SHORT COMMUNICATION: Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters (original) (raw)
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Environmental Toxicology and Pharmacology, 1996
The molecular and histological effects of tumor promoters on gap junctional intercellular communication (GJIC) were studied in three mouse epidermal cell types, representing different stages of tumor formation. GJIC was inhibited by most of the studied compounds (L-ethionine, d-limonene, o-anisidine, clofibrate, Aroclor 1260 and 1,1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene)(DDT))except NaF and phenobarbital (PB). Whatever their effect on GJIC, most of the studied compounds increased the phosphorylation state of the gap junction protein expressed in these cells, connexin43 (Cx43), as shown by Western analysis. All agents with GJIC inhibiting capacity changed the intensity of the immunofluorescent staining of Cx43 on the membrane of the cells, whereas NaF and PB had no effect on Cx43 immunostaining. No association could be found between the type of change in Cx43 localization (changed membrane and/or cytosolic staining) and Cx43 phosphorylation or GJIC inhibition. Because the cell adhesion molecule E-cadherin also regulates GJIC, the effects of tumor promoters on E-cadherin protein and localization were studied. No quantitative change could be observed in E-cadherin protein content of cells treated with any of the selected agents. However, all agents which decreased GJIC, affected E-cadherin immunostaining of the membrane, while PB and NaF had no effect. These results show that an association exists between inhibition of GJIC and localization of both connexin43 and E-cadherin protein, but not with Cx43 phosphorylation.
Carcinogenesis, 1996
Polycyclic aromatic hydrocarbons (PAHs) are a broad class of ubiquitous environmental pollutants with known or suspected carcinogenic properties. Tumor promotion is a cell-proliferative step of cancer that requires the removal of cells from growth suppression via the inhibition of gap-junctional intercellular communication (GJIC). Inhibition of GJIC measured with an in vitro WB-F344 rat liver epithelial cell system was used to assess the relative potencies of 13 PAHs suggested by the U.S. Environmental Protection Agency (EPA) as the principal contaminants and 22 other PAHs, most of them identified in environmental samples. Maximal inhibition of GJIC was detected after 30 min of exposure, followed by a recovery in intercellular communication after an additional 30 min of exposure, suggesting a transient character of inhibition. Although M concentrations of PAHs were required to reach the inhibition level equal to the model tumor promoter phorbol 12-myristate 13-acetate (IC 50 ؍ 8 nM), 12 of the PAHs under study were found to be strong inhibitors of GJIC (strongest effects were observed with fluoranthene, picene, 5-methylchrysene and nine additional PAHs). The other nine PAHs, including benzo[a]pyrene, inhibited GJIC only up to 50-75% of the control level. Interestingly, several high molecular weight PAHs with known strong carcinogenic properties possessed only weak (dibenzopyrenes) or no inhibition potency (dibenzofluoranthenes, naphtho[2,3-a]pyrene and benzo[a]perylene). Based on the IC 50 values related to the reference PAH benzo[a]pyrene, we suggested arbitrary values of inhibition equivalency factors (GJIC-IEFs) ranging from 0 (noninhibiting PAHs) to 10.0 (strongest inhibitors), suitable for the purposes of environmental risk assessment.
Carcinogenesis, 1997
A series of cells representing normal, non-tumorigenic cell lines, as well as differentiating neoplastic and undifferentiated neoplastic rat tracheal epithelial cell populations were evaluated for their ability to establish homologous and/or heterologous cell-cell gap junction communication in culture. Gap junction communication was evaluated by flow cytometric quantitation of the transfer of the fluorescent dye calcein from a donor to a recipient cell population via gap junctions. The data indicate that normal primary cultures of rat tracheal epithelial cells, as well as non-tumorigenic cell lines and squamous cell carcinomas cell populations, retain the ability to establish both homologous and heterologous gap junction communication. In all cases an average of >48% of recipient cells had acquired calcein label during a 5-h interval of co-culture of donor and recipient cells at confluent densities. Cells harvested directly from squamous cell carcinoma tumors exhibited similar levels of cell-cell communication. In contrast, cells giving rise to undifferentiated carcinomas, as well as cells harvested from undifferentiated carcinomas, exhibited very low levels or no homologous or heterologous cell-cell communication. Cell populations exhibiting distinctly different communication phenotypes were evaluated by Northern blot analysis for expression of connexins (Cx 26, 32 and 43) and E-cadherin. Neither communicating nor non-communicating cells expressed connexin 32. Those cell populations, which established functional gap junctions, expressed E-cadherin as well as connexin 26 and/or 43. In contrast, those cell populations that lacked the ability to communicate universally lacked expression of E-cadherin, and a quarter also lacked expression of detectable levels of connexin.
Carcinogenesis, 1996
The effects of five non-mutagenic carcinogens-Aroclor 1260, benzoyl peroxide (BP), phenobarbital (PB), 12-0tetradecanoyl-phorbol-13-acetate (TPA) and l,l'-(2,2,2trichloroethylidene)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 rf-limonene-were also tested for their effects on GJIC. Finally sodium fluoride (NaF), whose carcinogenic property is still unclear, was tested for its effects on GJIC in the 3PC cell line. Four of the five selected tumor promoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC between these initiated epidermal cells. The four non-mutagenic carcinogens with tumorpromoting activity in vivo (o-anisidine, clofibrate, L-ethionine and rf-llmonene) all inhibited GJIC, whereas NaF had no effect. Seven compounds (o-anisidine, Aroclor 1260, BP, DDT, L-ethionine, rf-limonene and TPA) had a dosedependent as well as time-dependent inhibitory effect on GJIC. Under the experimental conditions used, clofibrate showed only a dose-related inhibition of GJIC. PB showed no inhibitory effect on GJIC in the 3PC cell line. In order to determine the role of biotransformation in the tumor-promoting activity of PB, its effect on GJIC was also examined in the presence of an Aroclor 1254-induced rat liver homogenate (S9 mix) and in the hepatoma cell line HepG2. In the presence of rat liver homogenate PB decreased GJIC In the 3PC cell line, whereas in the HepG2 cells PB showed a time-and dose-dependent inhibitory effect To study the potential differences in susceptibility of cells representing different stages in the process of tumor formation, the effect of the selected tumor promoters on GJIC was also investigated in primary mouse keratinocytes and hi a mouse skin carcinoma-derived cell line (CA3/7). Primary keratinocytes were sometimes more (BP and clofibrate) and sometimes less sensitive (ethionine and limonene) for inhibitory effects on GJIC compared to the effects in the cell line 3PC. Except for TPA and anisidin, GJIC between the CA3/7 cells was less affected by the selected agents compared to the 3PC cell line. These results show that, during the process of tumor formation the susceptibility of cells to inhibition of GJIC by tumor promoters Is variable. Overall the CA3/7 cells are less
Carcinogenesis, 1996
Differences in calcium-mediated regulation of gap junctional intercellular communication (GJIC) between a cell line consisting of mouse epidermal initiated cells (3PC) and a mouse epidermal carcinoma-derived cell line (CA3/7) were studied. Under low extracellular calcium (Ca 2+ j) conditions (0.05 mM) CA3/7 cells showed a low level of GJIC compared with 3PC cells. High Ca 2+ e (1.20 mM) raised GJIC between CA3/7 cells to the GJIC level of 3PC cells, which in turn remained unchanged under these conditions. Raising the free intracellular calcium concentration (Ca 2+ |), using a calcium ionophore (ionomycin) or the Ca 2+-ATPase inhibitor thapsigargin under low Ca 2+ e conditions, did not affect the GJIC level between 3PC cells, and increased GJIC between CA3/7 cells. Intracellular calcium chelation in 3PC cells under low Ca 2+ e conditions by ethylene glycol-bis({i-amino-ethyl ether) N,N,N',N'tetra-acetic acid acetoxy-methyl ester (EGTA-AM) decreased GJIC in this cell line. High Ca 2+ e conditions protected both cell lines from a decreased GJIC by EGTA-AM exposure. Inhibition of calmodulin (CaM) by calmidazolium (CDZ) or A/-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide (W-7) under low Ca 2+ e conditions, inhibited GJIC in 3PC cells and increased GJIC in CA3/7 cells. Inhibition of Ca 2+ /CaM-dependent protein kinase (Ca 2+ /CaM-PK) by l-(5-iodonaphthalene-l-sulfonyl)-lHhexahydro-l,4-diazepine (ML-7) decreased GJIC in both cell h'nes. Western analysis showed that Cx43 was more phosphorylated in both cell lines in concurrence with different effects on the GJIC level. Under conditions in which GJIC was inhibited, a decreased immunostaining of Cx43 on the plasma membrane was found. The level of immunostaining of the cell adhesion molecule E-cadherin on the plasma membranes of both cell types remained unchanged under conditions in which GJIC was changed by modulaters of (Ca 2+),, CaM activity, or the Ca 2+ /CaM-PK activity. These results indicate that differences exist between 3PC cells and CA3/7 cells in the GJIC regulation by intracellular calcium and calmodulin.
Differential Induction of Connexins 26 and 30 in Skin Tumors and Their Adjacent Epidermis
Journal of Histochemistry and Cytochemistry, 2006
Gap junctions (GJs) have been shown to play a role in tumor progression including a variety of keratinocyte-derived and non-keratinocyte-derived skin tumors. Here we show that the synthesis of the GJ proteins connexin 26 and connexin 30 (Cx26 and Cx30) is induced in keratinocyte-derived epithelial skin tumors whereas there is either no change or a downregulation of Cx43. Cx26, Cx30, and Cx43 are absent in non-epithelial skin tumors. Further, Cx26 and Cx30 are induced in the epidermis adjacent to malignant melanoma but absent in the epidermis adjacent to benign non-epithelial skin lesions (melanocytic nevi and angioma). The keratinocyte-derived skin tumors are very heterogeneous regarding the Cx26/Cx30 pattern in the epidermis at the periphery of the tumors. We did not observe any difference in the localization of the very similar proteins Cx26 and Cx30 but a variation in intensity of immunoreactivity. As the staining patterns of Cx26 and Cx30 antibodies are not identical to those of...
is a characteristic of cancer cells. Since a coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis, the present study was designed to test the hypothesis that skin-derived tumor cells may modulate homologous and heterologous GJIC. While homologous GJIC of human dermal fibroblasts as well as epidermal keratinocytes was detected, no communication was measured between SCL-1 cells derived from squamous cell carcinoma of human skin. Interestingly, co-cultures of dermal fibroblasts and SCL-1 tumor cells in serum-containing medium resulted in a 52±70% lowering of the number of communicating fibroblasts. Furthermore, incubation of confluent fibroblast cultures with serum-free supernatant fractions (20±30 kDa) from tumor cells, termed the 20/30 fraction, lowered the homologous gap junction communication of fibroblasts by 490%. This novel aspect of down-regulated homologous GJIC of dermal fibroblasts, which is reversible, was neither mediated by alteration of the expression of con-nexin43, the major gap junctional protein of dermal fibroblasts, nor by aberrant localization of connexin43 in the plasma membrane. Furthermore, post-translational modifications of connexins, such as phosphorylation, was not measured by mobility shift studies. Tumor cell-mediated GJIC down-regulation between fibroblasts was suppressed using EGTA-containing serum-free tumor cell-derived supernatants suggesting that calcium ions (Ca 2 ) might mediate the transduction of this effect. The involvement of Ca 2 in down-regulation of homologous GJIC of fibroblasts was supported by an increase in fluorescence intensity of the intracellular calcium-sensitive indicator Fura-2 upon treatment of fibroblasts with the active 20/30 fraction. In conclusion, these data establish homologous GJIC of (stromal) fibroblasts as a parameter modulated by a paracrine acting factor(s) of epithelial tumor cells during tumor±stroma interaction of skin cells.
Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity
Critical Reviews in Biochemistry and Molecular Biology, 2009
Cultured HOSE cells exhibited extensive fluorescent dyecoupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N 6 ,2Ј-O-dibutyryladenosine 3Ј,5Ј-cyclic monophosphate and alltrans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 1996
is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in viva tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homocypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals; they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms. COMP BIOCHEM PHYSIOL 113C, 247-256, 1996.