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Akira Ikari - One of the best experts on this subject based on the ideXlab platform.

  • chrysin enhances anticancer drug induced toxicity mediated by the reduction of claudin 1 and 11 expression in a spheroid culture model of lung squamous cell carcinoma cells
    Scientific Reports, 2019
    Co-Authors: Ryohei Maruhashi, Satoshi Endo, Takumi Furuta, Toshiyuki Matsunaga, Hiroaki Eguchi, Risa Akizuki, Shohei Hamada, Kenji Ichihara, Akira Ikari
    Abstract:

    The aberrant expression of claudins (CLDNs), which are tight junctional proteins, is seen in various solid tumors, but the regulatory mechanisms and their pathophysiological role are not well understood. Both CLDN1 and CLDN11 were highly expressed in human lung squamous cell carcinoma (SCC). Chrysin, found in high concentration in honey and propolis, decreased CLDN1 and CLDN11 expression in RERF-LC-AI cells derived from human lung SCC. The phosphorylation level of Akt was decreased by chrysin, but those of ERK1/2 and c-Jun were not. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited the phosphorylation of Akt and decreased the expression levels of CLDN1 and CLDN11. The association between phosphoinositide-dependent kinase 1 (PDK1) and Akt was inhibited by chrysin, but the phosphorylation of PDK1 was not. Immunoprecipitation and quartz-crystal microbalance assays revealed that biotinylated-chrysin binds directly to Akt. The knockdown of CLDN1 and CLDN11 using small interfering RNAs increased the transepithelial flux of doxorubicin (DXR), an anthracycline anticancer drug. Similarly, both chrysin and LY-294002 increased DXR flux. Neither CLDN1 knockdown, CLDN11 knockdown, nor chrysin changed the anticancer drug-induced cytotoxicity in a two-dimensional culture model, whereas they enhanced cytotoxicity in a spheroid culture model. Taken together, chrysin may bind to Akt and inhibit its phosphorylation, resulting in the elevation of anticancer drug-induced toxicity mediated by reductions in CLDN1 and CLDN11 expression in RERF-LC-AI cells. We suggest that chrysin may be useful as an adjuvant chemotherapy in lung SCC.

  • decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin 1 in spheroid culture models of human lung adenocarcinoma a549 cells
    Biochimica et Biophysica Acta, 2018
    Co-Authors: Risa Akizuki, Satoshi Endo, Takumi Furuta, Toshiyuki Matsunaga, Ryohei Maruhashi, Hiroaki Eguchi, Kazuki Kitabatake, Mitsutoshi Tsukimoto, Akira Ikari
    Abstract:

    Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.

  • claudin 5 7 and 18 suppress proliferation mediated by inhibition of phosphorylation of akt in human lung squamous cell carcinoma
    Biochimica et Biophysica Acta, 2017
    Co-Authors: Risa Akizuki, Toshiyuki Matsunaga, Satoshi Endo, Shun Shimobaba, Akira Ikari
    Abstract:

    Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. However, it is unknown which subtype plays a key role in the regulation of proliferation in cancer cells. The expression of CLDN3-5, 7, and 18 in human lung squamous carcinoma tissues was lower than that in normal tissue. Here, we examined which combination of exogenous CLDNs expression inhibits proliferation and the molecular mechanism using human lung squamous RERF-LC-AI cells. Real-time polymerase chain reaction and western blotting showed that CLDN3-5, 7, and 18 are little expressed in RERF-LC-AI cells. In the exogenously transfected cells, CLDN5, 7, and 18 were distributed in the cell-cell contact areas concomitant with ZO-1, a tight junctional scaffolding protein, whereas CLDN3 and 4 were not. Cell proliferation was individually and additively suppressed by CLDN5, 7, and 18. The expression of these CLDNs showed no cytotoxicity compared with mock cells. CLDN5, 7, and 18 increased p21 and decreased cyclin D1, resulting in the suppression of cell cycle G1-S transition. The expression of these CLDNs inhibited phosphorylation of Akt without affecting phosphorylated ERK1/2. Furthermore, these CLDNs inhibited the nuclear localization of Akt and its association with 3-phosphoinositide-dependent protein kinase-1 (PDK1). The suppression of G1-S transition caused by CLDN5, 7, and 18 was rescued by the expression of constitutively active-Akt. We suggest that the reduction of CLDN5, 7, and 18 expression loses the suppressive ability of interaction between PDK1 and Akt and causes sustained phosphorylation of Akt, resulting in the disordered proliferation in lung squamous carcinoma cells.

Yves Mely - One of the best experts on this subject based on the ideXlab platform.

  • characterisation of endogenous claudin 1 expression motility and susceptibility to hepatitis c virus in crispr knock in cells
    Biology of the Cell, 2020
    Co-Authors: Camille M H Cleme, Maika Deffieu, Cristina M. Dorobantu, Yves Mely, Thomas F Aume, Nilda Vanesa Ayalanunez
    Abstract:

    BACKGROUND INFORMATION: Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. RESULTS: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs. CONCLUSIONS: The Huh7.5.1 TagRFP-CLDN1+/+ edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging. SIGNIFICANCE: Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.

  • Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.
    Biology of the Cell, 2020
    Co-Authors: Camille M H Clément, Maika Deffieu, Cristina M. Dorobantu, Thomas F. Baumert, Nilda Vanesa Ayala-nunez, Yves Mely, Philippe Ronde, Raphael Gaudin
    Abstract:

    Background Information: Claudin‐1 (CLDN1) is a four‐span transmembrane protein localised at cell–cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up‐regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. Results: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN‐1, allowing dynamic monitoring of CLDN‐1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP‐T protein fused at the N‐terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV‐G‐decorated pseudoparticles. Nonetheless, the TagRFP‐CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild‐type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP‐CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP‐CLDN1 proteins were stalled at TJs. Conclusions: The Huh7.5.1 TagRFP‐CLDN1+/+ edited cell line showed physiological features comparable to that of non‐edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging.

Stephen B Howell - One of the best experts on this subject based on the ideXlab platform.

  • regulation of the epithelial mesenchymal transition by claudin 3 and claudin 4
    PLOS ONE, 2013
    Co-Authors: Xinjian Lin, Xiying Shang, Gerald Manorek, Stephen B Howell
    Abstract:

    The mechanisms that control intracellular adhesion are central to the process of invasion and metastasis. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are major structural molecules of the tight junctions that link epithelial cells. Our prior work has demonstrated that knockdown of the expression of either CLDN3 or CLDN4 produces marked changes in the phenotype of ovarian carcinoma cells including increases in growth rate in vivo, migration, invasion, metastasis, and drug resistance, similar to those produced by the epithelial-to-mesenchymal transition (EMT). We postulated that these changes may result from the ability of CLDN3 or CLDN4 to suppress EMT. In this study we found that knockdown of either CLDN3 or CLDN4 increased cell size and resulted in flattened morphology. While knockdown of CLDN3 or CLDN4 did not alter the expression of vimentin, it significantly down-regulated the level of E-cadherin and up-regulated N-cadherin expression. Conversely, over-expression of CLDN3 or CLDN4 in a cell line that does not express endogenous CLDN3 or CLDN4 decreased N-cadherin expression. Re-expression of E-cadherin in the CLDN3 or CLDN4 knockdown cells reduced migration, invasion and tumor growth in vivo. Loss of either CLDN3 or CLDN4 resulted in activation of the PI3K pathway as evidenced by increased Akt phosphorylation, elevated cellular PIP3 content and PI3K activity as well as up-regulation of the mRNA and protein levels of the transcription factor Twist. Taken together, these findings suggest that CLDN3 and CLDN4 function to sustain an epithelial phenotype and that their loss promotes EMT.

  • claudin 3 and claudin 4 regulate sensitivity to cisplatin by controlling expression of the copper and cisplatin influx transporter ctr1
    Molecular Pharmacology, 2013
    Co-Authors: Xiying Shang, Xinjian Lin, Gerald Manorek, Stephen B Howell
    Abstract:

    Claudin-3 (CLDN3) and claudin-4 (CLDN4) are the major structural molecules that form tight junctions (TJs) between epithelial cells. We found that knockdown of the expression of either CLDN3 or CLDN4 produced marked changes in the phenotype of ovarian cancer cells, including an increase in resistance to cisplatin (cDDP). The effect of CLND3 and CLDN4 on cDDP cytotoxicity, cDDP cellular accumulation, and DNA adduct formation was compared in the CLDN3- and CLDN4-expressing parental human ovarian carcinoma 2008 cells and CLDN3 and CLDN4 knockdown sublines (CLDN3KD and CLDN4KD, respectively). Knockdown of CLDN3 or CLDN4 rendered human ovarian carcinoma 2008 cells resistant to cDDP in both in vitro culture and in vivo xenograft model. The net accumulation of platinum (Pt) and the Pt-DNA adduct levels were reduced in CLDN3KD and CLDN4KD cells. The endogenous mRNA levels of copper influx transporter CTR1 were found to be significantly reduced in the knockdown cells, and exogenous expression of CTR1 restored their sensitivity to cDDP. Reexpression of an shRNAi-resistant CLDN3 or CLDN4 up-regulated CTR1 levels, reversed the cDDP resistance, and enhanced TJ formation in the knockdown cells. Baseline copper (Cu) level, Cu uptake, and Cu cytotoxicity were also reduced in CLDN3KD and CLDN4KD cells. Cu-dependent tyrosinase activity was also markedly reduced in both types of CLDN knockdown cells when incubated with the substrate l-DOPA. These results indicate that CLDN3 and CLDN4 affect sensitivity of the ovarian cancer cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1.

  • tight junction proteins claudin 3 and claudin 4 control tumor growth and metastases
    Neoplasia, 2012
    Co-Authors: Xiying Shang, Xinjian Lin, Gerald Manorek, Edwin A Alvarez, Stephen B Howell
    Abstract:

    The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling.

  • recombinant cpe fused to tumor necrosis factor targets human ovarian cancer cells expressing the claudin 3 and claudin 4 receptors
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Xiaoqin Yuan, Xinjian Lin, Gerald Manorek, Isao Kanatani, Lawrence H Cheung, Michael G Rosenblum, Stephen B Howell
    Abstract:

    Using gene expression profiling, others and we have recently found that claudin-3 (CLDN3) and claudin-4 (CLDN4) are two of the most highly and consistently up-regulated genes in ovarian carcinomas. Because these tight junction proteins are the naturally occurring receptors for Clostridium perfringens enterotoxin (CPE), in this study, we used the COOH-terminal 30 amino acids of the CPE (CPE(290-319)), a fragment that is known to retain full binding affinity but have no cytolytic effect, to target tumor necrosis factor (TNF) to ovarian cancers. We constructed a pET32-based vector that expressed the fusion protein, designated here as CPE(290-319)-TNF, in which CPE(290-319) was fused to TNF at its NH(2)-terminal end. Western blotting confirmed presence of both CPE(290-319) and TNF in the fusion protein. The TNF component in CPE(290-319)-TNF was 5-fold less potent than free TNF as determined by a standard L-929 TNF bioassay. However, the CPE(290-319)-TNF was >6.7-fold more cytotoxic than free TNF to 2008 human ovarian cancer cells, which express both CLDN3 and CLDN4 receptors. shRNAi-mediated knockdown of either CLDN3 or CLDN4 expression in 2008 markedly attenuated the cytotoxic effects of CPE(290-319)-TNF. The fusion construct was efficiently delivered into target cells and located in both cytosol and vesicular compartments as assessed by immunofluorescent staining. We conclude that CPE(290-319) effectively targeted TNF to ovarian cancer cells and is an attractive targeting moiety for development of CPE-based toxins for therapy of ovarian carcinomas that overexpress CLDN3 and CLDN4.

Risa Akizuki - One of the best experts on this subject based on the ideXlab platform.

  • chrysin enhances anticancer drug induced toxicity mediated by the reduction of claudin 1 and 11 expression in a spheroid culture model of lung squamous cell carcinoma cells
    Scientific Reports, 2019
    Co-Authors: Ryohei Maruhashi, Satoshi Endo, Takumi Furuta, Toshiyuki Matsunaga, Hiroaki Eguchi, Risa Akizuki, Shohei Hamada, Kenji Ichihara, Akira Ikari
    Abstract:

    The aberrant expression of claudins (CLDNs), which are tight junctional proteins, is seen in various solid tumors, but the regulatory mechanisms and their pathophysiological role are not well understood. Both CLDN1 and CLDN11 were highly expressed in human lung squamous cell carcinoma (SCC). Chrysin, found in high concentration in honey and propolis, decreased CLDN1 and CLDN11 expression in RERF-LC-AI cells derived from human lung SCC. The phosphorylation level of Akt was decreased by chrysin, but those of ERK1/2 and c-Jun were not. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited the phosphorylation of Akt and decreased the expression levels of CLDN1 and CLDN11. The association between phosphoinositide-dependent kinase 1 (PDK1) and Akt was inhibited by chrysin, but the phosphorylation of PDK1 was not. Immunoprecipitation and quartz-crystal microbalance assays revealed that biotinylated-chrysin binds directly to Akt. The knockdown of CLDN1 and CLDN11 using small interfering RNAs increased the transepithelial flux of doxorubicin (DXR), an anthracycline anticancer drug. Similarly, both chrysin and LY-294002 increased DXR flux. Neither CLDN1 knockdown, CLDN11 knockdown, nor chrysin changed the anticancer drug-induced cytotoxicity in a two-dimensional culture model, whereas they enhanced cytotoxicity in a spheroid culture model. Taken together, chrysin may bind to Akt and inhibit its phosphorylation, resulting in the elevation of anticancer drug-induced toxicity mediated by reductions in CLDN1 and CLDN11 expression in RERF-LC-AI cells. We suggest that chrysin may be useful as an adjuvant chemotherapy in lung SCC.

  • decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin 1 in spheroid culture models of human lung adenocarcinoma a549 cells
    Biochimica et Biophysica Acta, 2018
    Co-Authors: Risa Akizuki, Satoshi Endo, Takumi Furuta, Toshiyuki Matsunaga, Ryohei Maruhashi, Hiroaki Eguchi, Kazuki Kitabatake, Mitsutoshi Tsukimoto, Akira Ikari
    Abstract:

    Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.

  • claudin 5 7 and 18 suppress proliferation mediated by inhibition of phosphorylation of akt in human lung squamous cell carcinoma
    Biochimica et Biophysica Acta, 2017
    Co-Authors: Risa Akizuki, Toshiyuki Matsunaga, Satoshi Endo, Shun Shimobaba, Akira Ikari
    Abstract:

    Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. However, it is unknown which subtype plays a key role in the regulation of proliferation in cancer cells. The expression of CLDN3-5, 7, and 18 in human lung squamous carcinoma tissues was lower than that in normal tissue. Here, we examined which combination of exogenous CLDNs expression inhibits proliferation and the molecular mechanism using human lung squamous RERF-LC-AI cells. Real-time polymerase chain reaction and western blotting showed that CLDN3-5, 7, and 18 are little expressed in RERF-LC-AI cells. In the exogenously transfected cells, CLDN5, 7, and 18 were distributed in the cell-cell contact areas concomitant with ZO-1, a tight junctional scaffolding protein, whereas CLDN3 and 4 were not. Cell proliferation was individually and additively suppressed by CLDN5, 7, and 18. The expression of these CLDNs showed no cytotoxicity compared with mock cells. CLDN5, 7, and 18 increased p21 and decreased cyclin D1, resulting in the suppression of cell cycle G1-S transition. The expression of these CLDNs inhibited phosphorylation of Akt without affecting phosphorylated ERK1/2. Furthermore, these CLDNs inhibited the nuclear localization of Akt and its association with 3-phosphoinositide-dependent protein kinase-1 (PDK1). The suppression of G1-S transition caused by CLDN5, 7, and 18 was rescued by the expression of constitutively active-Akt. We suggest that the reduction of CLDN5, 7, and 18 expression loses the suppressive ability of interaction between PDK1 and Akt and causes sustained phosphorylation of Akt, resulting in the disordered proliferation in lung squamous carcinoma cells.

Maika Deffieu - One of the best experts on this subject based on the ideXlab platform.

  • characterisation of endogenous claudin 1 expression motility and susceptibility to hepatitis c virus in crispr knock in cells
    Biology of the Cell, 2020
    Co-Authors: Camille M H Cleme, Maika Deffieu, Cristina M. Dorobantu, Yves Mely, Thomas F Aume, Nilda Vanesa Ayalanunez
    Abstract:

    BACKGROUND INFORMATION: Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. RESULTS: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs. CONCLUSIONS: The Huh7.5.1 TagRFP-CLDN1+/+ edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging. SIGNIFICANCE: Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.

  • Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.
    Biology of the Cell, 2020
    Co-Authors: Camille M H Clément, Maika Deffieu, Cristina M. Dorobantu, Thomas F. Baumert, Nilda Vanesa Ayala-nunez, Yves Mely, Philippe Ronde, Raphael Gaudin
    Abstract:

    Background Information: Claudin‐1 (CLDN1) is a four‐span transmembrane protein localised at cell–cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up‐regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. Results: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN‐1, allowing dynamic monitoring of CLDN‐1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP‐T protein fused at the N‐terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV‐G‐decorated pseudoparticles. Nonetheless, the TagRFP‐CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild‐type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP‐CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP‐CLDN1 proteins were stalled at TJs. Conclusions: The Huh7.5.1 TagRFP‐CLDN1+/+ edited cell line showed physiological features comparable to that of non‐edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging.