Bystander effect in uracil phosphoribosyltransferase/5-fluorouracil-mediated suicide gene therapy is correlated with the level of intercellular communication

We examined whether a suicide gene/prodrug system using the uracil phosphoribosyltransferase (UPRT) of E. coli origin and 5-fluorouracil (5-FU) could achieve a bystander effect in two rodent tumor cell lines, murine colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells. Cytotoxicity tests of mixed populations consisting of parent and transduced cells showed that the bystander effect was not produced in Colon 26 cells in either the UPRT/5-FU system or the herpes simplex virus-thymidine kinase/ganciclovir system but a strong bystander effect was evidenced by both suicide gene systems in 9L cells. The expression level of connexin 43, a protein that constitutes gap junctions, was high in 9L but low in Colon 26 cells. A gap junction-permeable fluorescein dye could be transferred among 9L cells but hardly at all among Colon 26 cells. Taken together, the efficacy of the bystander effect in the UPRT/5-FU system can be affected by gap junction-mediated intercellular communication.     

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.      

Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication

Antitumor suicide gene therapy is one of the emerging strategies against cancer. It consists of the introduction into cancer cells of a gene capable of converting a nontoxic prodrug into a cytotoxic drug. Because this therapeutic gene cannot be easily introduced into the whole cell population of a tumor, the successful eradication of tumors depends on a phenomenon called the "bystander effect," by which the introduced gene can affect even cells in which it is not itself present. From a therapeutic point of view, it may be crucial to enhance this phenomenon through various means to achieve tumor eradication. One such suicide gene, the thymidine kinase gene from the herpes simplex virus, in combination with the prodrug ganciclovir, has been extensively and successfully used in some animal models exhibiting a strong bystander effect. Among the mechanisms involved in this phenomenon, gap junctional intercellular communication (GJIC) is directly involved in the transfer of the toxic metabolites of ganciclovir, which pass directly from herpes simplex virus thymidine kinase-expressing cells to surrounding cells that do not express it. Because GJIC appears to be a mediator of the bystander effect both in vitro and in vivo, here we review possible molecular strategies for enhancing the extent of tumor cell death by increasing the intratumoral GJIC capacity.     

Suppressed gap junctional intercellular communication in carcinogenesis of endometrium.

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

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

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

Changes in gap junctional intercellular communication in mouse skin carcinogenesis

Gap junction intercellular communication (GJIC) has been measured in cell lines that represent different stages of chemically induced mouse skin carcinogenesis. No significant difference in GJIC, as measured by dye spread, was found in cultures of normal keratinocyte, papilloma or squamous carcinoma cell lines. There was no correlation, in this system, between the presence of a mutant Ha-ras gene and down- regulation of communication. There was, however, a marked decrease in GJIC (80-90%) on progression from squamous to spindle carcinoma cells. Measurement of GJIC in somatic cell hybrids shows that the genetic defect responsible for this down-regulation is recessive and is common to two independently isolated spindle cell lines. No abnormalities were found in the spindle cells in expression of connexin 43, a cell component involved in gap junction formation and permeability. However, expression of E-cadherin, a cell-cell adhesion molecule implicated in the process of gap junction formation, was missing in the spindle carcinoma cells. Introduction of an E-cadherin cDNA into the spindle cells partially restored junctional communication without causing any noticeable alterations in cell morphology. During the study a non- tumourigenic keratinocyte line, a sub-clone of a normal keratinocyte line, was also found to have a low level of GJIC. However, the defect in this line was shown, by genetic complementation in somatic cell hybrids, to be different from that in the spindle carcinoma cell lines. Consistent with these data, analysis by immunofluorescence shows an abnormal distribution of connexin 43 in these cells.      

Effect of DDT on hepatic gap junctional intercellular communication in rats

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

依托咪酯对神经胶质瘤细胞缝隙连接通讯的影响

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

Modulation of phenobarbitone-induced loss of gap junctional intercellular communication in hepatocyte couplets

Phenobarbitone (PB) produced a dose-and time-dependent decreasein gap junctional intercellular communication (GJIC) (up to25.0 ± 5.3% inhibition) in rat hepatocyte couplets (4h cultures). The effect was reversible and independent of proteinsynthesis. This inhibition was exacerbated (to 53.3 ±5.4% inhibition) by depletion of intracellular glutathione followingpretreatment with diethylmaleate (0.5 µM, 15 min). Inhibitionwas also significantly enhanced by addition of the cytochromeP450 inhibitors SKF 525A (25 µM) and metyrapone (20 nM).In contrast, hepatocyte couplets derived from rats pretreatedwith PB (0.1% w/v in drinking water) for up to 28 days werefully functional regarding GJIC and were found to be refractoryto the effects of PB added in vitro. This, coupled with thelack of effect of p-hydroxy-PB, suggests that an active metaboliteof PB is not involved in the inhibition of GJIC which may, instead,be through an oxidative stress, which is prevented by glutathione.     

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.     

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.     

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

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

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

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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.     

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

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

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.     

Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells

Modulation of gap junctional intercellular communication (GJIC)was studied in a multistep model of human renal epithelial carcinogenesis.We report that the majority of primary human kidney epithelialcells (NHKE) grown from fetal kidney explants did not communicatethrough gap junctions. Communication could, however, be observedwithin a subpopulation of the cells. Ni(II)-immortalized cells(IHKE) showed GJIC at a level of 10–20 communicating cells,but with heterogeneous regions on the dish, with regard to bothcommunication and distribution of connexin43. The heterogeneitywas less pronounced in a ras-transfected tumourigenic cell line(THKE), which also showed communication of     

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

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

Potential role of the human Ha-ras oncogene in the inhibition of gap junctional intercellular communication

The modulation of gap junctional intercellular communication (GJIC) plays an important role during tumor promotion. Several tumor-promoting agents are known to inhibit this form of cellular coupling. In addition, tumor cells and cells expressing certain oncogenic products have been shown to exhibit inhibited or reduced GJIC. The Ha-ras oncogene is expressed in a wide variety of human tumors from different tissues. Its p21 product is a membrane-bound polypeptide, the function of which is not fully characterized. We tested the effects of the expression of the human c-Ha-ras-1 oncogene, derived from the EJ/T4 bladder carcinoma cell line, on the ability of the Chinese hamster V79 cells to conduct gap Junctional communication. The Junctional competence was studied by two different methods, the scrape-loading/dye transfer technique and the metabolic cooperation assay. The results indicate a strong correlation between the expression of p21 ras protein and the inhibition of gap Junctional function. Assuming that reversible inhibition of intercellular communication plays a role during tumor promotion and stable inhibition during the tumor progression phase of carcinogenesis, our data suggest that, while chemical tumor promoters and the ras oncogenes might work by different biochemical mechanisms, they both affect a critical cellular function; namely, GJIC.