61 and Nov. Cyr61 has previously been shown to increase adhesion, migration and growth in many cell types, whereas NOV has growth suppressive capacities. Cyr61 RNA expression is shown here to respond to serum in quiescent cells in an immediate early gene fashion, independent of Cx43 expression. In contrast, Nov RNA levels remain constant, reflective of transfected Cx43 expression. Furthermore, confocal microscopy indicates that NOV colocalizes with Cx43 plaques at the cell membrane. These findings provide insight into the possible role of Nov and Cyr61 in tumour cells.
Cell Communication & Adhesion 02/2001; 8(4-6):441-5. · 1.18 Impact Factor
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ABSTRACT: Recent advances in genetic technology have provided a new platform on which the simultaneous analysis of a large number of genes is possible in a rapid and efficient fashion. To assess the differential expression of human genes during neuronal differentiation, we compared the transcript profiles of undifferentiated, partially differentiated, and fully differentiated NT2/D1 cultures with cDNA expression arrays. Approximately 75 genes (13% of the gene array pool) were differentially expressed during neuronal development of NT2/D1 cells. Genes coding for pyruvate kinase M2 isozyme, clathrin assembly proteins, calmodulin, fibronectin, laminin, thymosin beta-10, and many others were upregulated as NT2/D1 cells differentiated into neurons. In contrast, several kinases, phosphatases, and G-protein coupled receptor genes showed downregulation upon neuronal differentiation. The information provided here is an invaluable reference for characterizing the phenotype of these cells. This information can also be used in cell therapy and transplantation in which the graft microenvironment and interaction with the host tissue is crucial.
Biochemistry and Cell Biology 02/2001; 79(4):387-98. · 2.67 Impact Factor
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G S Goldberg, J F Bechberger,
Y Tajima,
M Merritt,
Y Omori,
M A Gawinowicz,
R Narayanan,
Y Tan,
Y Sanai,
H Yamasaki,
C C Naus,
H Tsuda,
B J Nicholson
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ABSTRACT: Gap junction expression has been reported to control the growth of a variety of transformed cells. We undertook parallel analysis of connexins Cx32 and Cx43 in glioma cells, which revealed potential mechanisms underlying this phenomenon and led to several novel findings. Cx43, but not Cx32, suppressed C6 glioma cell growth. Paradoxically, Cx32 transfection resulted in severalfold more dye transfer than Cx43. However, Cx43 transfectants shared endogenous metabolites more efficiently than Cx32 transfectants. Interestingly, a significant portion of Cx43 permeants were incorporated into macromolecules more readily than those that transferred via Cx32. Cx43 induced contact inhibition of cell growth but in contrast to other reports, did not affect log phase growth rates. Cell death, senescence, or suppression of growth factor signaling was not involved because no significant alterations were seen in cell viability, telomerase, or mitogen-activated protein kinase activity. However, suppression of cell growth by Cx43 entailed the secretion of growth-regulatory factors. Most notably, a major component of conditioned medium that was affected by Cx43 was found to be MFG-E8 (milk fat globule epidermal growth factor 8), which is involved in cell anchorage and integrin signaling. These results indicate that Cx43 regulates cell growth by the modulation of extracellular growth factors including MFG-E8. Furthermore, the ability of a Cx to regulate cell growth may rely on its ability to mediate the intercellular transfer of endogenous metabolites but not artificial dyes.
Cancer Research 12/2000; 60(21):6018-26. · 7.86 Impact Factor
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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).
Brain Research Reviews 05/2000; 32(1):259-66. · 10.34 Impact Factor
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ABSTRACT: Gap junctions are intercellular channels which provide for the passage of small ions and molecules (MW <1200 D) among adjacent cells. The NTera2/clone D1 (NT2/D1) cells are CNS precursors which differentiate into NT2-N neurons upon treatment with retinoic acid (RA) and antiproliferative agents. In this study, the effects of gap junction blockers 18 alpha-glycyrrhetinic acid (GRA) and carbenoxolone (CBX) have been compared with those of oleanolic acid (OLA) and glycyrrhizic acid (GZA), GRA analogs with no blocking effects. Both control and experimental cultures showed reduction of Cx43 protein after 4 weeks of RA induction. A major reduction was also observed in expression of cytokeratin, vimentin, and nestin in control cells at this time point while the cultures treated with the blockers did not show any significant change. The average number of MAP2-positive NT2-N differentiated neurons per field of view in the cultures treated with the blockers was less than 7% of that of control cultures. NT2-N cells were negative for Cx43, cytokeratin, vimentin, and nestin. The blockers did not appear to be operating through inhibition of RA signaling, as their presence did not affect the expression of retinoic acid receptors (RARalpha and RARgamma) nor did they inhibit RA-mediated gene transcription. These results, together, show that the blockage of gap junctions interferes with neuronal differentiation of NT2/D1 cells.
Experimental Neurology 03/1999; 156(1):16-32. · 4.70 Impact Factor
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ABSTRACT: Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated by channels composed primarily of connexin43. To examine some of the functions of this intercellular communication in glial cells, we have used three approaches. The first involves transfection of glioma cells, which are deficient in connexin expression and gap junctional communication, with connexin cDNAs to examine changes in cellular phenotype following increased gap junctional communication. Using differential display, we have identified several genes which appear to be regulated by GJIC. The second is to study astrocytes cultured from embryonic mice with a null mutation in the connexin43 gene. These homozygous null astrocytes are devoid of connexin43 and also deficient in intercellular dye transfer. Markers of glial differentiation appear similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. The growth rate of astrocytes lacking connexin43 was reduced compared to wild-type astrocytes. The third approach employs the use of gap junction blockers in a model of neuronal and glial differentiation, namely P19 mouse embryonal carcinoma cells treated with retinoic acid. In this case, blocking GJIC during the differentiation protocol prevents the appearance of neuronal and astrocytic phenotypes. Taken together, these data suggest an important role for GJIC in glial function and differentiation.
Advances in experimental medicine and biology 02/1999; 468:373-81. · 1.09 Impact Factor
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ABSTRACT: Invasion of the uterus by first trimester human placental extravillous trophoblast (EVT) cells depends on mechanisms shared by malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is stringently controlled in situ by local molecules such as transforming growth factor (TGF)beta. Since EVT cells possess active invasion-associated genes but are nontumorigenic, our objective was to induce premalignant and then malignant phenotype into a normal EVT cell line in order to identify the molecular basis of tumor progression. Simian virus 40 large T antigen (SV40 Tag) was introduced into a normal human first trimester invasive EVT cell line, HTR8, established in our laboratory. Since the HTR8 line has a limited in vitro lifespan of 12-15 passages, SV40 Tag-transformed cells were selected on the basis of extended lifespan. A long-lived line, RSVT-2, was produced and an immortalized subclone, RSVT2/C, was further derived under a forced crisis regimen. We examined transformation-induced alterations in proliferative and invasive abilities, responses to the invasion and proliferation-regulating growth factor TGFbeta and changes in gene expression for invasion-associated enzymes or enzyme inhibitors. RSVT-2 and RSVT2/C cell lines were hyperproliferative and hyperinvasive when compared with the parental HTR8 cell line. They were also variably resistant to the anti-proliferative and anti-invasive signals from TGFbeta. Since both cell lines remained non-tumorigenic in nude mice, these properties indicate that they attained a premalignant phenotype. Both cell lines showed reduced expression of tissue inhibitor of metalloproteases (TIMP)-1, while TIMP-2 and plasminogen activator inhibitor (PAI)-I expression was was also reduced in RSVT2/C cells, thus contributing to their hyperinvasiveness. Their resistance to the anti-invasive action of TGFbeta was explained by the failure of TGFbeta to upregulate TIMPs and PAI-I, in contrast to the TGFbeta-induced upregulation noted in parental HTR8 cells.
International Journal of Cancer 08/1998; 77(3):429-39. · 5.44 Impact Factor
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ABSTRACT: 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 Tag transformation: 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 beta (TGFbeta). 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 TGFbeta 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.
International Journal of Cancer 08/1998; 77(3):440-8. · 5.44 Impact Factor
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ABSTRACT: Astrocytes are characterized by extensive intercellular communication mediated primarily by gap junction channels composed of connexin43. To examine this junctional protein in astrocytic functions, astrocytes were cultured from embryonic mice with a null mutation in the connexin43 gene (Reaume et al.: Science 267:1831-1834, 1995). Using anti-Cx43 antibodies, immunoblotting and immunostaining indicated that homozygous null astrocytes were devoid of Cx43. They are also deficient in intercellular dye transfer. Astrocytes cultured from heterozygous embryos express significantly lower Cx43 compared to wild type, and their dye coupling is reduced. Markers of glial differentiation, such as glial fibrillary acidic protein and S100, appeared similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. In fact, the calcium response in homozygous astrocytes was similar to that observed in wild-type astrocytes in the presence of a gap junction blocker. The growth rate of astrocytes lacking Cx43 was reduced compared to wild-type astrocytes. These results suggest that gap junctional intercellular communication mediated by Cx43 is not critical for astrocyte differentiation but is likely involved in the regulation of intercellular calcium signaling and cell growth.
Journal of Neuroscience Research 10/1997; 49(5):528-40. · 2.74 Impact Factor
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ABSTRACT: Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, -32, and -43. Connexin26 and -32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and -32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells.
Journal of Neuroscience Research 08/1997; 49(1):19-31. · 2.74 Impact Factor
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ABSTRACT: The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.
Developmental Genetics 02/1997; 21(3):187-200.
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ABSTRACT: Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.
Developmental Brain Research 01/1997; 97(2):293-6. · 1.78 Impact Factor
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ABSTRACT: Transfection of C6 glioma cells with connexin43 (Cx43) cDNA under a constitutive promoter resulted in expression of Cx43 protein, an increase in functional gap junctions, and reduced growth under in vitro and in vivo conditions (D. Zhu et al., Proc. Natl. Acad. Sci. USA, 88: 1883-1887, 1991). To allow for precise temporal and quantitative control of Cx43 gene expression, the Cx43 cDNA was inserted into an expression vector [pSV2M(2)6] containing a modified metallothionein promoter. Upon transfection of this vector into C6 cells, clones were isolated that expressed increased levels of inducible Cx43 protein and dye coupling. The level of induction of Cx43 expression increased with increasing concentration of Zn2+, thus enabling the use of the same clone with different levels of gap junctions present. Although we observed no change in cell growth under in vitro conditions following exposure to Zn2+ or Cd2+, clones with inducible expression of Cx43 were characterized by reduced growth in vivo. Within tumors, the level of expression of Cx43 mRNA and protein corresponded to that seen in vitro following the addition of Zn2+. The suppression of tumor growth in vivo correlated with the level of induced Cx43 expression.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 11/1996; 7(10):1403-13.
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ABSTRACT: Gap junctions form between rat endometrial stromal cells as they undergo decidualization. We have examined the steady-state levels of the gap junction transcripts, connexins 26 and 43 (cx26 and cx43), during artificially induced decidualization in vivo and found that they have a temporal pattern similar to that observed in pregnancy. An in vitro model of decidualization was then used. Endometrial stromal cells from rat uteri sensitized for decidualization were cultured for 24, 48, or 72 h before total RNA was extracted and subjected to Northern blot analyses to determine the steady-state levels of cx26 and cx43 transcripts. The analyses revealed that cx26 transcript steady-state levels decreased, whereas those for cx43 increased, from 24 to 72 h. Using an anti-cx43 antibody, punctate immunofluorescent signals were observed around the periphery of the cells, suggesting that cx43 had been assembled into membrane plaques. The presence of functional gap junctions between the cells was determined in vitro by two dye-coupling methods: preloading and scrape-loading. Calcein (995 Da) and a membrane-bound dye, dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (933 Da), were preloaded into 5% of the endometrial stromal cells before plating. The percentage of preloaded cells that transferred calcein to adjacent cells increased from 10% at 3 h after plating to 40% at 6 h. To determine whether or not cells maintain the ability to dye-couple throughout the culture period, carboxyfluorescein (CF; 376 Da) and rhodamine dextran (9.3 kDa) were introduced into cells by scraping the cells with a scalpel, and the distribution of dyes was determined 20 min later. In cells cultured for 24, 48, or 72 h, only CF was transferred to cells distal to the scrape line. The results from these experiments suggest that stromal cells can dye-couple throughout the culture period (3-72 h) and indicate that functional gap junctions form between endometrial stromal cells as they undergo decidualization in vitro.
Biology of Reproduction 05/1996; 54(4):905-13. · 4.01 Impact Factor
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ABSTRACT: Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.
Experimental Cell Research 02/1996; 222(1):48-53. · 3.58 Impact Factor
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ABSTRACT: We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.
BioTechniques 04/1995; 18(3):490-7. · 2.67 Impact Factor
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ABSTRACT: C6 glioma cells do not express the gap junction protein connexin32 or its corresponding mRNA. Very low levels of connexin43 protein and mRNA, as well as weak intercellular coupling, have been detected. Studies investigating the role of gap junctions in cell proliferation and tumorigenesis have shown that C6 cells transfected with connexin43 have increased levels of intercellular coupling and reduced cell growth (D. Zhu et al., Proc. Natl. Acad. Sci. USA, 88:1883-1887, 1991). To determine whether this growth inhibition is observed with other connexins, a full-length cDNA for connexin32 was used to transfect C6 cells. A number of transfected clones, expressing various levels of connexin32 mRNA, were obtained. Further analysis of several of these clones has shown that they have a corresponding increase in both the amount of connexin32 immunoreactivity and intercellular coupling. Thus, transfection of the C6 glioma cell line with connexin32, a gene which is normally expressed in the rat brain but not in C6 cells, produces both a functional mRNA and protein. Growth of the transfected clones was reduced in vivo. In vitro, growth of the various clones was not correlated to either levels of connexin32 expression or intercellular coupling. This is in contrast to findings in the previous study, in which cell growth was reduced in response to connexin43 expression both in vivo and in vitro in the transfected cells. These clones provide a unique system to study the role of gap junctions in cell proliferation and other tumor characteristics.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 03/1994; 5(2):179-86.
