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.     

Inhibition of gap junctional intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat hepatocytes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent rodenthepatic tumor promoter. Unlike observations with the majorityof tumor promoting chemicals studied to date, most investigationshave failed to demonstrate down- regulation of gap junctionalintercellular communication (GJIC) in cultured cells by TCDD.The present study examined the effect of TCDD on GJIC in rathepatocytes in primary culture. At non-cytolethal doses TCDDinhibited GJIC In a time- (1, 4, 24 and 48 h) and concentration(1x10^–8–1x10^–14M)-dependent manner. This inhibitionoccurred within 4 h of treatment at doses of 1x10^–81x10–^–12MTCDD and persisted for up to 48 h, despite removal of TCDD.Treatment of rat hepatocytes with TCDD resulted in a decreasein hepatocyte connexin 32 mRNA, but had no apparent effect onconnexin 26 mRNA. Co-incubation of rat hepatocytes with TCDDand     

Decreased gap junctional intercellular communication in hexachlorobenzene-induced gender-specific hepatic tumor formation in the rat

Hexachlorobenzene (HCB), an epigenetic carcinogen, HCB induces the formation of liver tumors in female rats, whereas only a small percentage of males are responsive. Intercellular communication via gap junctions is decreased in carcinogenesis. Gap junctions are composed of proteins termed connexins (Cxs). The objectives of this study were (i) to determine if HCB-induced tumor development is associated with a loss of gap junctional communication; (ii) to assess if HCB causes a gender-specific decrease in the expression of Cx32 and Cx26; and (iii) to establish if these effects result from gender differences in the constitutive expression of these Cxs. Rats were given HCB by gavage for five consecutive days. In the first experiment, control and HCB-treated female rats were sampled on day 100. Intercellular communication was significantly decreased in HCB-treated females compared to controls. To investigate if changes in Cx levels occur prior to day 100, experiments done using male and female rats sampled on day 50. Hepatic mRNA levels for Cx26 and Cx32 were significantly lower only in HCB-treated females as compared to controls. Cx26 mRNA levels were 3-fold higher and Cx32 mRNA levels were 8-fold lower in females compared with males. In a third experiment, ovariectomy abolished any differences between male and female controls for both Cxs, while estradiol had a partial role in the regulation of Cx32. This suggests that the sexual dimorphism in hepatic Cx levels is determined by the ovarian hormones. However, the HCB-induced decrease in Cx32 and Cx26 mRNA levels was maintained in ovariectomized rats, suggesting that the HCB effects are not mediated via an ovary-dependent pathway. Overall results show that HCB exposure induces gender-specific long-term alterations in intercellular gap junctional communication in female rat liver. This effect appears to be a critical mechanism of HCB-induced liver carcinogenesis and tumor promotion.     

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.      

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.     

Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication

Breast cancer progresses toward increasingly malignant behavior in tumorigenic and metastatic stages. In the series of events in the metastatic stage, tumor cells leave the primary tumor in breast and travel to distant sites where they establish secondary tumors, or metastases. In this report, we demonstrate that cell-cell communication via gap junctions is restored in the metastatic human breast carcinoma cell line MDA-MB-435 when it is transfected with breast metastasis suppressor 1 (BRMS1) cDNA. Furthermore, the expression profile of connexins (Cxs), the protein subunits of gap junctions, changes. Specifically, the expression of BRMS1 in MDA-MB-435 cells increases Cx43 expression and reduces Cx32 expression, resulting in a gap junction phenotype more similar to normal breast tissue. Taken together, these results suggest that gap junctional communication and the Cx expression profile may contribute to the metastatic potential of these breast cancer cells.     

ACCELERATED PAPER: Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbituratesand this may involve their ability to inhibit hepatocyte gapjunctional intercellular communication (GJIC). In order to addressthe mechanisms and specificity of action of barbiturates onhepatocyte gap junctions, we have compared the effects of livertumor-promoting barbiturates (phenobarbital, sodium barbitaland amobar-bital: PB, SB and AB, respectively) and a non-livertumor-promoting barbiturate (barbituric acid: BA) on primarycultured rat hepatocyte GJIC and connexin32 (Cx32) expressionafter short (1–24 h) and long (2–14 days) treatmentGJIC was evaluated by fluorescent dye microin-jection (dye-coupling);Cx32 expression was monitored by Northern blot, Western blotand immunohistochemistry. Both parameters were maintained athigh levels over 14 days by coculture of the cells with WB-F344rat liver epithelial cells in the presence of dexamethasone.Treatment with PB (2 mM) for 1 h sharply reduced dye-couplingfrom     

Cannabinoids inhibit gap junctional intercellular communication and activate ERK in a rat liver epithelial cell line

Many tumor promoters suppress the immune system; however, the direct effect of immunosuppressants on the tumorigenic pathways of nonimmune cells in solid tissue has not been well documented. Cannabinoids were chosen to explore this question further. Cannabinoids are immune modulators that affect specific intracellular signaling pathways in leukocytes. Since these compounds are nongenotoxic, any tumorigenic effect that might be associated with these compounds would need to occur through an epigenetic mechanism. Therefore, we determined the effect of Delta(9)-THC and CBN, 2 plant-derived cannabinoids, on 2 key epigenetic markers of tumor promotion: inhibition of GJIC, which is essential in removing a cell from growth suppression, and activation of the ERK-MAPK pathway, which is crucial in activating the appropriate genes for mitogenesis. Both Delta(9)-THC and CBN reversibly inhibited GJIC at noncytotoxic doses (15 microM) in a normal diploid WB rat liver epithelial oval cell line within 20 min and activated ERK1 and ERK2 within 5 min. Inhibition of MEK with PD98059 prevented the inhibition of GJIC by either cannabinoid, suggesting that inhibition of GJIC was MEK-dependent. Based on RT-PCR analysis and employment of an antagonist of CB1 and CB2, the effects on GJIC and MAPK were independent of both cannabinoid receptors. Cannabinoids affected crucial epigenetic pathways associated with cell proliferation in a rodent liver epithelial cell model system.     

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.      

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

Tumor necrosis factor     

Increased gap junctional intercellular communication capacity and connexin 43 and 26 expression in rat bladder carcinogenesis

Many reports have suggested that gap junctional intercellularcommunication or gap junction proteins (connexins) could havetumor suppression characteristics. We investigated gap junctionalintercellular communication capacity and connexin 26, 32 and43 mRNA expression in four rat bladder cell lines and the resultswere compared to their tumorigenicity. We also examined connexinexpression in rat bladder carcinomas induced by 3,2'-dlmethyl-4-aminobiphenylor N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) and in normalbladders. There was clear tendency that cell lines with greatercommunication had stronger tumorigenicity and more expressionof connexin 26 or 43. We could not detect connexin 32 in thesecell lines. In normal bladder tissue, connexin 43 expressionwas barely detectable and there was no detectable connexin 26.However, in rat bladder carcinomas, especially the EHBNinducedcarcinomas, abundant expression of both connexins was observed.These results indicate that increased gap junctional intercellularcommunication capacity or increased connexin(s) expression maygive a growth advantage in rat bladder carcinogenesis.     

The dietary charred meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine acts as both a tumor initiator and promoter in the rat ventral prostate

Exposure of Fisher344 rats to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine in cooked meat, causes cancer in the rat ventral prostate, while sparing the dorsolateral and anterior lobes. Uncovering the molecular mechanisms of the lobe specificity of PhIP-induced rat prostate cancer may provide clues to the pathogenesis of human prostate cancer, which is also lobe selective. We examined the prostate and other organs for mutation frequencies using transgenic Fisher344 rats (Big Blue rats) after PhIP treatment. After PhIP treatment for as early as 4 weeks, the colon, spleen, seminal vesicles, and all lobes of the prostate had significantly elevated mutation frequencies compared with the saline-treated control group, and the differences became even greater after 8 weeks. G:C --> T:A transversions were the predominant type of mutation. After 8 weeks of treatment with PhIP, the Ki-67 index was increased (P < 0.001) in the ventral prostate, but not in the dorsolateral or anterior prostate. An increase in the number of stromal mast cells and macrophages was seen in the ventral prostate, but not in the other prostatic lobes. The apoptotic index also increased in the ventral lobe only. The increased proliferation and cell death in response to PhIP indicates that in addition to PhIP acting as an "initiator" of cancer, PhIP is also acting like an organ- and lobe-specific tumor "promoter." The prostate lobe-specific infiltration of mast cells and macrophages in response to PhIP suggests a potential new mechanism by which this dietary compound can increase cancer risk-by prompting inflammation.     

Phosphatase inhibitors, gap junctional intercellular communication and [125l)-EGF binding in hamster fibroblasts

A number of phosphatase inhibitors (okadaic acid, calyculinA, aluminium fluoride, sodium molybdate, sodium orthovanadate,pervanadate and vanadyl sulphate) were investigated for theireffects on gap junctional intercellular communication (GJIC)and [¹²⁵I-epidermal growth factor (EGF) binding in early passageSyrian hamster embryo cells (mainly fibroblast-like cells) andin V79 Chinese hamster lung fibroblasts. Only pervanadate decreasedGJIC significantly. After the initial pervanadateinduced decreasethe GJIC recovered rapidly. Only pervanadate was able to changethe band pattern of the gap junction protein connexin43 (cx43)in Western blots. Together this may indicate either that thereis a low turnover of phosphate groups in cx43 under basal conditionsor that the putative phosphatases are not sensitive to mostof 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 andthe EGF receptor, concomitantly with a decrease in GJIC and[¹²⁵I)-EGF binding. These effects are reversible after removalof TPA. It could be imagined that other phosphatases would acton cx43 and the EGF receptor after the forced phosphorylationof the two molecules. Thus TPA was used to downregulate GJICand [¹²⁵I]-EGF binding and phosphatase inhibitors were appliedin the upregulation phase. Only pervanadate affected the upregulationof GJIC, and pervanadate, orthovanadate and molybdate affectedthe upregulation of [¹²⁵I)-EGF binding. Thus it is not an identicalcomplement of phosphatases that act on cx43 and the EGF receptor.All the downregulating agents are assumed to be phosphotyrosinephosphatase inhibitors.     

Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro

To understand the initiating/promoting actions of pentachlorophenol (PCP), a non-mutagenic hepatocarcinogen, and its metabolite, tetrachlorohydroquinone (TCHQ), we investigated the effects of each chemical on gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape-loading and dye transfer method. After treatment with PCP, the GJIC was initially inhibited at 4 h but was restored in 6–8 h, followed by a second phase of inhibition between 16 and 24 h. Both the first and second inhibitions were concentration-dependent and were restored by 2–4 h after removal of PCP. The phosphorylation state of connexin 43 (CX43) and its localization on the plasma membrane were unchanged up to 24 h after treatment; however, this was accompanied by a decrease in the CX43 protein level. No inhibitory effect was apparent on the GJIC of cells treated with TCHQ. These results suggest that PCP may play a critical role of promoting activity via non-mutagenic mechanisms.     

Inhibition of gap junctional intercellular communication and malignant transformation of rat liver epithelial cells by neu oncogene

A retrovirus containing a neu oncogene was introduced into aFischer F344 rat liver epithelial cell line (WB-F344) to studythe effect of the expression of neu oncoprotein on gap junctionalintercellular communication (GJIC), the ability to form coloniesin soft agar and the ability to form tumors in rat liver bythese cells. After viral infection, five different neu-transducedepithelial clones were randomly selected for further analysis.Southern blot analysis of HindIII-digested genomic DNA hybridizedwith a neu specific probe indicated that the neu oncogene carriedby the retrovirus was integrated into different chromosomallocations in the five different neu-transduced WB cell lines.Using the fluorescence recovery after photobleaching (FRAP)assay, we found that GJIC was significantly reduced in neu-transducedWB clones, compared with control virus-infected and parentalWB cells. Western blot analysis of connexin 43 in the neu-transducedcell lines showed altered phosphorylation patterns comparedwith the normal WB-rat liver cell line. Confocal image analysisof the neu-transduced cells showed that the connexin 43 protein,as detected by fluorescent immunostaining, was localized inthe cell nucleus. The neu-transduced WB cell lines also acquiredthe ability to grow in soft agar. Furthermore, cells from threeof the five neu-transduced cell lines, when injected into theliver of Fischer F344 rats through the portal vein, were highlytumorigenic (multiple focal hepatic tumors developed within2 weeks). Cells derived from the tumor were shown to be G-418resistant, demonstrating that the tumor was derived from theinjected WB-neu cells. The results of this study demonstratethat the expression of the neu oncogene is able to block GJICand to induce tumorigenicity in the rat liver WB-F344 cell line.     

The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication

The effects of five non-mutagenic carcinogens—Aroclor1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate(TPA) and 1, 1'-(2, 2, 2-trichloroethylidene)bis[4-chlorobenzene](DDT)—on gap junctional intercellular communication (GJIC)were tested in a cell line consisting of initiated cells (3PC).Four agents suspected of tumor promotion activity—O-anisidine,clofibrate, L-ethionone and d-limonene—were also testedfor their effects on GJIC. Finally sodium fluoride (NaF), whosecarcinogenic property is still unclear, was tested for its effectson GJIC in the 3PC cell line. Four of the five selected tumorpromoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC betweenthese initiated epidermal cells. The four non-mutagenic carcinogenswith tumor-promoting activity in vivo (o-anisidine, clofibrate,L-ethionine and d-limonene) all inhibited GJIC, whereas NaFhad no effect. Seven compounds (o-anisidine, Aroclor 1260, BP,DDT, L-ethionine, d-limonene and TPA) had a dose-dependent aswell as time-dependent inhibitory effect on GJIC. Under theexperimental conditions used, clofibrate showed only a dose-relatedinhibition of GJIC. PB showed no inhibitory effect on GJIC inthe 3PC cell line. In order to determine the role of biotransformationin the tumor-promoting activity of PB, its effect on GJIC wasalso examined in the presence of an Aroclor 1254-induced ratliver homogenate (S9 mix) and in the hepatoma cell line HepG2.In the presence of rat liver homogenate PB decreased GJIC inthe 3PC cell line, whereas in the HepG2 cells PB showed a time-anddose-dependent inhibitory effect. To study the potential differencesin susceptibility of cells representing different stages inthe process of tumor formation, the effect of the selected tumorpromoters on GJIC was also investigated in primary mouse keratinocytesand in a mouse skin carcinoma-derived cell line (CA3/7). Primarykeratinocytes were sometimes more (BP and clofibrate) and sometimesless sensitive (ethionineand limonene) for inhibitory effectson GJIC compared to the effects in the cell line 3PC. Exceptfor TPA and anisidin, GJIC between the CA3/7 cells was lessaffected by the selected agents compared to the 3PC cell line.These results show that, during the process of tumor formationthe susceptibility of cells to inhibition of GJIC by tumor promotersis variable. Overall the CA3/7 cells are less sensitive comparedto 3PC cells. The susceptibility of primary keratinocytes isvariable compared to 3PC cells, depending on the agent used.These results also show that GJIC is a valid parameter for testingthe tumor-promoting activity of compounds. Finally, this studydemonstrates that mouse keratinocyte cell lines could serveas an in vitro model for the detection of non-mutagenic carcinogenswith diverse target organs in vivo. For this use the cell lineconsisting of initiated cells (3PC) is more sensitive than thecarcinoma-derived cell line CA3/7.     

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

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

Breast cancer metastatic potential: correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells

The breast cancer metastasis-suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA-MB-435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell-to-cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell-to-cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis-capable and -suppressed cell lines.     

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.