Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells

We examined gap junctional intercellular communication (GJIC)of primary human mesothelial cells and cell lines of asbestos-associatedhuman pleural mesotheliomas, and the effect of asbestos andother mineral fibres on these cells. In homologous cultures,the GJIC capacity of six out of seven tumour cell lines wasmarkedly less than for primary mesothelial cells. This defectin GJIC appeared not to be at the expression level of mRNA andprotein of the gene encoding the 43 kDa gap junction protein.In heterologous cocultures of tumour cells and primary mesothelialcells, however, 80–90% of the tumour cell/normal cellcontacts were functional. Exposure of primary mesothelial cellsto TPA, a phorbol ester tumour promoter, resulted in markedinhibition of GJIC, being an action common to numerous tumourpromoters. Such an effect though was not observed with the carcinogenicmesothelioma-inducing mineral fibres chrysotile and amosite,neither with glass wool. These results suggest that a permanentdefect in GJIC capacity is a common feature of human mesotheliomacells, but how mineral fibres are involved in the process ofmesotheliomagenesis is still unclear.     

Inhibitory effect of testosterone on gap junctional intercellular communication of human transitional cell carcinoma cell lines

A dye transfer method was applied to investigate the effect of testosterone on gap junctional intercellular communication (IC) of two kinds of human transitional cell carcinoma cell lines, JTC-30 and JTC-32. When JTC-30 cells were cultured with testosterone at nontoxic concentrations (17-69 microM), a dose and time dependent inhibition of dye transfer was observed. More than 90% inhibition occurred after exposure to 69 microM testosterone for 96 h. The inhibition was reversed rapidly after testosterone deprivation. Similar results were obtained with JTC-32 cells. 17 beta-Estradiol showed no inhibitory effect on IC of both transitional cell carcinoma cell lines even at toxic levels. Testosterone exhibited no inhibitory effect on IC of human fibroblasts. The inhibitory effect of 5 alpha-dihydrotestosterone was almost similar to that of testosterone. At concentrations examined, cyproterone acetate influenced neither dye transfer nor the inhibitory effect of testosterone, suggesting a mechanism of testosterone action different from that of the known receptor system. Since blockage of IC has been indicated as one reliable evidence for tumor promotion, current results suggest that testosterone is a possible endogenous promoter of the bladder carcinoma and may therefore possibly play a role on the sexually different incidence of bladder carcinoma.     

Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells

Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 microM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox, did not prevent the inhibition of GJIC by H2O2, which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione- sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH.      

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 epithelialcells (NHKE) grown from fetal kidney explants did not communicatethrough gap junctions. Communication could, however, be observedwithin 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 bothcommunication and distribution of connexin43. The heterogeneitywas less pronounced in a ras-transfected tumourigenic cell line(THKE), which also showed communication of     

Altered gap junctional intercellular communication in neoplastic rat esophageal epithelial cells

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

Inhibition of gap junctional intercellular communication between primary human smooth muscle cells by tumor necrosis factor {alpha}

Tumor necrosis factor     

Heavy-ion-induced bystander killing of human lung cancer cells: Role of gap junctional intercellular communication

The aim of the present study was to clarify the mechanisms of cell death induced by heavy-ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy-ions. In this experiment, the limited number of cells (0.0001–0.002%, 5–25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8–14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy-ions was induced mainly by direct cell-to-cell communication, such as GJIC, which might play important roles in bystander responses. ( Cancer Sci 2009; 100: 684–688)     

依托咪酯对神经胶质瘤细胞缝隙连接通讯的影响

背景与目的：细胞缝隙连接(gap junction,GJ)通讯能增强放疗或化疗药物对肿瘤细胞的细胞毒性作用。以往研究发现部分麻醉药物能够改变GJ功能从而影响肿瘤细胞对放疗的敏感性。该研究拟观察依托咪酯对神经胶质瘤细胞由缝隙连接蛋白Cx43组成的缝隙连接功能的影响,为麻醉药对化疗敏感性影响的机制研究提供线索。方法：采用磺酰罗丹明B法观察依托咪酯对神经胶质瘤细胞的抑制作用;细胞接种荧光法观察依托咪酯对神经胶质瘤细胞GJ功能的影响。结果：0.1、0.5、1和5μmol/L依托咪酯在作用4 h时间内均对细胞生长无抑制作用,将不影响细胞缝隙的数量;取药物浓度接近血药浓度的依托咪酯作用细胞4 h,与对照组相比,0.1μmol/L依托咪酯作用细胞4 h后,神经胶质瘤细胞GJ通讯的荧光传递功能无明显变化;而0.5和1μmol/L依托咪酯作用细胞4 h后,神经胶质瘤细胞的荧光传递功能明显减弱。结论：依托咪酯能抑制神经胶质瘤细胞Cx43组成的GJ功能。     

Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells

Two flavones, apigenin and tangeretin, were studied for theirability to modulate gap junctional intercellular communication(GJIC) in the rat liver epithelial cell line REL. Their cytotoxicitywas first determined by cell density and neutral red uptakeassays: neither apigenin nor tangeretin are cytotoxic at 10and 25 µM, the concentrations used in our experiments.We then studied GJIC using the dye transfer assay and we observedthat both apigenin and tangeretin enhance it, the maximum stimulation(x 1.7–1.8) being achieved at 25 µM for 24 h. Whenthe dye transfer was enhanced, the amount of connexin 43 increased,which was demonstrated by Western blot and immunofluorescenceanalysis. For apigenin only, Northern blot analysis showed anaccumulation of connexin 43 mRNA. In addition, the incubationof REL cells with the two compounds, for 1 or 24 h, preventedthe inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate(1or 10 ng/ml). The enhancement of GJIC by apigenin could be oneof the major mechanisms responsible for apigenin's anti-tumourpromoting action in vivo. As for tangeretin, its capacity toenhance GJIC completes its potential protective properties towardsthe post-initiation process.     

Effects of SV40 transformation on intercellular gap junctional communication in human fibroblasts

The effect of SV40 viral transformation of human fibroblasts on intercellular gap junctional communication (IJC) was investigated using a short-term quantitative assay. IJC was measured using metabolic cooperation in a coculture system of argininosuccinate synthetase- and argininosuccinate lyase-deficient human fibroblasts. These cell lines were transformed with origin-defective adenovirus/SV40 recombinant virions, and IJC was determined both between transformed cells (homologous IJC) and between transformed and untransformed cells (heterologous IJC). At equivalent cell densities, homologous IJC between transformed cells was reduced to 25-55% of the level between untransformed cells. Intermediate levels of IJC (50-70% of normal) were observed in heterologous cocultures of transformed with untransformed cells. Transformed and untransformed cells were equally sensitive to inhibition of IJC by phorbol esters and by glycyrrhetinic acid, and also did not differ in the degree of upregulation of IJC by forskolin. We conclude that SV40 transformation of human fibroblasts leads to a partial impairment of IJC which is additive when both communicating partners are transformed.     

Suppressed gap junctional intercellular communication in carcinogenesis of endometrium.

To examine whether and at which stage of endometrial carcinogenesis decreased connexin expression occurs, we investigated changes in the expression of the gap junction proteins, connexin 26 (Cx26), Cx32 and Cx43, in human endometrial hyperplasia and cancer samples. Forty-eight endometrial tissue samples (15 endometrial hyperplasias and 33 endometrial cancers) were subjected to immunofluorescence and RT-PCR analysis. In endometrial hyperplasia, Cx26 was aberrantly expressed in all samples as revealed immunohistochemically. There was weak or negative expression in 12 samples (80.0%) and diffuse expression in cytoplasm in 3 samples (20.0%). Cx32 expression in those samples was similar to that of Cx26; there was weak or negative expression in 11 samples (73.3%) and diffuse expression in 4 samples (26.7%). In endometrial cancer, Cx26 was expressed weakly or negatively in 25 samples (75.8%), diffusely in 6 samples (18.2%) and normally in 2 samples (6.1%), while Cx32 was expressed weakly or negatively in 26 samples (78.8%), diffusely in 5 samples (15.2%) and normally in 2 samples (6.1%). It was confirmed that weak staining of Cx26 and Cx32 was due to poor expression of their mRNA. All samples showed weak Cx43 protein expression as revealed by immunohistochemical analysis. In the majority of samples, concomitant expression levels of Cx26 and Cx32 protein were observed, confirming our long-term hypothesis that Cx26 and Cx32 are both abnormally regulated in a coordinated fashion in the endometrium. Our results indicate that during endometrial carcinogenesis, loss of gap junctional intercellular communication (GJIC) may occur due to the suppressed expression and the aberrant localization of connexin at relatively early stages.     

洛伐他汀对 MCF-7细胞增殖分化功能及间隙连接细胞通讯的影响

背景与目的:洛伐他汀( lovastatin)是细胞内源性胆固醇合成的抑制剂,临床已普遍用于治疗高脂血症.现有研究报道,洛伐他汀具有抗肿瘤作用,但分子机制尚不清楚.本文探讨洛伐他汀对人乳腺癌细胞 MCF-7增殖功能以及间隙连接细胞通讯( gap junctional intercellular communication,GJIC)的影响.方法:分别以 4、 8、 16 μ mol/L洛伐他汀处理 MCF-7细胞 24～ 72 h后,流式细胞仪检测细胞周期的时相分布及细胞凋亡率,硝基蓝四氮唑( NBT)还原实验鉴定细胞分化,并采用划痕标记染料示踪技术观察洛伐他汀对 MCF-7细胞 GJIC功能的影响.结果:不同浓度洛伐他汀处理细胞不同时间后, MCF-7细胞的增殖明显受抑,抑制率最高可达 75.8%,且同一处理时相点各组间比较差异均有显著性 (P< 0.05);细胞周期分析显示,各处理组内 G0/G1期细胞明显增多,处理 72 h后可高达 80%左右;同时洛伐他汀能明显促进 MCF-7细胞分化、各处理组间比较差异均有显著性 (P< 0.01),但洛伐他汀诱导该细胞凋亡的作用不明显.另外,洛伐他汀有上调或恢复 MCF-7细胞 GJIC的作用; 16 μ mol/L洛伐他汀处理细胞 72 h后,荧光染料传输的范围可达 4～ 5层细胞.以上作用均有浓度-效应和时间依赖关系.结论:洛伐他汀可抑制 MCF-7细胞增殖,使细胞生长阻滞于 G0/G1期,并能促进细胞分化,该作用可能与洛伐他汀上调或恢复 MCF-7细胞的 GJIC功能有关.     

STUDIES ON THE GAP JUNCTIONAL INTERCELLULARCOMMUNICATION OF HUMAN NASOPHARYNGEALCARCINOMA CELLS AND THE EFFECT OF RII

ＳＴＵＤＩＥＳＯＮＴＨＥＧＡＰＪＵＮＣＴＩＯＮＡＬＩＮＴＥＲＣＥＬＬＵＬＡＲＣＯＭＭＵＮＩＣＡＴＩＯＮＯＦＨＵＭＡＮＮＡＳＯＰＨＡＲＹＮＧＥＡＬＣＡＲＣＩＮＯＭＡ　ＣＥＬＬＳＡＮＤＴＨＥＥＦＦＥＣＴＯＦＲＩＩＨａｎＬｉｑｕｎ韩立群；ＧａｏＪｉｎ高进；...     

SHORT COMMUNICATION: Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) andbenzoyl peroxide (BoP) on gap junctional intercellular communication(GJIC) and the amount and localization of E-cadherin was studiedin initiated mouse epidermal cells (3PC) and in carcinoma cells(CA3/7) originating from the same cell type. In addition, thelocalization and phos-phorylation of connexin43 was studiedin both cell lines and in primary keratinocytes. GJIC inhibitionby TPA and BoP was stronger in primary keratinocytes comparedwith both cell lines. BoP strongly decreased the amount of E-cadherinprotein and the level occurring in the membranes in both celllines, whereas TPA caused a transloca-tion of E-cadherin fromthe membrane towards the cytosol, without decreasing the totalamount of E-cadherin present. The effect of both tumor promoterson connexin43 phos-phorylation and localization was agent aswell as cell dependent. These results show for the first timethat tumor promoters can decrease the quantity and membranelocalization ofE-cadherin in different cell types.     

Effective asymmetry in gap junctional intercellular communication between populations of human normal lung fibroblasts and lung carcinoma cells

The dysfunction of homologous and/or heterologous gap junctional intercellular communication (GJIC) has been implicated in tumorigenesis of many kinds of cells. Here we have characterized GJIC and the expression of connexins in six human lung carcinoma cell lines and normal lung fibroblasts (HLF). Compared with HLF, all the carcinoma cells showed reduced or little homologous GJIC. They expressed remarkably reduced connexin(Cx)43 mRNA and variable levels of Cx45 mRNA, but neither Cx43 nor Cx45 protein could be detected. However, using a preloading assay, transfer of calcein was observed between donor HLF cells and first order neighboring recipient tumor cells (recipient cells in 1000-fold excess). Transfer from tumor to HLF cells under the same conditions was not seen, although increasing the ratio of donor tumor cells to recipient HLF cells and plating the cells at low density did reveal weak transfer from tumor cells to HLF. Transfection of Cx43 into giant cell carcinoma PG cells increased homologous communication and eliminated the rectifying behavior of heterologous communication. This indicates that the apparent rectification of dye transfer between normal and tumor cells was a product of low rates of heterologous transfer linked to (i) rapid dilution of the dye to below detectable limits through a very well coupled cell population (tumor to HLF) and (ii) concentration of dye in immediate neighbors in a poorly coupled cell population (HLF to tumor cells). These results suggest that the coupling levels may need to exceed a certain threshold to allow propagation of signals over a sufficient distance to affect behavior of a cell population. We propose that the relative rates of heterologous and homologous coupling of cell populations and the 'pool size' of shared metabolites in tumor cells and the surrounding normal tissue are likely to be very important in the regulation of their growth.     

Overexpression of estrogen receptor-alpha gene suppresses gap junctional intercellular communication in endometrial carcinoma cells

Stimulation of the endometrium by estrogens without the differentiating effect of progestins is the primary etiological factor associated with the development of endometrial hyperplasia and adenocarcinoma. However, the correlation between sex steroids and gap junctional intercellular communication (GJIC), which is considered to play an important role in the control of cell growth and differentiation, is not well known in endometrial carcinoma. In this study, we focused on the influence of estrogen and its receptor in connexin (Cx) expression and GJIC in endometrial carcinoma cells, established stable clone IK-ER1 overexpressing ER-alpha to transfect the expression vector and analysed them in various hormonal conditions. The growth of IK-ER1 was accelerated by 17beta-estradiol and the acceleration of the 5-bromo-25-deoxyuridine labeling index was observed. GJIC was assayed by scoring the number of dye-coupled cells after microinjection of single cells with Lucifer-Yellow, and subcellular localization of Cx26 and Cx32 was analysed by immunocytochemistry. In the presence of estradiol, dye-coupled cells of IK-ER1 were significantly reduced compared to those without estradiol and the reduction was completely inhibited by adding ICI182.780, a pure antiestrogen substrate. Cxs were detected as only small spots by immunocytochemistry, and Western blotting showed that the expression was decreased. These results suggest that activation of ER-alpha by estrogen results in tumor progression by stimulating cell growth and suppressing GJIC via suppression of the expression of Cxs in endometrial carcinogenesis.     

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

The effects of K₂CrO₄, H₂O₂, benzoyl peroxide, menadione, KBrO₃and UV_365nm on gap junctional intercellular communication (GJIC)have been studied in the 12-O-tetra-decanoylphorbol-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 GJICwas found for the oxidative agents studied. Hydrogen peroxidewas used as a model compound in the subsequent studies. Theincrease in GJIC was reversible, and it was not due to an increasednon-junctional permeability. Hydrogen peroxide counteractedthe TPA-induced decrease in GJIC, regardless of whether thecells were exposed to the compounds simultaneously or the cellswere pre-exposed to TPA before addition of H₂O₂. The GJIC enhancementby H₂O₂ was slightly reduced by the addition of the hydroxylradical scavenger dimethylsulphoxide or by the inhibition ofcatalase by amitrole. The cAMP/ protein kinase A system is theonly characterized signal transduction system that is knownto increase GJIC in most cell types. Hydrogen peroxide did notincrease the amount of cAMP (or cGMP) in BPNi cells, while forskolinand a phosphodiesterase inhibitor had to increase the cAMP levelseveral-fold to affect GJIC to the same degree as the oxidativeagents. Some inhibitors of protein kinase A were assayed fortheir ability to inhibit the increases in GJIC caused by H₂O₂and forskolin. Staurosporine inhibited the forskolin-inducedincrease in GJIC, with much less effect on the H₂O₂-inducedincrease. H8, H88 and H89 had less effect than staurosporineon the forskolin-induced increase in GJIC. The results suggestthat the cAMP/protein kinase A system may not be involved inthe increase in GJIC caused by H₂O₂, although this cannot becompletely ruled out.     

Effect of DDT on hepatic gap junctional intercellular communication in rats

The effects of in vivo exposure to DDT on hepatic gap junctionalintercellular communication (GJIC) and connexin gene/proteinexpression in Sprague-Dawley rats were examined by in vivo/in vitro dye-transfer assay, immunohistochemical staining, andby Western and Northern blot analyses. In the dose-responsestudy, three dose levels of DDT (5, 25 and 50 mg/kg/day) wereadministered orally to rats once a day for 2 weeks. The averagesize of the dye spread after injection of Lucifer Yellow andthe area of Cx32 spots per hepatocyte decreased in a dose-dependentmanner, but there was no effect on the number of Cx32 spotsper hepatocyte. In the time-course study, DDT (50 mg/kg/day)was administered orally once a day for up to 6 weeks. HepaticGJIC decreased at week 1 but recovered at week 6. The averagearea of Cx32 spots per hepatocyte gradually decreased at weeks2 and 4, and remained at the same level at week 6, correlatingwith the decreased Cx32 protein level in plasma membranes. Theaverage area of Cx26 spots per hepatocyte in the peripheralzones clearly decreased at week 1, but quickly recovered atweek 2 and increased at week 6; however, no clear change ofthe Cx26 protein level in plasma membranes was observed. Nochanges of Cx32 and Cx26 mRNA levels were observed in DDT groups.These results suggest that DDT, a liver tumor-promoting agent,inhibits hepatic GJIC in vivo dose-dependently in rats and thataberrant Cx32 and Cx26 protein expression and/or localizationmay be responsible for this effect.     

Inhibitory effects of endosulfan on gap junctional intercellular communication in WB-F344 rat liver cells and primary rat hepatocytes.

The chlorinated cyclodiene insecticide endosulfan is a potent inhibitor of gap junctional intercellular communication (GJIC) in vitro, a property shared by many tumour promoters and suggested to indicate an intrinsic tumour-promoting potential. However, endosulfan did not act as a tumour promoter in an altered hepatic foci assay in the rat in vivo, and ambiguous results regarding the carcinogenic potential of endosulfan have emerged from long-term studies in rodents. In the present study the GJIC-inhibitory potentials of the two isomers of endosulfan were investigated in WB-F344 rat liver epithelial cells and primary rat hepatocytes. The results show that both isomers are inhibitors of GJIC. However, beta-endosulfan (ENDO beta) is a more potent inhibitor of GJIC in primary rat hepatocytes than alpha-endosulfan (ENDO alpha), whereas the two isomers were equally potent as inhibitors of GJIC in WB-F344 rat liver cells. In primary rat hepatocytes membrane-permeant dibutyryl cyclic AMP (dB-cAMP) counteracts the inhibitory effect of ENDO beta without affecting the effect of ENDO alpha. However, in WB-F344 rat liver cells dB-cAMP failed to prevent the inhibitory effects of either ENDO alpha or ENDO beta. In addition, studies in WB-F344 rat liver cells show that ENDO alpha beta does not decrease the intracellular cAMP concentration. Thus, it is unlikely that ENDO alpha beta or its isomers and metabolites inhibit GJIC by lowering the intracellular cAMP concentration. Furthermore, comparison of the effective doses and recovery times imply that GJIC in WB-F344 rat liver cells is more sensitive to treatment by ENDO alpha beta, its isomers and metabolites than GJIC in primary rat hepatocytes. Thus, the present results demonstrate significant differences between primary rat hepatocytes and WB-F344 rat liver cells in the response of their GJIC to endosulfan.