Suppression Of Gap Junctional Intercellular Communication Research Articles

Abstract A01: Environmentally prevalent polycyclic aromatic hydrocarbons elicit cocarcinogenic properties in human and mouse lung cells

Abstract Rationale: Polycyclic aromatic hydrocarbons (PAHs) are prevalent contaminants in our environment, workplace, and in first- and secondhand smoke from both cigarettes and marijuana. The toxicity of low molecular weight (LMW) PAHs compared to high molecular weight PAHs (e.g., benzo[a]pyrene, B[a]P) is not well studied in lung cells. Our overall hypothesis was that LMW PAHs act as cocarcinogens in the presence of a known carcinogen (B[a]P) in human and mouse lung epithelial cells. Gap junctions are commonly suppressed during tumor promotion, while DNA-adduct formation and micronuclei (MN) are observed during the initiation stage of cancer. We used these endpoints together as markers of carcinogenicity in Beas2B and C10 cells. Methods: Beas2B, a human bronchial epithelial cell line, and C10 cells, a nontumorigenic type II alveolar pneumocyte cell line, were used. LMW PAHs (1-methylanthracene, 1-MeA, and fluoranthene, Flthn) were applied in a 1:1 ratio mixture based on previous studies and B[a]P as a classic carcinogen. Cells were treated for 30 min.-24 h with the LMW PAH mixture, B[a]P, or both, and assessed for the following endpoints: cytotoxicity, genotoxicity (MN), apoptosis, proliferation, gap junctional intercellular communication (GJIC) by the lucifer yellow scalpel-loaded/dye-transfer assay, and DNA adducts of B[a]P by HPLC with fluorescence detection in C10 cells. Results: LMW PAHs are more cytotoxic in the human Beas2B cells than the mouse C10 cells. Significant toxicity is observed in Beas2B cells at 0.3 uM B[a]P combined with the LMW PAH mixture (0.1-1 uM), while no toxicity is observed in C10 cells at identical doses. No significant apoptosis was observed in Beas2B cells while significant increases in S-phase were observed in response to B[a]P combined with LMW PAH mixture compared to B[a]P alone. B[a]P in combination with the LMW PAH mixture significantly inhibited GJIC in C10 and Beas2B cells compared to B[a]P or the LMW PAH mixture alone, supporting a role for GJIC suppression in cancer development. B[a]P diol-epoxide (BPDE)-DNA adduct levels were also significantly induced in C10 cells at 1.0 uM B[a]P with the LMW PAH mixture at several doses (less than or equal to 1.0 uM) and significant increases in BPDE-DNA adduct formation compared to B[a]P alone were observed. Lastly, MN were also observed in both cell lines and significantly differed between B[a]P alone and in combination with the LMW PAH mixture. DNA adduct formation and genotoxicity coincided with GJIC inhibition. Conclusion: These studies provide evidence that LMW PAHs in combination with B[a]P can elicit increased carcinogenic potential in human and mouse cells. Support: Flight Attendant Medical Research Institute (CIA130022, AKB); the National Institute for Environmental Health Sciences (R15ES024893, AKB). Citation Format: Alison K. Bauer, Katelyn J. Siegrist, Sabine Plöttner, Thomas Brüning, Heiko U. Käfferlein. Environmentally prevalent polycyclic aromatic hydrocarbons elicit cocarcinogenic properties in human and mouse lung cells [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A01.

Benzo[a]pyrene-decreased gap junctional intercellular communication via calcium/calmodulin signaling increases apoptosis in TM4 cells.

The mechanism of male reproductive toxicity induced by benzo[a]pyrene (BaP) is poorly understood. Gap junctional intercellular communication (GJIC) is known to play a critical role in maintaining spermatogenesis. The aim of the present study was to determine the toxic effects of BaP in Sertoli cells, and to explore the possibility and potential mechanisms of BaP-induced changes in the level of GJIC, and the relationship between GJIC and BaP-induced apoptosis. We treated mouse Sertoli cell lines (TM4) with different concentrations (0.1-100μm) of BaP for 1-48hours, and found that GJIC exhibited a dose- and time-dependent downregulation. Treatment with 10μm BaP increased apoptosis, intracellular Ca2+ level ([Ca2+ ]i ) and calmodulin (CaM) protein expression, and decreased the protein level of connexin 43 (Cx43) (also known as gap junction α-1 protein [GJA1]) in TM4 cells. However, BaP had no effect on the phosphorylation of Cx43 at Ser279/282, Ser255, Ser368 or Ser262. Downregulation of [Ca2+ ]i by BAPTA-AM significantly attenuated the BaP-induced GJIC suppression, Cx43 protein decrease and CaM protein increase. Interestingly, inhibition of CaM expression by W7 partially recovered BaP-induced GJIC inhibition, but had no effect on BaP-induced Cx43 protein decrease. Pretreatment with the GJIC activator retinoic acid significantly mitigated BaP-induced apoptosis. In conclusion, these results suggest that BaP can decrease GJIC via Ca2+ /CaM signaling, and that BaP-induced GJIC suppression increases apoptosis in TM4 cells.

23. Constitutively Active Signal Transducer and Activator of Transcription-3; an Oncogene that Increases Gap Junctional, Intercellular Communication


 Gap junctions are specialized intercellular channels that connect the cytoplasm of adjacent cells and mediate the direct exchange of small ions and molecules between them. Gap junctional intercellular communication (GJIC) is usually blocked, or down regulated, in cells transformed by oncogenes, such as Src. One of the Src effector pathways leading to transformation and GJIC suppression is the Ras/Raf/MEK/ERK pathway that has a prominent etiological role in cell proliferation, differentiation and cell survival; inhibition of this pathway in vSrc transformed cells restores GJIC. In addition, the distinct downstream effector of Src that is obligatory for neoplasia is the signal transducer and activator of transcription-3 (Stat3). Stat3 is up regulated in a range of tumors, and a modified version, the constitutively active form of Stat3 (Stat3C), has been shown to function as an oncogene. To examine the role of Stat3 upon the Src-mediated, GJIC suppression, Stat3 was down regulated in rat liver epithelial T51B cells expressing activated Src. The extent of GJIC was determined by the migration of the fluorescent dye, Lucifer Yellow, through adherent cells subsequent to electroporation. The results demonstrate that, contrary to inhibition of the Ras pathway, Stat3 inhibition in cells expressing activated Src does not restore GJIC. On the contrary, Stat3 inhibition in normal cells with high GJIC levels eliminated junctional permeability. Interestingly, our results also demonstrate that expression of Stat3C T51B cells and human lung cancer SK-LuCi6 cells, which have extensive communication and low Src levels, increased GJIC. Therefore, Stat3 is actually required for and increases junctional permeability.

Environmentally prevalent polycyclic aromatic hydrocarbons can elicit co-carcinogenic properties in an in vitro murine lung epithelial cell model

Low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAH) are the most abundant PAHs environmentally, occupationally, and are in cigarette smoke; however, little is known about their carcinogenic potential. We hypothesized that LMW PAHs act as co-carcinogens in the presence of a known carcinogen (benzo[a]pyrene (B[a]P)) in a mouse non-tumorigenic type II cell line (C10 cells). Gap junctions are commonly suppressed and inflammation induced during tumor promotion, while DNA-adduct formation is observed during the initiation stage of cancer. We used these endpoints together as markers of carcinogenicity in these lung adenocarcinoma progenitor cells. LMW PAHs (1-methylanthracene and fluoranthene, 1–10 µM total in a 1:1 ratio) were used based on previous studies as well as B[a]P (0–3 µM) as the classic carcinogen; non-cytotoxic doses were used. B[a]P-induced inhibition of gap junctional intercellular communication (GJIC) was observed at low doses and further reduced in the presence of the LMW PAH mixture (P < 0.05), supporting a role for GJIC suppression in cancer development. Benzo[a]pyrene diol-epoxide (BPDE)-DNA adduct levels were significantly induced in B[a]P-treated C10 cells and additionally increased with the LMW PAH mixture (P < 0.05). Significant increases in cyclooxygenase (Cox-2) were observed in response to the B[a]P/LMW PAH mixture combinations. DNA adduct formation coincided with the inhibition of GJIC and increase in Cox-2 mRNA expression. Significant cytochrome p4501b1 increases and connexin 43 decreases in gene expression were also observed. These studies suggest that LMW PAHs in combination with B[a]P can elicit increased carcinogenic potential. Future studies will further address the mechanisms of co-carcinogenesis driving these responses.

Differential effects of polyoma virus middle tumor antigen mutants upon gap junctional, intercellular communication

Gap junctions are channels that connect the cytoplasm of adjacent cells. Oncogenes such as the middle Tumor antigen of polyoma virus (mT) are known to suppress gap junctional, intercellular communication (GJIC). mT associates with and is tyrosine-phosphorylated by cSrc family members. Specific mT phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) or the SH2 domain of the regulatory subunit of the phosphatidylinositol-3 kinase (PI3k, mT-tyr315). Binding results in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI3 kinase/Akt, respectively, both of which are needed for full neoplastic transformation.To examine the effect of mT-initiated pathways upon gap junctional communication, GJIC was quantitated in rat liver epithelial T51B cells expressing mT-mutants, using a novel technique of in situ electroporation. The results demonstrate for the first time that, although even low levels of wild-type mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr-250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI3k pathway is not required for GJIC suppression, indicating that GJIC suppression is independent of full neoplastic conversion and the concomitant morphological changes. Interestingly, expression of a constitutively active, myristylated form of the catalytic subunit of PI3k, p110, or the constitutively active mutants E545K and H1047R increased GJIC, while pharmacological inhibition of PI3k eliminated communication. Therefore, although PI3k is growth promoting and in an activated form it can act as an oncogene, it actually plays a positive role upon gap junctional, intercellular communication.

Abstract 3114: Constitutively active signal transducer and activator of transcription-3: an oncogene that increases gap junctional intercellular communication.

Abstract Gap junctions are specialized intercellular channels that connect the cytoplasm of adjacent cells and mediate the direct exchange of small ions and molecules between them. Gap junctional intercellular communication (GJIC) is usually blocked, or downregulated, in cells transformed by oncogenes, such as Src. One of the Src effector pathways leading to transformation and GJIC suppression is the Ras/Raf/MEK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) pathway that has a prominent etiological role in cell proliferation, differentiation and cell survival; inhibition of this pathway in vSrc transformed cells restores GJIC. In addition, the distinct downstream effector of Src that is obligatory for neoplasia is the signal transducer and activator of transcription-3 (Stat3). Stat3 has the intrinsic characteristic of being upregulated in a range of tumors, and a modified version, the constitutively active form of Stat3 (Stat3C), has been shown to function as an oncogene. Conversely, inhibition of Stat3 can suppress tumor growth. To examine the role of Stat3 upon the Src-mediated, GJIC suppression, Stat3 was downregulated in rat liver epithelial T51B cells expressing activated Src through treatment with the CPA7, Stat3 inhibitor, or through infection with a retroviral vector expressing a Stat3-specific shRNA. The extent of GJIC was determined by electroporating the fluorescent dye, Lucifer yellow, into cells grown on two co-planar electrodes of electrically conductive, optically transparent, indium-tin oxide, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. The results demonstrate that, contrary to inhibition of the Ras pathway, Stat3 inhibition in cells expressing activated Src does not restore GJIC. On the contrary, Stat3 inhibition in normal cells with high GJIC levels eliminated junctional permeability. Interestingly, our results also demonstrate that expression of Stat3C in T51B cells and human lung cancer SK-LuCi6 cells, which have extensive communication and low Src levels, increased GJIC. Therefore Stat3, although it is generally growth promoting and in an activated form can act as an oncogene, its function is actually required for and increases junctional permeability. Citation Format: Aaron Trotman-Grant, Mulu Geletu, Leda Raptis. Constitutively active signal transducer and activator of transcription-3: an oncogene that increases gap junctional intercellular communication. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2013-3114

Abstract 31: Activated phosphatidylinositol-3 kinase: An oncogene that increases gap junctional, intercellular communication

Abstract Gap junctions are channels that connect the cytoplasm of adjacent cells. Gap junctional, intercellular communication (GJIC) is blocked in cells transformed by oncogenes such as the middle Tumor antigen of polyoma virus (mT), an oncoprotein which associates with and is tyrosine-phosphorylated by cSrc family members. Specific phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) and the SH2 domain of the phosphatidylinositol 3-kinase (mT-tyr315), resulting in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI-3 kinase/Akt, respectively. To examine the effect of these mT-initiated pathways upon gap junctional communication and neoplasia, GJIC was assessed in mT-mutant-expressing, rat liver epithelial T51B cells which normally have extensive GJIC, using a novel technique of in situ electroporation we developed. The results show that although low levels of wt-mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI-3 kinase pathway is not required for GJIC suppression. It is remarkable that T51B cells expressing a mutant deficient in binding of Shc, hence activation of the Ras pathway, are still morphologically transformed and do grow into tumors in syngeneic rats, albeit with an altered morphology. These results indicate that GJIC suppression requires Ras but not PI-3 kinase/Akt signalling, and is independent of full neoplastic conversion in rat liver epithelial cells. Since PI3k activation by mT increases GJIC, we examined whether PI3k might have a positive role upon GJIC. The results showed that PI3k pharmacological inhibition eliminates GJIC, concomitant with apoptosis induction. We next expressed a form of PI3k rendered constitutively active through the addition of a myristylation signal (myr-PI3k). The results demonstrated that myr-PI3k causes an increase in GJIC. Therefore PI3k, although in an activated form it can act as an oncogene, it plays a positive role upon gap junctional, intercellular communication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 31. doi:1538-7445.AM2012-31

Abstract 3123: Differential effects of polyoma virus middle tumor antigen mutants upon gap junctional, intercellular communication

Abstract Gap junctions are channels that connect the cytoplasm of adjacent cells. Gap junctional, intercellular communication (GJIC) is blocked in cells transformed by oncogenes such as the middle Tumor antigen of polyoma virus (mT), an oncoprotein which associates with and is tyrosine-phosphorylated by cSrc family members. Specific phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) and the SH2 domain of the phosphatidylinositol 3-kinase (mT-tyr315), resulting in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI-3 kinase/Akt, respectively. To examine the effect of these mT-initiated pathways upon gap junctional communication and neoplasia, GJIC was assessed in mT-mutant-expressing, rat liver epithelial T51B cells which normally have extensive GJIC, using a novel technique of in situ electroporation we developed. The results show that although low levels of wt-mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI-3 kinase pathway is not required for GJIC suppression. It is remarkable that T51B cells expressing a mutant deficient in binding of Shc, hence activation of the Ras pathway, are still morphologically transformed and do grow into tumors in syngeneic rats, albeit with an altered morphology. These results indicate that GJIC suppression requires Ras/Erk but not PI-3 kinase/Akt signalling, and is independent of neoplastic conversion in rat liver epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2011-3123

Stat3 Activity Is Required for Gap Junctional Permeability in Normal Rat Liver Epithelial Cells

Neoplastic transformation by oncogenes such as activated Src is known to suppress gap junctional, intercellular communication (GJIC). One of the Src effector pathways leading to GJIC suppression and transformation is the Ras/Raf/Mek/Erk, so that inhibition of this pathway in vSrc-transformed cells restores GJIC. A distinct Src downstream effector required for neoplasia is the signal transducer and activator of transcription-3 (Stat3). To examine the role of Stat3 upon the Src-mediated, GJIC suppression, Stat3 was downregulated in rat liver epithelial cells expressing activated Src through treatment with the CPA7, Stat3 inhibitor, or through infection with a retroviral vector expressing a Stat3-specific shRNA. GJIC was examined by electroporating the fluorescent dye, Lucifer yellow, into cells grown on two coplanar electrodes of electrically conductive, optically transparent, indium-tin oxide, followed by observation of the migration of the dye to the adjacent, nonelectroporated cells under fluorescence illumination. The results demonstrate that, contrary to inhibition of the Ras pathway, Stat3 inhibition in cells expressing activated Src does not restore GJIC. On the contrary, Stat3 inhibition in normal cells with high GJIC levels eliminated junctional permeability. Therefore, Stat3's function is actually required for the maintenance of junctional permeability, although Stat3 generally promotes growth and in an activated form can act as an oncogene.

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.

STUDY ON GAP JUNCTIONAL INTERCELLULAR COMMUNICATION INHIBITION BY ELF MAGNETIC FIELDS USING FRAP METHOD

Inhibition of gap junctional intercellular communication (GJIC) is an important event in the multistage process of carcinogenesis. Our previous study showed that extremely low frequency (ELF) magnetic fields (MFs) inhibit GJIC, and enhance the suppression of GJIC induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) using a microinjection technique. In the present study, the inhibition of GJIC by ELF MFs and its threshold were further studied with fluorescence recovery after photobleaching (FRAP) technique. The results indicated that the FRAP technique is more sensitive in detecting the changes of GJIC than microinjection, and the threshold level is 0.4 mT for GJIC suppression by 50 Hz MFs. In addition, 0.2 mT, or more than 0.2 mT ELF can enhance the inhibition of GJIC induced by TPA. We concluded that MFs thus might act as a cancer promoter or work in synergy with other cancer promoters. The data also provide grounds to revise the reference standard of ELF MFs exposure.

ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells.

The effects of extremely low frequency (ELF) magnetic field on gap junctional intercellular communication (GJIC), protein levels, and phosphorylation of connexin43 (Cx43) were studied in NIH3T3 cells. The suppression of GJIC by 24 h, 50 Hz, 0.8 mT ELF magnetic field, 2 h, 3 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or ELF combined with TPA treatment was confirmed by the fluorescence recovery after photobleaching (FRAP) analysis with a confocal microscope. The results showed that ELF or TPA exposure induced 50-60% inhibition of GJIC (P < 0.01). ELF combined with TPA enhanced the inhibition of GJIC. Western blot analysis using Cx43 specific antibodies showed obviously decreasing non phosphorylated Cx43 (P(0)) induced by ELF and/or TPA exposure. On the other hand, cells treated with ELF and/or TPA displayed a hyperphosphorylated Cx43 band (P(3)). However, there was no obvious changes in the level of Cx43 protein. The results implied that the P(3) band appeared to result from phosphorylation of P(0). But it remains possible that upon the ELF exposure P(0) is converted to P(1), P(2) or both and that P(3) is formed from P(1) or P(2) resulting in the observed hyperphosphorylation pattern. From the present study, we conclude that ELF magnetic field inhibits GJIC and the main mechanism is the hyperphosphorylation of Cx43.

The Mechanism of Suppression of GAP Junctional Intercellular Communication by 50-Hz Magnetic Fields

In our previous studies, we found that a 50-Hz magnetic field exposure inhibited gap junctional intercellular communication (GJIC). A pulsed magnetic field (PMF) is more effective than a 50-Hz sinusoidal magnetic field in downregulation of the GJIC. Protein kinase C (PKC) is known to phosphorylate connexins and to downregulation gap junctions. Several investigators have noted that extremely low-frequency magnetic field exposure activates PKC. We hypothesized that PKC may be involved in the suppression of GJIC by a PMF. An experiment with the PKC inhibitor stawosporine (STS) or palmitoyl carnitine (PMC) was carried out. Chinese hamster lung (CHL) cells were exposed to 50 Hz at an average flux density of 0.8 mT for 24 h. They were treated with PKC inhibitor (STS or PMC) at different concentrations during the last hour. The results showed that the suppression of GJIC induced by PMF was dramatically inhibited in the presence of 10 nM STS or 10 μM PMC. We conclude that the inhibition of GJIC induced by a PMF m...

Exposure to 50-HZ Electromagnetic Fields: Effects of Time and Field Strength on GAP Junctional Intercellular Communications

The relationship between a dose of 50-Hz magnetic field (MF) and its effects on suppression of gap junctional intercellular communication (GJIC) function were studied. An experiment was conducted on the effect of exposure to different intensities of 50-Hz MFs with the same exposure duration, and to different exposure durations of MF at the same intensity on GJIC in Chinese hamster lung (CHL) cells. Lucifer yellow, a fluorescent dye, was inserted into single CHL cells by iontophoretic injection (dye transfer assay). The number of dye-coupled cells (DCC) 5 min after injection was used as an indicator of GJIC function. The results showed that suppression of GJIC by 50-Hz MFs depended on the magnetic flux density. Lower MF flux densities at 0.05, 0.2, and 0.4 mT for 24 h did not inhibit GJIC function. Higher MF flux densities (0.8 and 1.6 mT) did inhibit. No' “amplitude window effect” was found with MF exposure at 0-1.6 mT. MF exposure at a fixed intensity of 0.8 mT for 5 min had no effects, but a 1-h exposur...