Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells

High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca²⁺ oscillations are generated. Both GJIC blockade and Ca²⁺ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.     

Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.     

Restoration of gap-junctional intercellular communication in a communication-deficient rat liver cell mutant by transfection with connexin 43 cDNA

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC. © 1993 Wiley-Liss, Inc.     

v-Src requires Ras signaling for the suppression of gap junctional intercellular communication

Cell transformation by v-Src causes suppression of gap junctional intercellular communication (GJIC). Although tyrosine phosphorylation of connexin43 (Cx43), a gap junctional component, appears to be necessary for the suppression, involvement of other signaling remains unclear. We investigated the role of Ras signaling in the suppression of GJIC by v-Src. Conditional expression of either S17N Ras or mtGap1m dramatically recovered GJIC in v-Src-transformed cells. Although expression of S17N Ras or mtGap1m substantially decreased the levels of active Ras, tyrosine phosphorylation of cellular proteins including Cx43 remained unchanged. Similarly, treatment of v-Src-transfomed cells with a Ras farnesyltransferase inhibitor, manumycin A, restored GJIC, whereas tyrosine phosphorylation of Cx43 remained unchanged. Thus, these results strongly suggest that, in addition to Cx43 phosphorylation, constitutive activation of Ras signaling is required for the suppression of GJIC by v-Src.     

Intracellular distribution of 73,000 and 72,000 dalton heat shock proteins in HeLa cells

Intracellular localization of 73,000 and 72,000 dalton heat shock proteins (HSP73/72) in HeLa cells that were heat shocked or treated with chemical stressors was investigated using indirect immunofluorescent staining. The antiserum used specifically recognized the HSP73/72 in HeLa cells, and HSPs were increased by heating cells at 42 degrees C for 2 or 4 h and by prior treatment with chemical stressors (sodium arsenite, cadmium chloride, 8-hydroxyquinoline and ethanol). There was diffuse cytoplasmic staining at 37 degrees C, whereas nucleoli were stained brightly when cells were heated at 42 degrees C for 2 h. This rapid accumulation of HSP73/72 in the nucleoli was not inhibited by cycloheximide (50 micrograms/ml). Translocation of HSPs to the nucleoli was specific for heat because no translocation was induced by treatment with chemical stressors. When the cells were returned to 37 degrees C after heating, the HSPs in their nucleoli disappeared rapidly and diffuse cytoplasmic staining was present after 6-9 h. Our results suggest that the transient accumulation of HSP73/72 in HeLa cell nucleoli that is induced by heat shock is not correlated with the development of thermotolerance obtained in other cell systems.     

Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication

The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA.     

The preventive effect of green tea on the gap junction intercellular communication in renal epithelial cells treated with a renal carcinogen

OBJECTIVE: Clinical studies imply that (-)-epigallocatechin-3-gallate (EGCG), a main ingredient of green tea catechins, has a chemopreventive action against cancers and suppresses the proliferation of cancer cells. However, there is no report about its chemopreventive effect for renal cancer. We previously determined that renal carcinogens suppressed the gap junction intercellular communication (GJIC) of renal epithelial cells. In this study, we investigated the effect of EGCG on the GJIC of renal epithelial cells treated with a renal carcinogen. MATERIALS AND METHODS: Mardin-Darby canine kidney (MDCK) cells were used to determine the protective effects of EGCG on dimethylnitrosamine-induced alteration of GJIC and connexin 43 (Cx 43). The maximum concentration of EGCG was determined by the lactate dehydrogenase assay method. The scrape-loading dye transfer method was used to assess the expression and cellular localization of Cx 43. The phosphorylation status of Cx 43 was determined by Western blot analysis. RESULTS: The optimal noncytotoxic concentration of EGCG was determined to be 10 microg/ml. The levels of GJIC and Cx 43 expression were markedly decreased in MDCK cells exposed to dimethylnitrosamine. A 12-h pretreatment with EGCG greatly ameliorated the GJIC-inhibitory effects of dimethylnitrosamine. CONCLUSION: These results suggest that the preservation of GJIC may indicate the chemopreventive effect of green tea on renal epithelial cells treated with a renal carcinogen in vitro.     

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap-junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myclras all displayed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src- and neu-transformed cells, but not in the ras- and myclras-transformed cells. Nuclei isolated from WB-neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB-neo control cells, supporting the idea that the nuclear-localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC-deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane-localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane-associated Cx43 immunostaining. These data suggest that altered localization of the gap-junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells. © 1996 Wiley-Liss, Inc.     

Exogenous CXCL12 activates protein kinase C to phosphorylate connexin 43 for gap junctional intercellular communication among confluent breast cancer cells

Despite ongoing attempts to improve the overall breast cancer (BC) survival rate, BC cells’ (BCCs) predilection for metastasizing to the bone marrow has enabled BCCs to not only remain dormant, but also evade detection. BCCs are able to acquire quiescence by establishing gap junctional intercellular communication (GJIC) with the stroma through the assembly of connexins (Cxs). The chemoattractant CXCL12 also appears to play a role in GJIC based on its tendency to decrease when GJIC is formed between BCCs and bone marrow stroma. This study investigates the role CXCL12 has on Cx43 expression and PKC-mediated Cx43 phosphorylation. Cx43 gene reporter assays revealed that as the BCCs come in contact with each other and establish GJIC, there is an inverse relationship between CXCL12 level and Cx43 expression. Immunoblot analyses confirmed this relationship at the level of protein, showing decreased Cx43 and reduced Cx43 phosphorylation at higher CXCL12 concentrations. However, real-time PCR studies revealed little change in Cx43 mRNA levels, despite stimulation with different concentrations of CXCL12, indicating CXCL12’s effect on Cx43 is post-translational, through phosphorylation. Immunoblot analyses and functional dye exchange studies showed activation of PKC by exogenous CXCL12 in the phosphorylation, which in turn, increased intercellular communication. These findings elucidate the importance of considering the microenvironment’s role in micrometastasis in clinical studies pertaining to prospective breast cancer treatment.     

All-trans retinoic acid enhances gap junctional intercellular communication among renal epithelial cells in vitro treated with renal carcinogens

Epidemiological and clinical studies imply that retinoids have a chemopreventative action against cancer and can suppress the growth of cancer cells. The regulation of connexin (Cx) expression by retinoids varies among tissues and organs. In this study, we investigated whether all-trans retinoic acid (ATRA) upregulates gap junctional intercellular communication (GJIC) in renal epithelial cells exposed to renal carcinogens. Madin Darby canine kidney (MDCK) cells were incubated with ATRA for 3 days, then briefly exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or renal carcinogens potassium bromate (KBrO₃) and dimethylnitrosamine (DMN). ATRA increased the expression of connexin 43 mRNA and protein without affecting Cx 43 phosphorylation and prevented inadequate Cx 43 localisation caused by TPA/KBrO₃ or DMN. Consequently, ATRA prevented the disruption of GJIC in MDCK cells. These data suggest that ATRA enhanced GJIC by upregulating Cx 43 expression and that ATRA might be useful for prevention of renal cell carcinoma.     

Heat shock protein 72 does not modulate ionizing radiation-induced apoptosis in U1810 non-small cell lung carcinoma cells

Heat shock proteins (HSP) have been shown to interfere with apoptosis signaling, suggesting that there might be a role for these proteins as mediators of resistance to ionizing radiation (IR)-induced apoptosis. Protein expression of the stress inducible heat shock proteins, HSP72 and HSP27, was analyzed in a panel of lung carcinoma cell lines displaying various degrees of radiosensitivity. Expression of HSP72 was high in all cell lines investigated while HSP27 was present in all non-small cell lung carcinoma (NSCLC) and 6/9 small cell lung carcinoma (SCLC) cell lines. Heat shock, but not IR, induced or further increased the expression of HSP27 and HSP72. Moreover, elevation of heat shock protein level prior to irradiation did not attenuate IR-induced apoptotic signaling or the induction of apoptosis. Protein level of HSP72 was downregulated in a radioresistant NSCLC cell line by RNA interference. However, this did not sensitize cells to treatment with DNA-damaging agents such as IR, cisplatin or VP16. Thus, the results from this first study on the relationship between stress-inducible HSP expression and IR-induced apoptosis in lung cancer cells do not support a role for HSP 27 and 72 in the radioresistance of NSCLC cells.     

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.      

Thermotolerance and heat shock protein induction by slow rates of heating

The magnitude of thermotolerance and the level of heat shock protein (HSP) expression have been measured in Chinese hamster ovary cells after gradual temperature transients from 37 degrees or 39 degrees to 42 degrees or 43 degrees C. When the level of thermotolerance was measured by clonogenic survival after challenging temperatures between 42 degrees and 43 degrees, substantial thermotolerance was observed. However, when the challenging temperature was raised to 45 degrees C, proportionally less thermotolerance was apparent. Heat shock proteins were quantitated by scanning densitometry of radiographs and, in the case of HSP 70, by immunoassay. Scanning densitometry revealed that low levels of heat shock proteins were synthesized during the heating gradients, but less than after a heat shock at 45 degrees C that delivered an equivalent heat dose. The immunoassay of HSP 70 levels measures both pre-existing and newly synthesized protein, and showed that there was net increase in HSP 70 during two of the heating gradients tested, despite the increase in synthesis noted on the gels. Higher turnover of HSP 70 at the elevated temperatures possibly accounted for the failure to detect a net gain in total protein. In contrast, the total amount of HSP 70 doubled during the 6 hr following a heat shock of 45 degrees for 10 min, an equivalent heat dose to one of the gradients where no net increase in HSP 70 was measured by immunoassay. It appears, then, that tolerance to hyperthemia at 43 degrees C or below may occur under some conditions in the absence of elevated levels of HSP 70, but tolerance to higher temperatures is more closely correlated with increased levels of heat shock proteins. However, even at higher temperatures, our data show disparities between the levels of HSP measured and the thermotolerance expressed.     

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C

In this study, we investigated whether the tumor promoters, 12-O- tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1- bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB- F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin- specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.      

Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells

PURPOSE: Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents. METHODS: We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA. RESULTS: Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged. CONCLUSIONS: This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.     

转染连接蛋白基因对人脑胶质瘤细胞间隙连接通讯及其生长变化的研究

目的 研究连接蛋白（Cx)基因对人脑胶质瘤细胞的细胞间隙连接通讯（GJIC）及其增殖的抑制作用,探索以Cx43基因治疗胶质瘤的可行性。方法 将含Cx43cDNA的质粒,以脂质体介导转染Cx43表达缺失的TJ905人胶质母细胞瘤细胞,通过Northern印染杂交、原位杂交及免疫组化染色检测Cx43mRNA及蛋白表达,划痕标记荧光染料示踪技术（SLDT）检测GJIC,MTT法测定细胞增殖率,核仁组成区嗜银蛋白（AgNOR)染色检测细胞增殖活性,TUNEL法检测细胞凋亡。结果 转染后TJ905细胞有不同程度的Cx43mRNA和蛋白表达及GJIC恢复。Cx43表达水平高的克隆细胞增殖明显下降,细胞凋亡并未增加。结论 Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用,可能成为恶性胶质瘤基因治疗的优选靶的之一。     

Inhibition of intrinsic gap-junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant-negative connexin 43 mutant

The tumor-suppressive property of the connexin gap-junction proteins was postulated from the fact that their function of cell coupling is impaired in most cancer cells. However, in conflict with this notion, certain cancer cells are able to communicate through gap junctions despite their malignancy. To explain this phenomenon, we studied by using a dominant-negative strategy the effect on tumorigenicity of loss of intrinsic gap-junction intercellular communication (GJIC) in the rat bladder carcinoma cell line BC31, which shows both expression of connexin 43 (Cx43) and intercellular communication. In cells transfected with a mutant Cx43 with seven residues deleted from the internal loop at positions 130–136 (Cx43Δ), transport of the resulting connexin protein to the plasma membrane occurred normally, but the GJIC of the cells was effectively abolished at the level of permeability of established gap junctions. Dominant-negative inhibition of GJIC by Cx43Δ accelerated growth of BC31 cells in nude mice. In contrast, when GJIC in BC31 cells was artificially enforced by transfection of wild-type Cx43, the cells lost the capacity to grow in vivo. Decreased phosphorylation of Cx43Δ suggested close interaction of the internal loop of connexin with its commonly phosphorylated domains in the C-terminal tail and involvement of this interaction in gap-junction permeability. Therefore, we conclude that the intrinsic GJIC observed in cancer cells should be considered a tumor-suppressor factor and that its level may influence malignant growth capacity. Mol. Carcinog. 23:254–261, 1998. © 1998 Wiley-Liss, Inc.     

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner

Many lines of evidence support the hypothesis that connexins form a family of tumor-suppressor genes. Transfection of connexin43 (Cx43) into rat C6 glioma cells have revealed that Cx43 functions as a growth- and tumor-suppressor in C6 cells. In previous studies, we and others have reported that several mutant connexins can inhibit gap junctional intercellular communication (GJIC) realized by the wild type in a dominant-negative manner. We have now examined dominant-negative effects of Cx43 mutants on cell growth control exerted by wild-type Cx43 in C6 cells. When 2 Cx43 mutants (L160M and A253V) were transfected into Cx43-transfected C6 cells, they restored anchorage-independent growth capacity and reinforced the tumorigenicity of these cells, meaning that these 2 mutants can inhibit growth-suppressive function of wild-type Cx43 in a dominant-negative manner. Neither of the mutants appeared to affect phosphorylation states and subcellular localization of Cx43 proteins. Intriguingly, the mutant A253V did not suppress GJIC capacity, implying a growth-suppressive pathway mediated by Cx43 may not be related to GJIC.Int. J. Cancer 78:446–453, 1998. © 1998 Wiley-Liss, Inc.     

Hyperthermia induces differentiation without apoptosis in permissive temperatures in human erythroleukaemia cells.

PURPOSE: The aim of the present study was to investigate whether induction of differentiation by hyperthermia is accompanied by apoptosis and necrosis to further evaluate the benefits of using hyperthermia as a differentiation inducing physical modality. MATERIALS AND METHOD: Differentiation was evaluated in K562 erythroleukaemia cells by measuring haemoglobin synthesis and flow cytometric measurement of glycophorin A expression. Apoptosis was measured by Annexin-V-FITC and Propidium Iodide (PI) double staining assay. Apoptosis and necrosis was also evaluated morphologically using staining with acridine orange/ethidium bromide (AO/EtBr) by fluorescence microscopy. Heat shock protein 70 (HSP70) level was measured by ELISA kit. RESULTS: Hyperthermia (43 degrees C) induced differentiation as judged by increased haemoglobin synthesis and glycophorin A expression. No sign of apoptosis or necrosis could be detected at this temperature. Cell viability did not change due to heat treatment, and cellular proliferation was reduced in a dose (heating time) dependent manner. At 45 degrees C, hyperthermia induced apoptosis and necrosis with minimal or no sign of differentiation. HSP70 level was significantly increased at 43 degrees C along with differentiation of leukaemic cells, while at 45 degrees C no significant effect on HSP70 production could be observed. CONCLUSIONS: The encouraging results obtained here indicate that by heat treatment at 43 degrees C, hyperthermia can be used alone or in combination with other modalities as a differentiation inducing agent without any detectable apoptotic activity. Positive correlation between HSP70 production and induction of differentiation and lack of apoptosis by hyperthermia confirm the possible role of HSP70 in the heat-induced differentiation and apoptosis in leukaemic cells.     

Effects of peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on intercellular communication and connexin43 in two hamster fibroblast systems

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5–15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding. Int. J. Cancer 73:240–248, 1997. © 1997 Wiley-Liss, Inc.