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PubMed1991

Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation.

A spontaneously immortalized clonal granulosa cell line (SIGC) derived from primary rat ovarian granulosa cell cultures was developed as a model system to explore the process of transformation using an epithelial cell type. SIGC has an epithelial morphology and grows in culture without undergoing luteinization. The cell line is thought to represent an intermediate step in carcinogenesis because it seems to grow indefinitely in culture but does not form clones in soft agar or tumors in nude mice. Indirect immunofluorescence and Western blot analysis verified the constitutive expression of the recessive oncogene product p53 in the cell line, thereby suggesting a possible mechanism of immortalization. Ultrastructural studies indicated that SIGC cells are characterized by an undifferentiated phenotype with prominent intermediate filaments, desmosomes, and gap junctions. The identification of cytokeratin by indirect immunofluorescence and Western blot analysis suggests that SIGC functions as an epithelial cell type. Functional studies of cell-cell communication by a dye transfer technique (fluorescence recovery after photobleaching) showed reduced communication compared to normal primary granulosa cells in culture. SIGC cells were transfected with early region genes of SV40 virus in an attempt to generate fully transformed cell lines. The resulting cell line SV-SIGC expressed T-antigen, was anchorage independent, formed tumors in nude mice, and had reduced intercellular communication as compared to SIGC cells. Explants from the tumors in nude mice were used to generate another cell line (T-SV-SIGC), which exhibited further reduction in both the incidence and the rate of communication. These results clearly demonstrated a progressive loss of functional communication during multistep transformation of an ovarian cell type. These data demonstrate that this assay system based on an epithelioid cell type can be used to study the relationship between intercellular communication and the multistep process of carcinogenesis.

Animals↗
PubMed2002

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.

Animals↗
PubMed1998

Gap junction disappearance in astrocytes and leptomeningeal cells as a consequence of protozoan infection.

Trypanosoma cruzi and Toxoplasma gondii are protozoan parasites capable of causing infections of the nervous system. In order to determine effects of infection by these organisms on intercellular communication in the brain, dye coupling and connexin abundance and distribution were examined in leptomeningeal cells and astrocytes infected with T. cruzi or T. gondii. For both cell types infected with either type of protozoan parasite, intercellular diffusion of intracellularly injected Lucifer Yellow was dramatically reduced. Immunocytochemistry with antibodies specific for connexin43 (in astrocytes) or both connexin43 and connexin26 (for leptomeningeal cells) demonstrated that punctate gap junctional staining was much reduced in infected cells, although uninfected neighbors could display normal connexin abundance and distribution. Western blot analyses revealed that connexin43 abundance in both cell types infected with either parasite was similar to that in uninfected cells. Phosphorylation state of connexin43 (inferred from electrophoretic mobility of connexin43 isoforms) was not significantly affected by the infection process. Immunocytochemistry of whole brains from animals acutely infected with either parasite also showed a marked reduction in connexin43 expression. We conclude that infection of both types of brain cells with either protozoan parasite results in a loss of intercellular communication and organized gap junction plaques without affecting expression levels or posttranslational processing of gap junction proteins. Presumably, these changes in gap junction distribution result from altered targeting of the junctional protein to the plasma membrane, and/or from changes in assembly of subunits into functional channels.

Animals↗
PubMed2004

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

Adaptation, Physiological↗
PubMed2005

Bystander effects may modulate ultraviolet A and B radiation-induced delayed mutagenesis.

Ultraviolet irradiation of cells can induce a state of genomic instability that can persist for several cell generations after irradiation. However, questions regarding the time course of formation, relative abundance for different types of ultraviolet radiation, and mechanism of induction of delayed mutations remain to be answered. In this paper, we have tried to address these questions using the hypoxanthine phosphoribosyl transferase (HPRT) mutation assay in V79 Chinese hamster cells irradiated with ultraviolet A or B radiation. Delayed HPRT(-) mutations, which are indications of genomic instability, were detected by incubating the cells in medium containing aminopterin, selectively killing HPRT(-) mutants, and then treating the cells with medium containing 6-thioguanine, which selectively killed non-mutant cells. Remarkably, the delayed mutation frequencies found here were much higher than reported previously using a cloning method. Cloning of cells immediately after irradiation prevents contact between individual cell clones. In contrast, with the present method, the cells are in contact and are mixed several times during the experiment. Thus the higher delayed mutation frequency measured by the present method may be explained by a bystander effect. This hypothesis is supported by an experiment with an inhibitor of gap junctional intercellular communication, which reduced the delayed mutation frequency. In conclusion, the results suggest that a bystander effect is involved in ultraviolet-radiation-induced genomic instability and that it may be mediated in part by gap junctional intercellular communication.

Aminopterin↗
PubMed2003

Expression of CONNEXIN43 is highly sensitive to ionizing radiation and other environmental stresses.

To gain a greater understanding of the mechanisms underlying the cellular responses to low-dose ionizing radiation, gene expression profiles were examined by microarray analysis of cDNA from confluent human diploid fibroblast cultures exposed to very low fluences of alpha-particles. The data, supported by Northern and Western analyses, indicate that radiation induces a significant up-regulation of CONNEXIN43 expression. This phenomenon was observed in a variety of irradiated cell types. These findings are consistent with our previous observations that connexin43 (cx43)-mediated gap-junction intercellular communication is involved in the bystander response observed in cell cultures exposed to fluences of alpha-particles by which only a very small fraction of the cell nuclei is traversed by a particle track (E. I. Azzam et al., Proc. Natl. Acad. Sci. USA, 98: 473-478, 2001). Increased mRNA levels in cells from irradiated cultures correlated with increased cx43 protein levels by approximately 4 h after irradiation. The induction of cx43 was observed by mean alpha-particle doses as low as 0.16 cGy, and also in cells exposed to gamma-rays, t-butyl hydroperoxide, and hyperthermia. Exposure to these stresses also resulted in post-translational modification of cx43; increased phosphorylation and hyperphosphorylation of the protein was observed. Up-regulation of cx43 expression in ionizing radiation exposed cells correlated with functional communication through gap junctions, as evidenced by dye transfer from irradiated to nonirradiated cells. In contrast, the response after UV radiation varied and was cell type-dependent. Overall, these data suggest a critical role for genes involved in intercellular communication in mediating the cellular responses to a variety of stresses.

Alpha Particles↗
PubMed2005

Modulation of connexin43 alters expression of osteoblastic differentiation markers.

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43(-)). hFOB/Cx43(-) cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43(-) cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43(-) cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor alpha1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43(-). Osteopontin mRNA levels were increased in hFOB/Cx43(-) relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43(-) and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells.

Alkaline Phosphatase↗
PubMed1994

Limb mesenchymal cells inhibited from undergoing cartilage differentiation by a tumor promoting phorbol ester maintain expression of the homeobox-containing gene Msx1 and fail to exhibit gap junctional communication.

Tumor promoting phorbol esters are potent inhibitors of the chondrogenic differentiation of limb mesenchymal cells, but the mechanism by which these agents elicit their antichondrogenic effect is unknown. Here we report that limb mesenchymal cells inhibited from undergoing chondrogenesis by a tumor promoting phorbol ester exhibit deregulated expression of the homeobox-containing gene Msx1, a gene implicated in suppressing differentiation of limb mesenchymal cells, and fail to exhibit the extensive gap junctional intercellular communication that normally occurs at the onset of chondrogenesis. These results suggest that tumor promoting activity of phorbol esters may relate to their ability to modulate the expression of regulatory genes involved in controlling terminal differentiation, as well as to their ability to disrupt the intercellular communication involved in initiating the differentiated phenotype of cells.

Animals↗
PubMed1991

PDGF and the small inducible gene (SIG) family: roles in the inflammatory response.

A growing body of evidence suggests that cells responding to an initial growth, inflammatory or immune signal can respond by inducing the transcription of selective members of the SIG family. This family of related inducible cytokines are involved in the amplification, propagation and coordination of intercellular communication among cell types involved in the immune and inflammatory responses. The principal anti-inflammatory action of glucocorticoids may involve the transcriptional and translational inhibition of cytokines such as JE and other members of the SIG family to effectively disrupt the normal lines of intercellular communication which normally coordinates the immune and inflammatory response. The identification of new members of the family and the discovery of the functions of the known members will lead to a clearer understanding of the complicated processes which lead to normal and pathological immune and inflammatory responses.

Cytokines↗
PubMed2001

Antioxidants prevent gamma-hexachlorocyclohexane-induced inhibition of rat myometrial gap junctions and contractions.

Lindane (gamma-hexachlorocyclohexane) is a commonly used pesticide that bioaccumulates in mammalian adipose tissue. Lindane inhibits gap junctional intercellular communication and oscillatory contractions of pregnant rat myometrium in vitro. The present study investigated the role of oxidative stress in lindane's inhibition of myometrial function in mid-gestation pregnant rat uteri. Lucifer yellow dye was microinjected into cultured myocytes to assess gap junctional intercellular communication. Lindane exposure (100 microM) resulted in a time-dependent, biphasic inhibition of dye transfer. This pattern of inhibition was also seen upon cell exposure to the pro-oxidant, tert-butyl hydroperoxide (100 microM). Lindane's initial and secondary-onset dye transfer inhibitions were reversed by cotreatment and pretreatment with the antioxidants, alpha-tocopherol (25-100 microM), diphenyl-1,4-phenylene diamine (10-30 microM), and superoxide dismutase (100-400 U/ml). D-mannitol (100-300 mM) also reversed lindane's initial dye transfer inhibition. Nitro blue tetrazolium reduction to formazan (measured spectrophotometrically) was elevated upon exposure of cultured cells to lindane or tert-butyl hydroperoxide, indicating the presence of reducing agents. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was also elevated in lindane-exposed cell cultures. alpha-Tocopherol reversed this elevation. Finally, uterine contractility was assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. Pretreatment with alpha-tocopherol prevented lindane's abolishment of uterine contractions in vitro. These data support the hypothesis that lindane inhibits uterine contractility and myometrial gap junctions by establishing an oxidative stress environment.

Animals↗
PubMed1986

Extracellular matrix regulation of cell-cell communication and tissue-specific gene expression in primary liver cultures.

Epithelial-mesenchymal interactions are effected, in part, by extracellular matrix components. We have spent many years analyzing the influence of extracellular matrix, both as extracts of matrix and as purified matrix components, on the growth and differentiation of normal and neoplastic liver cells. Currently we are focused on analyzing the influence of the extracellular matrix components, glycosaminoglycans and proteoglycans. We have found that these factors induce dramatic morphological changes, are potent inducers of gap junction synthesis and can regulate tissue-specific gene expression. With respect to gap junctions: intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 hrs in primary rat hepatocytes cultured in serum supplemented media or within 24 hrs in cells in a serum free, hormonally defined medium designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. Addition of proteoglycans or glycosaminoglycans to hormonally defined medium after 24 hrs resulted in reexpression of electrical and dye coupling when assayed at 96 hrs of culture. The incidence of coupling was less than 5% in hormonally defined medium alone. Coupling incidence increased to 10-30% with the addition of 10 micrograms/ml of glycosaminoglycans (i.e., hyaluronic acid, dermatan sulfate, chondroitin 4- or 6-sulfate, and iota- or kappa-carrageenan) to hormonally defined medium. By contrast, the same concentrations of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or lambda-carrageenan resulted in dye coupling in more than 70% of the cells, with numerous cells showing dye spread from a single injected cell (in the case of the proteoglycans). The greatest effect of those tested was elicited by the dermatan sulfate proteoglycans, which induced cell-cell communication in 90-100% of the cells. Heparins gave intermediate responses (30-50%). Western blots demonstrated that the amounts of the main intrinsic gap junction polypeptide (27 KDa) extractable from cells correlated with the degree of electrical and dye coupling. Thus, proteoglycans and glycosaminoglycans appear to elicit the formation and function of gap junctions and may thus play a role in the regulation of intercellular communication under normal and pathological conditions. With respect to gene expression: normal rat hepatocytes maintained in culture on tissue culture plastic and in serum supplemented medium lose their tissue-specific functions within hours to a few days due to loss of synthesis and to rapid degradation of tissue-specific mRNAs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Regulation of the blood-biliary barrier: interaction between gap and tight junctions in hepatocytes.

Hepatocytes tightly connect with each other by intercellular junctions to form liver cell plates. The junctions composed of gap, tight, and adherens junctions and desmosomes concentrate around bile canaliculi. In particular, tight junctions serve as a barrier to keep bile in bile canaliculi away from the blood circulation. Thus, it is very reasonable to call tight junctions of hepatocytes the blood-biliary barrier. On the other hand, gap junctions of hepatocytes are considered to enable ordered contraction of bile canaculi from centrizonal to periportal hepatocytes by their function of intercellular communication. Gap and tight junctions may thus play a crucial role in bile secretion, one of the most differentiated functions of the liver. In intrahepatic cholestasis, a common pathological condition of the liver, downregulation of gap and tight junctional functions is seen, which results in impaired intercellular communication and in leaky tight junctions. Although the changes in gap and tight junctions had been considered to be independent of each other, recent findings that the tight junction-associated proteins ZO-1 and occludin bind to connexins indicate the possibility of either coordinate or reciprocal regulation of macromolecular complexes containing gap- and tight-junction proteins. In this review, we introduce the interaction and regulation between gap and tight junctions of hepatocytes in vitro and discuss the regulatory mechanisms of the "blood-biliary barrier" to study the molecular pathogenesis of cholestasis.

Actins↗
PubMed2000

Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone.

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.

Animals↗
PubMed1994

[Reflexions on the usage of information theory in biology].

For living beings, information is as fundamental as matter or energy. In this paper we show: a) inadequacies of quantitative theories of information, b) how a qualitative analysis leads to a classification of information systems and to a modelling of intercellular communication. From a quantitative point of view, the application in biology of information theories borrowed from communication techniques proved to be disappointing. These theories ignore deliberately the significance of messages, and do not give any definition of information. They refer to quantities, based upon arbitrarily defined probabilistic events. Probability is subjective. The receiver of the message needs to have 'meta-knowledge' of the events. The quantity of information depends on language, coding, and arbitrary definition of disorder. The suggested objectivity is fallacious. In common language, the word 'information' is synonymous with knowledge of order. Following common sense a message (letters, coded signals, etc.) is information just in case it is interpretable, i.e. if it fits to a previously acquired meaning (the words of an available language, etc.). The consequence is that calculation of quantities in the sense of Shannon can be used for transmissions, but it is itself meaningless (has no significance). In linguistics and semantics, information is composed of a 'signifier', a physical medium (letters, coded signals, etc.), and a 'signified' or significance. The nature of information is complex. The laws of linguistics and semantics are valid not only at the human, organismic level, but also at the cellular and molecular level. The physiology of sensations gives us many examples for application of a concept of information An electromagnetic wave of 0.7% give us the sensation of a red colour. Sensations have no physical reality. They are purely subjective. At the cellular level communication operates by means of chemical messengers (first messengers), which generally do not penetrate the plasmic membrane. Specific captors operate as transductors: external factors are converted into secondary messengers (cyclic AMP, Ca ion, etc.). Sometimes, electric signals (like depolarization waves) may also play a part in the intercellular communication. Such processes are characterized by changes in a sequence of different molecules carried by a physical signal. What is transmitted is the meaning of the message (significance) which can be memorized by the cell, providing a possible following use. At the molecular level one can find also the processes of linguistic nature. We know that the significance of a word is changed with changing the order of letters (ADD-->DAD, etc.).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1.

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.

Animals↗
PubMed1988

Transforming growth factor beta as a potent promoter in two-stage BALB/c 3T3 cell transformation.

We have tested transforming growth factor beta (TGF beta) in the two-stage BALB/c 3T3 cell transformation assay for possible tumor-promoting activity, since it has several effects similar to those of tumor-promoting phorbol esters. After initiation of BALB/c 3T3 cells with 3-methylchol-anthrene, treatment with TGF beta at 1 ng/ml alone or in combination with epidermal growth factor (EGF) for 4 weeks enhanced the number of transformed foci by 5- to 6-fold in comparison with uninitiated cells. Initiation treatment alone induced no or very few transformed foci in several assays. Treatment with phorbol-12,13-didecanoate (PDD) at 100 ng/ml for 4 weeks enhanced the number of transformed foci in initiated BALB/c 3T3 cells by 4- to 5-fold in comparison with uninitiated cells. Thus, TGF beta at 1 ng/ml is as potent as PDD at 100 ng/ml for tumor-promoting activity in the two-stage BALB/c 3T3 cell transformation assay. The enhancing effect of TGF beta was dose-related in the dose range tested (0.03-1 ng/ml) and was not reversible. Some of the foci induced by combined MCA-TGF beta-EGF treatment were cloned, and eight out of nine clones tested produced tumors in nude mice. TGF beta (1 ng/ml) plus EGF (2 ng/ml) increased the saturation density to a similar extent as PDD (100 ng/ml) but did not affect the growth of BALB/c 3T3 cells. We observed no change in junctional intercellular communication, as measured by the dye transfer method, when cells were treated with TGF beta during the two-stage BALB/c 3T3 cell transformation assay. Nevertheless, there was selective communication between transformed and surrounding nontransformed cells; MCA-TGF beta transformed cells intercommunicated among themselves but not with surrounding nontransformed cells. Our results indicate that TGF beta has potent tumor-promoting activity in vitro, but that this activity is not mediated by a complete blockage of intercellular communication, as is suggested for phorbol ester tumor promoters.

Animals↗
PubMed2001

Diabetes-induced disruption of gap junction pathways within the retinal microvasculature.

PURPOSE: Microvascular damage caused by diabetes is a leading cause of visual loss. Identifying events early in the course of diabetic retinopathy may help in understanding and, perhaps, preventing this disorder. The hypothesis that cell-to-cell communication within the retinal microvasculature may be affected soon after the onset of diabetes was tested. METHODS: Streptozotocin was used to induce diabetes in rats. To assess cell-to-cell coupling the gap junction-permeant tracer, Neurobiotin, was delivered via patch pipettes into pericytes located on microvessels freshly isolated from the retinas of diabetic and control animals. Subsequently, immunohistochemical methods revealed the extent of the intercellular spread of the tracer. Electrophysiological methods were also used to detect intercellular communication. RESULTS: In retinal microvessels of control rats, Neurobiotin spread hundreds of micrometers from the tracer-loaded pericytes. However, within days after the onset of diabetes, this cell-to-cell coupling was dramatically reduced. In contrast, microvessels of insulin-treated diabetic rats showed no significant loss of intercellular communication. Consistent with protein kinase C (PKC) playing a role in the diabetes-induced inhibition of gap junction pathways, exposure of microvessels to a PKC activator (phorbol myristate acetate) markedly reduced tracer coupling. CONCLUSIONS: Within retinal microvessels there is extensive cell-to-cell coupling, which is markedly reduced soon after the onset of streptozotocin-induced diabetes. The closure of gap junction pathways disrupts the multicellular organization of retinal microvessels and may contribute to vascular dysfunction.

Animals↗
PubMed1996

Ras is involved in gap junction closure in proliferating fibroblasts or preadipocytes but not in differentiated adipocytes.

A decrease in gap junctional, intercellular communication (GJIC) has been associated with cells neoplastically transformed by a variety of factors. To investigate the role of the Ras oncogene product in gap junction function, a panel of murine C3H10T1/2 (10T1/2) fibroblasts was constructed in which the levels of ras gene expression could be effectively up- or down-regulated. Intercellular communication was measured using a novel technique of in situ electroporation of adherent cells on a partly conductive slide. The introduction of increasing amounts of activated Ras(leu61) in mouse 10T1/2 fibroblasts proportionally reduced GJIC, while the downregulation of endogenous c-ras gene expression increased junctional permeability. These results indicate that Ras plays an important role in the junction closure pathway leading to the proliferation of normal cells. However, differentiation of c-Ras-deficient preadipocytes entirely abolished their initially extensive GJIC, indicating that junction closure in response to adipocytic differentiation is independent of Ras.

Adipocytes↗