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.     

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.     

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.     

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.     

Reduction in gap junction intercellular communication promotes glioma migration

Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. One of the molecular changes observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity. We used a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. We found that down-regulation of Cx43 expression in the U118 human glioma cell line increased migration by reducing cell-ECM adhesion, and changed the migration pattern from collective to single cell. In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Taken together our findings reveal an unexplored role of GJIC in facilitating collective migration.     

miR-221/222 is the regulator of Cx43 expression in human glioblastoma cells

The miR-221/222 cluster is significantly upregulated in malignant glioma cells and regulates the expression of multiple genes associated with glioma cell proliferation, invasion and apoptosis, which was shown in our previous studies. Cx43 has been identified as a tumor suppressor and major component for the establishment of gap junction intercellular communication (GJIC) in glial cells, which is frequently reduced or deleted in high-grade gliomas. According to bioinformatic analysis, connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the present study was to validate Cx43 as a target gene of miR-221/222 and to determine whether overexpression of miR-221/222 is one of the molecular mechanisms for the reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine method. Northern blot analysis was conducted to detect the expression of the miR-221/222 cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence staining. Scrape loading and dye transfer (SLDT) assays were used for examination of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell assays, respectively. Cell cycle kinetics and apoptosis were determined with flow cytometry. We found that expression of the miR-221/222 cluster was significantly reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222, and the GJIC deficiency in parental U251 cells was re-established. Moreover, the luciferase activity determined by the luciferase reporter assay was enhanced in AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed while apoptosis was induced. We conclude that miR-221/222 function as oncogenic microRNAs in human gliomas, at least in part, by targeting Cx43.     

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。     

间隙连接基因Cx43表达对肺癌细胞体内成瘤生长的抑制

目的探讨间隙连接基因表达和细胞通讯功能对肿瘤生长的抑制作用。方法以高转移性人肺癌ＰＧ细胞为材料,该细胞的间隙连接基因Ｃｘ４３表达抑制,细胞通讯功能缺陷。用Ｃｘ４３ｃＤＮＡ转染ＰＧ细胞,分离转染子克隆,与只转染空载体ｃＤＮＡ的对照组ＰＧ进行比较。用Ｎｏｒｔｈｅｒｎ分子杂交和染料传输方法检查间隙连接表达情况,并观察细胞在体外和裸鼠体内生长。结果空载体对照组与未转染组ＰＧ相似,Ｃｘ４３ｍＲＮＡ无表达,通讯功能缺陷,细胞生长快,在软琼脂内集落形成率高（１１．６％）,植入裸鼠体内２８天,平均瘤重３．４７ｇ。转染组细胞Ｃｘ４３ｍＲＮＡ表达升高,通讯功能增强,细胞生长慢,在软琼脂内集落形成率和在裸鼠体内生长速度明显低于对照组,抑制率分别为９０％和７５％。结论间隙连接基因Ｃｘ４３表达对肺癌细胞有抑瘤作用。     

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.      

Gap junctional intercellular communication and cellular response to heat stress

Gap junctional intercellular communication (GJIC) is essential in the maintenance of tissue homeostasis and has been implicated in tumor suppression. Recent studies have indicated that GJIC is also involved in cellular stress responses to low dose ionizing radiation, UV light and hydrogen peroxide. However, the contribution of GJIC to the heat stress response has not yet been elucidated. We here demonstrate a potential link between GJIC and the heat stress response. First, we investigated whether the abolition of GJIC by lindane affects heat sensitivity in normal human cells. Lindane potentiated cell killing by heat shock at 43 degrees C, whereas little or no cytotoxicity was observed at 37 degrees C. Nuclear translocation of heat shock protein 72 (HSP72) was interrupted by lindane, although its induction was not affected. These results indicate that lindane exacerbates hyperthermic lethality via disrupted nuclear translocation of HSP72 protein. Second, we assessed whether heat shock alters GJIC and phosphorylation of gap junction connexin (Cx) proteins in normal human cells. Persistent heat treatment augmented Cx43 phosphorylation in a heat- and time-dependent fashion and this phosphorylation was recovered after heat shock. GJIC was also disturbed by heat shock. These results indicate that heat shock augments Cx43 phosphorylation leading to GJIC abrogation. Our present results imply that GJIC contributes to protection against heat stress and that loss of GJIC function during carcinogenesis exacerbates hyperthermic lethality.     

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.     

Action of db-cAMP on the bystander effect and chemosensitivity through connexin 43 and Bcl-2-mediated pathways in medulloblastoma cells

Medulloblastoma (MB) is one of the most common malignant brain tumors of childhood and is associated with a poor prognosis. Gap-junctional intercellular communication (GJIC) is an important mode for cell-to-cell communication. Dysfunctional GJIC is exhibited in most cancer cells. There is significant evidence that GJIC is important in at least some prodrug/suicide gene systems by augmenting the bystander effect (BE). GJIC is made up of connexins (Cxs), among which Cx43 is present in most tissues. Bcl-2, an important apoptosis blocker, is closely associated with the sensitivity to anticancer drugs. Our study showed that dibutyryl cyclic adenosine monophosphate (db-cAMP) upregulated the Cx43 expression and GJIC function in Daoy medulloblastoma cells. It directly enhanced the BE using a herpes simplex virus thymidine kinase (HSV?tk)/ganciclovir (GCV) system, which was blocked by a Cx43 inhibitor. In addition, db-cAMP increased the cytotoxicity of temozolomide and teniposide, possibly by downregulating the Bcl-2 expression and inducing apoptosis. Taken together, we demonstrated the beneficial effect of db-cAMP in treating medulloblastoma depending on the upregulation of BE and chemosensitivity through Cx43 and Bcl-2-mediated pathways.     

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.     

Effects of the histone deacetylases inhibitors sodium butyrate and trichostatin A on the inhibition of gap junctional intercellular communication by H2O2- and 12-O-tetradecanoylphorbol-13-acetate in rat liver epithelial cells

The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and sodium butyrate (NaBu) are considered as potent therapeutic agents for cancer treatment presenting therapeutic benefits with less risk of side effects. The microbial metabolite, TSA is a potent reversible and highly specific inhibitor of mammalian histone deacetylases. NaBu causes hyperacetylation of core histones with effects similar to TSA but it is not a specific inhibitor of HDACs. The gap junction is a channel in the plasma membrane of most cell types which allows direct communication (gap junctional intercellular communication; GJIC) of small molecules and ions. Modulation of GJIC is a known cellular event associated with tumor promotion. The effects of NaBu and TSA on the H(2)O(2)- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced GJIC inhibition of WB cells and the mechanisms involved in the process were assessed. TSA and NaBu exerted differential preventive effects on the H(2)O(2) and TPA-induced inhibition of GJIC as well as hyperphosphorylation of connexin43 (Cx43) in WB-F344 rat liver epithelial cells (WB cells). NaBu prevented the TPA-induced GJIC inhibition via ERK1/2 inactivation whilst TSA restored the H(2)O(2)-induced GJIC inhibition and Cx43 hyperphosphorylation by preventing p38 MAP kinase. The inhibition of tyrosine phosphorylation and down-regulation of src protein observed may also contribute to Connexin 43 dephosphorylation and GJIC restoration by TSA and NaBu partly through depletion of src protein pool. Thus, TSA and NaBu exert differential effects on chemically induced GJIC inhibition via modulation of MAP kinases and partly, tyrosine kinases.     

Cytoplasmic accumulation of connexin32 protein enhances motility and metastatic ability of human hepatoma cells in vitro and in vivo

Connexins have long been believed to suppress tumour development during carcinogenesis by exerting gap junctional intercellular communication (GJIC). Although GJIC is abrogated in hepatocellular carcinoma (HCC), connexin32 (Cx32) protein tends to remain expressed in cytoplasm, but not in cell-cell contact areas; thus, it is incapable of forming gap junctions. Hypothesising that cytoplasmic Cx32 protein that has accumulated in HCC should have its proper functions, which are independent of GJIC, we established an inducible expression system of Cx32 in human HuH7 HCC cells, which were unable to support the formation of Cx32-mediated gap junctions, so that Cx32 protein could be overexpressed by doxycycline (Dox) withdrawal. Although the established clone HuH7 Tet-off Cx32 cells exhibited a 4-fold increase in Cx32 expression after Dox withdrawal, none of them were dye-coupled, and Cx32 protein was retained in the Golgi apparatus. However, the proliferation rate of the HuH7 Tet-off Cx32 cells was significantly higher in the Dox-free medium than in the Dox-supplemented one. Transwell assays also revealed that Dox withdrawal enhanced serum-stimulated motility and invasiveness into Matrigel of the HuH7 Tet-off Cx32 cells. Furthermore, when HuH7 Tet-off Cx32 cells were xenografted into the liver of SCID mice, only the mice to which no Dox was administered developed metastatic lesions, indicating that overexpression of cytoplasmic Cx32 protein induced metastasis of HuH7 cells. Our results suggest that, while Cx32-mediated GJIC suppresses the development of HCCs, cytoplasmic Cx32 protein exerts effects favourable for HCC progression, such as invasion and metastasis, once the cells have acquired a malignant phenotype.     

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.     

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.      

Effect of Cx43 gene-modified leukemic bone marrow stromal cells on the regulation of Jurkat cell line in vitro

We recently reported that Cx43 expression and gap junction intercellular communication (GJIC) between acute leukemic bone marrow stromal cells (BMSCs) were deficient, which could recovery after effective chemotherapy. However, the exact role of GJIC in the hematopoietic microenvironment in leukemic cell death and proliferation is not clear. We show here that following transfection with the Cx43 gene, GJIC function was increased between leukemic BMSCs. Furthermore, compared with leukemic cells alone, the proliferation and apoptosis of leukemic cells co-cultured with BMSCs were inhibited, the percentage of G0-phase cells was higher; and expression of p53 increased and bax decreased. However, after co-culturing leukemic cells with Cx43-modified BMSCs, the number of proliferative and spontaneously apoptotic Jurkat cells increased; the percentage of G0-phase cells decreased; the expression of p53 decreased; and bax increased. Compared with Jurkat cells co-cultured with BMSCs and Jurkat cells alone, the sensitivity of leukemic cells co-cultured with Cx43-modified BMSCs to chemotherapeutics increased. Our data suggests that GJIC between leukemia BMSCs is one of the impact factor to the proliferation, apoptosis and drug sensitivity of co-cultured leukemic cells. Up-regulating its function can inhibit the protective effects of leukemic BMSCS and enhance the efficacy of therapies in hematologic malignancies.     

A derivative of oleamide potently inhibits the spontaneous metastasis of mouse melanoma BL6 cells

We reported previously that the abnormally augmented expression of connexin 26 (Cx26) is responsible for the enhanced spontaneous metastasis of mouse BL6 melanoma cells, and that the exogenous expression of a dominant negative form of Cx26 inhibits the spontaneous metastasis of BL6. Here we show that daily intraperitoneal (i.p.) injections of oleamide, a sleep-inducing lipid hormone, weakly inhibited the spontaneous metastasis of BL6 cells. To obtain a more effective reagent, 19 oleamide derivatives were chemically synthesized and tested for their ability to inhibit the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of Cx26 or Cx43. One of these, denoted metastasis inhibitor-18 (MI-18), inhibited the GJIC formed by Cx26 as well as oleamide but unlike oleamide, which is a non-selective inhibitor of connexin, it did not inhibit the GJIC formed by Cx43. Daily i.p. injections of MI-18 potently blocked the spontaneous metastasis of BL6 cells down to 15% of that in the untreated control mice. MI-18 was safe because even after >7 weeks of daily injections, the survival rate of the mice was 93%. We propose that MI-18 may serve as a novel and clinically important prototype of a potent inhibitor of spontaneous metastasis.     

Gap junction expression following UVC-induced neoplastic transformation in human hybrid cell lines

Decreased connexin gene expression and loss of the capacity for either homologous or heterologous intercellular communication has been associated with neoplastic transformation. We tested the hypothesis that loss of gap junctional intercellular communication (GJIC) correlates with tumorigenic potential in the HeLa x skin fibroblast human hybrid cell system. Connexin gene expression, gap junction function and tumorigenicity were determined for the non-tumorigenic somatic hybrid cell line (CGL1) and a series of UVC-induced tumorigenic cell lines derived from CGL1. CGL1 and the parental skin fibroblasts express connexin43 (alpha1 gap junction gene) mRNA and protein, form gap junctional plaques and have functional gap junctions. UVC- irradiation of CGL1 cells produced a cell line (UV12) with an aggressive tumorigenic phenotype, which lost connexin43 expression as well as both homologous and heterologous GJIC and was in this respect similar to HeLa cells. However, the phenotype of UV12 cells exhibited some instability and revertants to a less aggressive tumorigenic phenotype were isolated. These cells expressed connexin43 mRNA and protein, and demonstrated homologous GJIC. Furthermore, cells reconstituted from a tumor derived from this revertant cell line retained significant connexin43 expression and homologous GJIC, although they exhibited an aggressive tumorigenic phenotype. Thus, functional homologous GJIC cannot be dissociated from tumorigenicity in this system. However, heterologous GJIC between these same UVC-induced tumorigenic cell lines and normal human skin fibroblasts was reduced, whereas the non-tumorigenic hybrid cells showed extensive heterologous GJIC. In summary, re-acquisition of connexin43 expression and homologous GJIC does not restore the non-tumorigenic phenotype in UVC- induced tumorigenic HeLa skin fibroblast human hybrid cells. However, reduction of heterologous GJIC does correlate with tumorigenicity in this cell system.