Decrease In Gap Junctional Intercellular Communication Research Articles

IL-22 Down-Regulates Cx43 Expression and Decreases Gap Junctional Intercellular Communication by Activating the JNK Pathway in Psoriasis

The roles of IL-22 in the pathomechanisms of psoriasis have been well demonstrated. Gap junctional intercellular communication (GJIC) is widely known for its involvement in multiple biological and pathological processes such as growth-related events, cell differentiation, and inflammation. Here, we show that IL-22 significantly decreased GJIC and down-regulated Cx43 expression in HaCaT cells. Cx43 overexpression markedly inhibited the proliferation of and increased GJIC in HaCaT cells, but the silencing of Cx43 exerted the opposite effects. Additionally, Cx43 overexpression effectively rescued the IL-22-induced decrease in GJIC in HaCaT cells. The IL-22-induced down-regulation of Cx43 expression and decrease in GJIC can be significantly blocked by the JNK inhibitor SP600125 and by the overexpression of IL-22RA2 (which specifically binds to IL-22 and inhibits its activity), but not by the NF-κB inhibitor BAY11-7082, in HaCaT cells. Furthermore, the IL-22-induced down-regulation of Cx43 expression mediated by the JNK signaling pathway was confirmed in a mouse model of IL-22-induced psoriasis-like dermatitis. Similarly, Cx43 expression was significantly lower in the lesional skin than in the nonlesional skin of patients with psoriasis. These results suggest that IL-22 decreases GJIC by activating the JNK signaling pathway, which down-regulates Cx43 expression; this process is a possible pathomechanism of keratinocyte hyperproliferation in psoriasis.

Temporal regulation of gap junctional communication between tenocytes subjected to static tensile strain with physiological and non-physiological amplitudes

The present study has been performed on temporal changes in gap junctional intercellular communication (GJIC) between tenocytes under static tensile strain with the magnitude of 0% (no strain), 4% (physiological magnitude) or 8% (overloading magnitude) during a 24-h culture period. Tenocytes were isolated from rabbit Achilles tendon and seeded on a stretchable microgroove substrate. GJIC was evaluated as intercellular diffusion coefficient of calcein (DGJ) using fluorescence loss in photobleaching (FLIP) protocol accompanied with a mathematical model of molecular diffusion both within the cell and between the cells. It was exhibited that the application of 4% strain for 1 h increased DGJ significantly. The increased level was maintained for 6 h, followed by returning to the pre-strain level at 24 h. This was associated with a transient increase in connexin 43 (Cx43) gene expression and protein localisation at 1 h, suggesting the increased GJIC may have involved new synthesis of gap junctions. By contrast, the application of 8% static strain reduced DGJ to the similar or lower level from 0% strain group for 6 h, associated with inhibited Cx43 gene expression. However, Cx43 protein localisation was not changed much, and thus, there seem no direct interactions among changes in GJIC, Cx43 gene expression and Cx43 localisation. The present findings highlight the differences in mechanical regulation of GJIC between physiological and non-physiological loadings, and thus the increase or the decrease in GJIC may affect tenocyte functions in different ways.

Inorganic arsenic trioxide induces gap junction loss in association with the downregulation of connexin43 and E-cadherin in rat hepatic “stem-like” cells

Chronic exposure to inorganic arsenic trioxide causes tumors of the skin, urinary bladder, lung, and liver. Several cancer initiators and promoters have been shown to alter cell–cell signaling by interference with gap junction intercellular communication (GJIC) and/or modulation of cell adhesion molecules, such as connexin43 (Cx43), E-cadherin, and β-catenin. The aim of this study was to determine whether the disruption of cell–cell interactions occurs in liver epithelial cells after exposure to arsenic trioxide. WB-F344 cells were treated with arsenic trioxide (6.25–50 μM) for up to 8 hours, and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, the changes in mRNA and protein levels of Cx43, E-cadherin, and β-catenin were determined. A significant dose- and time-dependent decrease in GJIC was observed when WB-F344 cells were exposed to arsenic trioxide (p < 0.05). Consistent with the inhibition of GJIC, cells' exposure to arsenic trioxide resulted in dose- and time-dependent decreases in Cx43 and E-cadherin mRNA expression and protein levels. However, arsenic trioxide did not alter the mRNA or protein levels of β-catenin. In an immunofluorescence study, nuclei were heavily stained with anti-β-catenin antibody, indicating significant nuclear translocation. In this study, we also demonstrated that arsenic trioxide-induced GJIC loss was a reversible process. Taken together, these data support the hypothesis that disruption of cell–cell communication may contribute to the tumor-promoting effect of inorganic arsenic trioxide.

Down‐regulation of connexin43 expression reveals the involvement of caveolin‐1 containing lipid rafts in human U251 glioblastoma cell invasion

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

Apoptosis and inhibition of gap-junctional intercellular communication induced by LA-12, a novel hydrophobic platinum(IV) complex

A new hydrophobic platinum(IV) complex, LA-12, a very efficient anticancer drug lacking cross-resistance with cisplatin (CDDP), is now being tested in clinical trials. Here we investigated the apoptogenic activity of LA-12 and its effect on gap-junctional intercellular communication (GJIC) in the rat liver epithelial cell line WB-F344. LA-12 induced apoptosis much more efficiently than did CDDP due to a combination of rapid penetration into the cell and attack on DNA, leading to fast activation of p53 and caspase-3. Exposure of WB-F344 cells to LA-12 led to rapid induction of the time- and dose-dependent decrease in GJIC. On the molecular level, loss of GJIC induced by LA-12 was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated by the use of inhibitors of ERK activation. Inhibition of GJIC was linked to rapid hyperphosphorylation of connexin-43 and disappearance of connexon clusters from membranes, which was not observed in the case of CDDP.

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.

Repeated Simulated Ischemia and Protection Against Gap Junctional Uncoupling

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The aim of this study was to investigate the effect of early and delayed preconditioning on gap junction communication, connexin abundance, and phosphorylation in cultured neonatal rat cardiac myocytes. Prolonged ischemia followed 5 minutes after preconditioning in the early protocol, whereas 20 hours separated preconditioning and prolonged ischemia in the delayed preconditioning protocol. Gap junctional intercellular communication (GJIC) was assessed by Lucifer yellow dye transfer. An initial reduction in communication in response to sublethal ischemia was observed. This may be one mechanism whereby neighboring cells are protected from damaging substances produced during the first phase of subsequent regional ischemia in early preconditioning protocols. With respect to delayed preconditioning, the transient decrease in GJIC disappeared prior to prolonged ischemia, indicating that other mechanisms are responsible for delayed protection. Both early and delayed preconditioning preserved intercellular coupling after prolonged ischemia and this correlated with presence of less connexin43 dephosphorylation assessed by immunoblot.

Effects of Gingko biloba Extract on Gap Junction Changes Induced by Reperfusion/Reoxygenation After Ischemia/Hypoxia in Rat Brain

Gap junction communication between astrocytes plays an important role in the brain. The purpose of this study was to investigate the effects of Gingko biloba extract (GBE) on the changes of connexin 43 (Cx43) mRNA and protein expression levels of rat cortex and hippocampus induced by ischemia-reperfusion and astrocyte gap junction intercellular communication (GJIC) induced by hypoxia-reoxygenation. After 2 hours of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion, there was obvious neurological deficit in rats. Cx43 mRNA and protein expression levels of rat cortex and hippocampus in the ischemia hemisphere were decreased significantly. When GBE at doses of 50 and 100 mg/kg body weight was administrated by p.o. daily for 7 days, the neurological deficit was improved, and lower Cx43 mRNA and protein expression levels induced by ischemia-reperfusion were recovered to normal. The i.p. injection of nimodipine (0.7 mg/kg weight body) also showed improvement on neurological deficit and Cx43 expression levels. Astrocyte GJIC was measured by the fluorescence recovery after photobleaching (FRAP). Hypoxia-reoxygenation induced a significant decrease in GJIC. Pretreatment with GBE (100 mg/l) and nimodipine (1.6 mg/l) significantly prevented the hypoxia-reoxygenation inhibition of GJIC. These results suggest that GBE could exert its neuroprotective effects by improvement of Cx43 expression and GJIC induced by hypoxia/ischemia-reoxygenation/ reperfusion injury.

Effects of cadmium on gap junctional intercellular communication in WB-F344 rat liver epithelial cells.

Cadmium has been associated with a number of tumors but its role in tumor promotion has not been elucidated clearly or the results obtained from various studies have been conflicting. This study was designed to investigate the effects of cadmium on the gap junctional intercellular communication (GJIC), number of gap junctions per cell, and cell proliferation in WB-F344 rat liver epithelial cells from the viewpoint of tumor promotion. GJIC was monitored by counting the cells stained with Lucifer yellow CH dye, using the scrape-loading and dye-transfer method. The numbers of gap junctions per cell were visually quantitated after an indirect immunostaining for gap junction protein using an antibody to connexin 43. Cell proliferation was assayed by direct counting of the living cells using the trypan blue dye exclusion method. In the time course study, cells treated with 200 microM CdCl2 showed rapid and nearly complete inhibition of GJIC (approximately 14% of the control) and a decrease in the number of gap junctions per cell (approximately 21% of the control) at 30 min, and the decrease continued up to 4 h without any changes in the cell viability. Treatment with CdCl2 (7.4-200 microM) for 4 h resulted in the decrease of GJIC and gap junction numbers per cell in a dose-response pattern without changes in the cell viability. In the long-term (14 days) exposure studies at doses of 0.01-7.4 microM CdCl2, an increase in cell proliferation was observed at low doses of 0.03-2.5 microM CdCl2, with GJIC also decreasing. These data demonstrate that cadmium inhibits GJIC, reduces the number of gap junctions per cell, and induces cell proliferation while decreasing the function of the gap junction.

Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells: relation to down-regulation of gap junctional intercellular communication

Pentachlorophenol (PCP), a promoter of murine hepatocarcinogenesis, inhibits gap junctional intercellular communication (GJIC) in rat liver epithelial cells in vitro. To test the hypothesis that both inhibition of GJIC and apoptosis contribute to tumor promotion, we investigated the effect of PCP on both GJIC and serum deprivation-induced apoptosis in v-myc-transfected rat liver epithelial cells. The results showed that PCP inhibited apoptosis, as measured by the TUNEL assay and DNA ladder formation. Inhibition of apoptosis was associated with a decrease in GJIC. The study demonstrated that PCP has a potential for inhibiting apoptosis and GJIC, supporting the hypothesis.

Hypoxia-reoxygenation inhibits gap junctional communication in cultured human umbilical vein endothelial cells.

We studied the change in gap junctional intercellular communication (GJIC) on human umbilical vein endothelial cells (HUVEC) under hypoxia-reoxygenation (H-R) conditions by the fluorescence redistribution after photobleaching (FRAP) method. Confluent HUVEC monolayers were exposed to hypoxia (pO2<0.1%) for 12 hours, and then were returned to normal atmospheric conditions for reoxygenation. Contrast microscopic observation showed no significant changes in the morphology of the HUVEC at any times after H-R. Reoxygenation following hypoxia caused time-dependent decrease in GJIC, that is, GJIC reduction was induced after 2 hours and reached maximum at 4-6 hours which recovered to normal levels after 18 hours. Oxidant sensitive fluorescence dye assay revealed that the generation of intracellular free radicals increased during the first 2 hours after reoxygenation. Hydroxyl radical scavengers (MCI-186, DMSO) and an iron chelator (deferoxamine) abolished the reduction of GJIC due to H-R. However, SOD, catalase and probucol were essentially inactive on this reduction. These data suggest that ischemia-reperfusion injury may be caused by a functional defect of GJIC induced by reactive oxygen radicals.

A characterization of permolybdate and its effect on cellular tyrosine phosphorylation, gap junctional intercellular communication and phosphorylation status of the gap junction protein, connexin43

Biological and analytical characterizations of permolybdate (a mixture of H 2O 2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H 2O 2 (3–10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H 2O 2:molybdate). The mixtures of molybdenum(V) chloride and H 2O 2 gave a maximum effect at 4:1 molar ratio (H 2O 2:molybdenum(V)). This can be explained by decomposition of H 2O 2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H 2O 2 to Mo(V) solutions, and no spectrum appeared when H 2O 2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H 2O 2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.

Changes in gap junctional intercellular communication in mouse skin carcinogenesis.

Gap junction intercellular communication (GJIC) has been measured in cell lines that represent different stages of chemically induced mouse skin carcinogenesis. No significant difference in GJIC, as measured by dye spread, was found in cultures of normal keratinocyte, papilloma or squamous carcinoma cell lines. There was no correlation, in this system, between the presence of a mutant Ha-ras gene and down-regulation of communication. There was, however, a marked decrease in GJIC (80-90%) on progression from squamous to spindle carcinoma cells. Measurement of GJIC in somatic cell hybrids shows that the genetic defect responsible for this down-regulation is recessive and is common to two independently isolated spindle cell lines. No abnormalities were found in the spindle cells in expression of connexin 43, a cell component involved in gap junction formation and permeability. However, expression of E-cadherin, a cell-cell adhesion molecule implicated in the process of gap junction formation, was missing in the spindle carcinoma cells. Introduction of an E-cadherin cDNA into the spindle cells partially restored junctional communication without causing any noticeable alterations in cell morphology. During the study a non-tumourigenic keratinocyte line, a sub-clone of a normal keratinocyte line, was also found to have a low level of GJIC. However, the defect in this line was shown, by genetic complementation in somatic cell hybrids, to be different from that in the spindle carcinoma cell lines. Consistent with these data, analysis by immunofluorescence shows an abnormal distribution of connexin 43 in these cells.

Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells.

Modulation of gap junctional intercellular communication (GJIC) was studied in a multistep model of human renal epithelial carcinogenesis. We report that the majority of primary human kidney epithelial cells (NHKE) grown from fetal kidney explants did not communicate through gap junctions. Communication could, however, be observed within a subpopulation of the cells. Ni(II)-immortalized cells (IHKE) showed GJIC at a level of 10-20 communicating cells, but with heterogeneous regions on the dish, with regard to both communication and distribution of connexin43. The heterogeneity was less pronounced in a ras-transfected tumourigenic cell line (THKE), which also showed communication of approximately 10-20 dye-coupled cells. Communication within the IHKE sub-clone K7 decreased from 55 dye-coupled cells communicating on day 1 after seeding to approximately 13 in cells grown for 4 days. Daily change of growth medium reduced the decrease in GJIC. EGF enhanced communication following a lag period which depended on days in culture. The largest increase in GJIC was observed in 2-day-old cultures, where the number of communicating cells in some experiments increased from approximately 45 to 130 dye-coupled cells 4 h following change to medium with EGF. The induction was concentration dependent and communication was enhanced gradually between 2 and 7 h after exposure to EGF. A 15 min pulse of EGF was sufficient to induce the GJIC increase if the total incubation period was unchanged. Cycloheximide completely blocked the EGF-induced enhancement of communication, while actinomycin D had no effect. EGF exposure resulted in an increase in the cellular level of connexin43 protein in parallel with the enhancement in communication. Together, these results indicate that the EGF-induced enhancement of GJIC in human kidney epithelial cells was mediated through translational control of connexin43 expression.

Regulation of gap junction intercellular Communication by pH in MC3T3-E1 osteoblastic cells

Gap junction intercellular communication (GJIC) may be related to coordinating the function of osteoblasts during bone mineralization. Since an alkaline pH supports mineral deposition while an acidic pH promotes mineral dissolution, it was investigated whether GJIC is altered by changes in extracellular pH (pHo) Functional GJIC was assessed by fluorescent dye transfer after microinjection, and connexin protein abundance was examined by immunoprecipitation and immunoblotting in MC3T3-E1 cells, a model of osteoblast-like cells. The percent of cells coupled by GJIC was found to be 40.7% (24 of 59 injected cells) at pH 6.9, 72.2% (26 of 36) at pH 7.2, and 92.8% (26 of 28) at pH 7.6. A decrease in GJIC was detectable by 30-60 minutes of exposure to a pHo of 6.9. Decreased gap junction communication was also found in cells after 3, 8, and 24 h of incubation in a bicarbonate-CO2 system at an ambient pH of 6.9. Connexin protein abundance experiments showed that at after exposure to a pH of 6.9 for 2.75 h, the specific band(s) at 41-43 kD were fainter compared with these same band(s) at pH 7.2 and 7.6. There was no significant difference in band densities at pH 7.2 and 7.6. Determination of intracellular pH (pHi) showed that it was similar to pHo after 2.75 h of incubation at each ambient pH. When pHi was clamped at 6.9 or 7.2, there was a time-dependent decrease in the gap junction coupling frequency at a pHi of 6.9 when pHo was 7.2. Steady-state mRNA levels were decreased at pHo 6.9 but were unchanged at either pHo 7.2 or 7.6. Our conclusions are that (1) longer incubations ( > or = 2.75 h) at low pHo decrease GJIC which in part may be due to a decrease in connexin protein abundance perhaps as a result of a decrease in connexin steady-state mRNA expression; (2) GJIC inhibition or augmentation found at low and high pHo, respectively, suggests that gating of the GJ channel by pH may also occur; (3) pho-induced alterations in GJIC in the MC3T3-E1 osteoblastic model are related to concomitant changes in pHi.

Increased gap junctional intercellular communication in Syrian hamster embryo cells treated with oxidative agents.

The effects of K2CrO4, H2O2, benzoyl peroxide, menadione, KBrO3 and UV365nm on gap junctional intercellular communication (GJIC) have been studied in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive Syrian hamster embryo (SHE) cell line BPNi. All agents were found to increase the level of GJIC by 50-100%. Also, in early passage SHE cells, a tendency for increased GJIC was found for the oxidative agents studied. Hydrogen peroxide was used as a model compound in the subsequent studies. The increase in GJIC was reversible, and it was not due to an increased non-junctional permeability. Hydrogen peroxide counteracted the TPA-induced decrease in GJIC, regardless of whether the cells were exposed to the compounds simultaneously or the cells were pre-exposed to TPA before addition of H2O2. The GJIC enhancement by H2O2 was slightly reduced by the addition of the hydroxyl radical scavenger dimethylsulphoxide or by the inhibition of catalase by amitrole. The cAMP/protein kinase A system is the only characterized signal transduction system that is known to increase GJIC in most cell types. Hydrogen peroxide did not increase the amount of cAMP (or cGMP) in BPNi cells, while forskolin and a phosphodiesterase inhibitor had to increase the cAMP level several-fold to affect GJIC to the same degree as the oxidative agents. Some inhibitors of protein kinase A were assayed for their ability to inhibit the increases in GJIC caused by H2O2 and forskolin. Staurosporine inhibited the forskolin-induced increase in GJIC, with much less effect on the H2O2-induced increase. H8, H88 and H89 had less effect than staurosporine on the forskolin-induced increase in GJIC. The results suggest that the cAMP/protein kinase A system may not be involved in the increase in GJIC caused by H2O2, although this cannot be completely ruled out.

Phosphatase inhibitors, gap junctional intercellular communication and [125I]-EGF binding in hamster fibroblasts.

A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.

Association of viral oncogene-induced changes in gap junctional intercellular communication and morphological transformation in BALB/c3T3 cells.

In order to study the relationship between altered gap junctional intercellular communication (GJIC) and induction of cell transformation by oncogenes, we transfected six viral oncogenes into BALB/c3T3 A31-1-1 cells. BALB/c3T3 cells with v-src, v-ras or polyoma middle T (PyMT) genes grew in soft agar and formed distinct transformed foci in the absence or presence of a vast excess of non-transfected cells. On the other hand, those with v-myc, v-fos or polyoma large T (PyLT) genes expressed less distinctly transformed phenotypes (less transformed morphology, higher saturation density than non-transfected counterparts and less growth in soft agar), and did not form distinct foci in coculture with non-transformed cells. When their homologous GJIC capacities were examined by the microinjection/dye transfer assay, no decrease in GJIC was observed in any of the v-onc-transformed cells. Non-transformed and all v-onc-transformed cell lines expressed similar levels of connexin 43 mRNA. v-myc-, v-fos- and PyLT-transformed cells, but not v-ras-, v-src- and PyMT-transformed cells were able to communicate heterologously with non-transformed cells. Tumor promoting phorbol esters strongly inhibited GJIC of non-transformed and all v-onc-transformed BALB/c3T3 cell lines. In cocultures of v-myc-, v-fos- or PyLT-transformed cells with non-transformed BALB/c3T3 A31-1-1 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the number of transformed foci. However, when these v-onc-transformed cells were co-cultured with non-transformed BALB/c3T3 A31-1-13 cells (which lose GJIC at growth confluence, as if TPA had been added), no morphologically transformed foci appeared. These results suggest that factors other than GJIC are involved in the suppression of oncogene-transformed cells by surrounding normal counterparts.

Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis: direct measurement of communication in vivo

We have developed a simple method to measure gap-junctional intercellular communication (GJIC), by means of microinjection/dye transfer assay, in liver slices freshly removed from the rat. Using this method and immunostaining of connexin 32 (cx32), the major liver gap junction protein, we studied sequential changes of GJIC during chemical hepatocarcinogenesis in male Fischer-344 rats under a modified Solt-Farber protocol (3 weeks 4 day exposure regimen). Four weeks after commencement of the protocol, there was a substantial decrease in GJIC in the liver parenchyma, which was free from focal lesions. The decrease in GJIC persisted up to at least the 15th week of treatment, while a decrease in the number of immunoreactive cx32 spots was evident only at 4 weeks of post-protocol commencement. Most enzyme-altered (GST-P-positive) focal lesions showed markedly lower GJIC and a significantly lower number of cx32-positive spots than surrounding hepatocytes. Most GST-P-positive foci showed a selective lack of GJIC with surrounding heptocytes. Hepatocellular carcinomas arising 1 year after the carcinogenic regimen had significantly reduced communicational capacity accompanied by a large decrease in cx32 expression. These results suggest that a progressive decrease in homologous as well as heterologous GJIC in preneoplastic lesions occurs during rat hepatocarcinogenesis, and that preneoplastic lesions with the most prominent disorders in GJIC may be more likely to develop into carcinomas.