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The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus

Expression of the DsRed-tagged N-cadherin cytoplasmic domain (DNCT) in MDCK cells increases cell proliferation.Clones showing tet-repressible expression of DNCT or DsRed were isolated and designated DNCT+ or DsRed+, respectively. (a) Morphology of cells after 3 d. DsRed+ cells grew in monolayer cultures as epithelial cell sheets with a typical cobblestone morphology, whereas expression of the DNCT protein produced dramatic changes in the DNCT+ cell cultures, including multilayering of the cells, and rounding of cells in the top layer (−Dox). Culturing DNCT+ cells in the presence of Dox (+Dox) completely reversed the morphological changes induced by DNCT expression. (b) Immunoblot analysis of confluent cell cultures revealed that p44/42 MAP kinase and Akt were phosphorylated (activated) in DNCT+ (−Dox) cells and that phosphorylation decreased upon Dox treatment (+Dox). DNCT expression also increased levels of cyclin D1. Quantifications obtained by densitometry are indicated below the corresponding panels. DNCT was detected with an anti-FLAG antibody. Vinculin was used as a loading control. (c) Growth and detachment of cells from the monolayer. DNCT+ cells were cultured with (+) or without (−) Dox on Transwell plates and cells shed into the medium and on the membranes were counted. Values indicate the mean ± S.E.; n = 3. Approximately 30% of the cells were shed into the medium during 3 d in culture. (d) DNCT expression increases the number of Ki-67–positive cells. DsRed+ or DNCT+ cells were cultured for 2 d and were stained with an anti–Ki-67 antibody and DAPI. There was a substantial increase in the number of proliferating (Ki-67–positive) cells in the DNCT+ but not DsRed+ cultures. With respect to the DsRed+ cells, the Ki-67–positive cells were located at the periphery of colonies. Reducing DNCT expression by incubating DNCT+ cells in the presence of Dox (+Dox) decreased the number of proliferating cells, and the Ki-67–positive cells were confined to cells in the periphery. ELAαC (an E-cadherin–α-catenin chimera) expression in DNCT+ cells (DNCT+ELAαC) decreased the number of Ki-67-positive cells, but ELA (a mutant E-cadherin) expression in DNCT+ cells (DNCT+ELA) had no effect. Bars, 25 μm.
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f1: Expression of the DsRed-tagged N-cadherin cytoplasmic domain (DNCT) in MDCK cells increases cell proliferation.Clones showing tet-repressible expression of DNCT or DsRed were isolated and designated DNCT+ or DsRed+, respectively. (a) Morphology of cells after 3 d. DsRed+ cells grew in monolayer cultures as epithelial cell sheets with a typical cobblestone morphology, whereas expression of the DNCT protein produced dramatic changes in the DNCT+ cell cultures, including multilayering of the cells, and rounding of cells in the top layer (−Dox). Culturing DNCT+ cells in the presence of Dox (+Dox) completely reversed the morphological changes induced by DNCT expression. (b) Immunoblot analysis of confluent cell cultures revealed that p44/42 MAP kinase and Akt were phosphorylated (activated) in DNCT+ (−Dox) cells and that phosphorylation decreased upon Dox treatment (+Dox). DNCT expression also increased levels of cyclin D1. Quantifications obtained by densitometry are indicated below the corresponding panels. DNCT was detected with an anti-FLAG antibody. Vinculin was used as a loading control. (c) Growth and detachment of cells from the monolayer. DNCT+ cells were cultured with (+) or without (−) Dox on Transwell plates and cells shed into the medium and on the membranes were counted. Values indicate the mean ± S.E.; n = 3. Approximately 30% of the cells were shed into the medium during 3 d in culture. (d) DNCT expression increases the number of Ki-67–positive cells. DsRed+ or DNCT+ cells were cultured for 2 d and were stained with an anti–Ki-67 antibody and DAPI. There was a substantial increase in the number of proliferating (Ki-67–positive) cells in the DNCT+ but not DsRed+ cultures. With respect to the DsRed+ cells, the Ki-67–positive cells were located at the periphery of colonies. Reducing DNCT expression by incubating DNCT+ cells in the presence of Dox (+Dox) decreased the number of proliferating cells, and the Ki-67–positive cells were confined to cells in the periphery. ELAαC (an E-cadherin–α-catenin chimera) expression in DNCT+ cells (DNCT+ELAαC) decreased the number of Ki-67-positive cells, but ELA (a mutant E-cadherin) expression in DNCT+ cells (DNCT+ELA) had no effect. Bars, 25 μm.

Mentions: We made a chimeric construct (DNCT) comprising a fluorescent protein, DsRed, the N-cadherin cytoplasmic domain (NCT), and a C-terminal FLAG tag25. The construct was expressed under the control of a Tet-repressible transactivator in an MDCK cell clone T23 expressing the tet repressor (DNCT+ cells). A FLAG-tagged DsRed construct was used as a control (DsRed+ cells). Cells expressing the control DsRed grew in monolayer cultures as epithelial clusters with a typical cobblestone morphology (Fig. 1a). By contrast, expression of the DNCT protein produced dramatic changes in the cultured cells, including multilayering of cells, rounding of cells in the top layer, and detachment of spherical cells from the monolayer (Fig. 1a). Culturing DNCT+ cells in the presence of Dox completely reversed these morphological changes. Immunoblot analysis of DNCT+ T23 cells cultured for 2 d with or without doxycycline showed that DNCT was detected in protein extracts from cells cultured without Dox, but was significantly reduced (~10%) in extracts from cells cultured with Dox (Fig. 1b). During culture of DNCT+ cells in the absence of Dox, we observed a number of floating cells (Fig. 1a, central panel), raising the possibility that DNCT expression induced cell shedding. To address this, cells were grown to confluence in the presence or absence of Dox and the cells shed into the medium were counted. The number of shed cells was markedly elevated in DNCT+ cells cultured in the absence of Dox, as compared to DNCT+ cells cultured in the presence of Dox (Fig. 1c).


The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

Expression of the DsRed-tagged N-cadherin cytoplasmic domain (DNCT) in MDCK cells increases cell proliferation.Clones showing tet-repressible expression of DNCT or DsRed were isolated and designated DNCT+ or DsRed+, respectively. (a) Morphology of cells after 3 d. DsRed+ cells grew in monolayer cultures as epithelial cell sheets with a typical cobblestone morphology, whereas expression of the DNCT protein produced dramatic changes in the DNCT+ cell cultures, including multilayering of the cells, and rounding of cells in the top layer (−Dox). Culturing DNCT+ cells in the presence of Dox (+Dox) completely reversed the morphological changes induced by DNCT expression. (b) Immunoblot analysis of confluent cell cultures revealed that p44/42 MAP kinase and Akt were phosphorylated (activated) in DNCT+ (−Dox) cells and that phosphorylation decreased upon Dox treatment (+Dox). DNCT expression also increased levels of cyclin D1. Quantifications obtained by densitometry are indicated below the corresponding panels. DNCT was detected with an anti-FLAG antibody. Vinculin was used as a loading control. (c) Growth and detachment of cells from the monolayer. DNCT+ cells were cultured with (+) or without (−) Dox on Transwell plates and cells shed into the medium and on the membranes were counted. Values indicate the mean ± S.E.; n = 3. Approximately 30% of the cells were shed into the medium during 3 d in culture. (d) DNCT expression increases the number of Ki-67–positive cells. DsRed+ or DNCT+ cells were cultured for 2 d and were stained with an anti–Ki-67 antibody and DAPI. There was a substantial increase in the number of proliferating (Ki-67–positive) cells in the DNCT+ but not DsRed+ cultures. With respect to the DsRed+ cells, the Ki-67–positive cells were located at the periphery of colonies. Reducing DNCT expression by incubating DNCT+ cells in the presence of Dox (+Dox) decreased the number of proliferating cells, and the Ki-67–positive cells were confined to cells in the periphery. ELAαC (an E-cadherin–α-catenin chimera) expression in DNCT+ cells (DNCT+ELAαC) decreased the number of Ki-67-positive cells, but ELA (a mutant E-cadherin) expression in DNCT+ cells (DNCT+ELA) had no effect. Bars, 25 μm.
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f1: Expression of the DsRed-tagged N-cadherin cytoplasmic domain (DNCT) in MDCK cells increases cell proliferation.Clones showing tet-repressible expression of DNCT or DsRed were isolated and designated DNCT+ or DsRed+, respectively. (a) Morphology of cells after 3 d. DsRed+ cells grew in monolayer cultures as epithelial cell sheets with a typical cobblestone morphology, whereas expression of the DNCT protein produced dramatic changes in the DNCT+ cell cultures, including multilayering of the cells, and rounding of cells in the top layer (−Dox). Culturing DNCT+ cells in the presence of Dox (+Dox) completely reversed the morphological changes induced by DNCT expression. (b) Immunoblot analysis of confluent cell cultures revealed that p44/42 MAP kinase and Akt were phosphorylated (activated) in DNCT+ (−Dox) cells and that phosphorylation decreased upon Dox treatment (+Dox). DNCT expression also increased levels of cyclin D1. Quantifications obtained by densitometry are indicated below the corresponding panels. DNCT was detected with an anti-FLAG antibody. Vinculin was used as a loading control. (c) Growth and detachment of cells from the monolayer. DNCT+ cells were cultured with (+) or without (−) Dox on Transwell plates and cells shed into the medium and on the membranes were counted. Values indicate the mean ± S.E.; n = 3. Approximately 30% of the cells were shed into the medium during 3 d in culture. (d) DNCT expression increases the number of Ki-67–positive cells. DsRed+ or DNCT+ cells were cultured for 2 d and were stained with an anti–Ki-67 antibody and DAPI. There was a substantial increase in the number of proliferating (Ki-67–positive) cells in the DNCT+ but not DsRed+ cultures. With respect to the DsRed+ cells, the Ki-67–positive cells were located at the periphery of colonies. Reducing DNCT expression by incubating DNCT+ cells in the presence of Dox (+Dox) decreased the number of proliferating cells, and the Ki-67–positive cells were confined to cells in the periphery. ELAαC (an E-cadherin–α-catenin chimera) expression in DNCT+ cells (DNCT+ELAαC) decreased the number of Ki-67-positive cells, but ELA (a mutant E-cadherin) expression in DNCT+ cells (DNCT+ELA) had no effect. Bars, 25 μm.
Mentions: We made a chimeric construct (DNCT) comprising a fluorescent protein, DsRed, the N-cadherin cytoplasmic domain (NCT), and a C-terminal FLAG tag25. The construct was expressed under the control of a Tet-repressible transactivator in an MDCK cell clone T23 expressing the tet repressor (DNCT+ cells). A FLAG-tagged DsRed construct was used as a control (DsRed+ cells). Cells expressing the control DsRed grew in monolayer cultures as epithelial clusters with a typical cobblestone morphology (Fig. 1a). By contrast, expression of the DNCT protein produced dramatic changes in the cultured cells, including multilayering of cells, rounding of cells in the top layer, and detachment of spherical cells from the monolayer (Fig. 1a). Culturing DNCT+ cells in the presence of Dox completely reversed these morphological changes. Immunoblot analysis of DNCT+ T23 cells cultured for 2 d with or without doxycycline showed that DNCT was detected in protein extracts from cells cultured without Dox, but was significantly reduced (~10%) in extracts from cells cultured with Dox (Fig. 1b). During culture of DNCT+ cells in the absence of Dox, we observed a number of floating cells (Fig. 1a, central panel), raising the possibility that DNCT expression induced cell shedding. To address this, cells were grown to confluence in the presence or absence of Dox and the cells shed into the medium were counted. The number of shed cells was markedly elevated in DNCT+ cells cultured in the absence of Dox, as compared to DNCT+ cells cultured in the presence of Dox (Fig. 1c).

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus