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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 DNCT in MDCK cells supports anchorage-independent growth.(a) Phase contrast micrographs of cells cultured on ultra-low attachment (PrimeSurfaceTM) plates to keep cells in suspension. A significant portion of DsRed+ cells die (dark cells) after 3 d in suspension culture, but DNCT+ cells remain viable (bright cells). (b) Quantitation of cell viability after 5 d in suspension culture. Cells expressing DNCT, DsRed, DNCTSA, DNCT and ELA (DNCT+ELA), DNCT and ELAαC chimera (DNCT+ELAαC), or DNCTNLS were cultured in the presence (+) or absence (−) of Dox on normal tissue culture plates (control) or on ultra-low attachment plates and were assayed for their viability using the WST-1 assay. The results are expressed as a percentage of the values obtained with attached (control) cells. Values indicate the mean ± S.E.; n = 3. DNCTSA is a DNCT derivative bearing alanine substitutions of the conserved eight serine residues in the catenin-binding site, which weakens its interactions with β-catenin. DNCTNLS is a DNCT derivative bearing an SV40 NLS signal at its C-terminus. (c) Cells undergo anoikis in suspension culture; DNCT expression protected against this cell death. Cells were cultured on ultra-low attachment plates for 3 d, and then stained with FITC-annexin V. (d) DNCT+ cells show anchorage-independent growth. Cells were cultured in soft agar in the presence (+Dox) or absence (−Dox) of Dox for 21 d. The number of colonies larger than 50 μm in diameter was counted and is shown at the bottom of the pictures. Values indicate the mean ± S.E.; n = 3. Bars, 25 μm.
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f2: Expression of DNCT in MDCK cells supports anchorage-independent growth.(a) Phase contrast micrographs of cells cultured on ultra-low attachment (PrimeSurfaceTM) plates to keep cells in suspension. A significant portion of DsRed+ cells die (dark cells) after 3 d in suspension culture, but DNCT+ cells remain viable (bright cells). (b) Quantitation of cell viability after 5 d in suspension culture. Cells expressing DNCT, DsRed, DNCTSA, DNCT and ELA (DNCT+ELA), DNCT and ELAαC chimera (DNCT+ELAαC), or DNCTNLS were cultured in the presence (+) or absence (−) of Dox on normal tissue culture plates (control) or on ultra-low attachment plates and were assayed for their viability using the WST-1 assay. The results are expressed as a percentage of the values obtained with attached (control) cells. Values indicate the mean ± S.E.; n = 3. DNCTSA is a DNCT derivative bearing alanine substitutions of the conserved eight serine residues in the catenin-binding site, which weakens its interactions with β-catenin. DNCTNLS is a DNCT derivative bearing an SV40 NLS signal at its C-terminus. (c) Cells undergo anoikis in suspension culture; DNCT expression protected against this cell death. Cells were cultured on ultra-low attachment plates for 3 d, and then stained with FITC-annexin V. (d) DNCT+ cells show anchorage-independent growth. Cells were cultured in soft agar in the presence (+Dox) or absence (−Dox) of Dox for 21 d. The number of colonies larger than 50 μm in diameter was counted and is shown at the bottom of the pictures. Values indicate the mean ± S.E.; n = 3. Bars, 25 μm.

Mentions: We noted that suspension culture of DsRed+, but not DNCT+, cells resulted in cell death (Fig. 2a). These findings implied that DNCT could inhibit anoikis resulting from the loss of cell–substrate adhesion. To test this hypothesis, DsRed+ cells and DNCT+ cells with or without Dox treatment were cultured in specialized tissue culture plates, called PrimeSurfaceTM, which prevent cell attachment. Expression of DNCT, but not DsRed alone, promoted the proliferation of cells under suspension culture conditions as measured by the WST-1 assay (Fig. 2b). However, a Dox-induced reduction in DNCT levels decreased the rate of cell proliferation to control (DsRed+) levels. Thus, DNCT expression in T23 MDCK cells protected against anoikis.


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

Ozawa M - Sci Rep (2015)

Expression of DNCT in MDCK cells supports anchorage-independent growth.(a) Phase contrast micrographs of cells cultured on ultra-low attachment (PrimeSurfaceTM) plates to keep cells in suspension. A significant portion of DsRed+ cells die (dark cells) after 3 d in suspension culture, but DNCT+ cells remain viable (bright cells). (b) Quantitation of cell viability after 5 d in suspension culture. Cells expressing DNCT, DsRed, DNCTSA, DNCT and ELA (DNCT+ELA), DNCT and ELAαC chimera (DNCT+ELAαC), or DNCTNLS were cultured in the presence (+) or absence (−) of Dox on normal tissue culture plates (control) or on ultra-low attachment plates and were assayed for their viability using the WST-1 assay. The results are expressed as a percentage of the values obtained with attached (control) cells. Values indicate the mean ± S.E.; n = 3. DNCTSA is a DNCT derivative bearing alanine substitutions of the conserved eight serine residues in the catenin-binding site, which weakens its interactions with β-catenin. DNCTNLS is a DNCT derivative bearing an SV40 NLS signal at its C-terminus. (c) Cells undergo anoikis in suspension culture; DNCT expression protected against this cell death. Cells were cultured on ultra-low attachment plates for 3 d, and then stained with FITC-annexin V. (d) DNCT+ cells show anchorage-independent growth. Cells were cultured in soft agar in the presence (+Dox) or absence (−Dox) of Dox for 21 d. The number of colonies larger than 50 μm in diameter was counted and is shown at the bottom of the pictures. Values indicate the mean ± S.E.; n = 3. Bars, 25 μm.
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Related In: Results  -  Collection

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Show All Figures
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f2: Expression of DNCT in MDCK cells supports anchorage-independent growth.(a) Phase contrast micrographs of cells cultured on ultra-low attachment (PrimeSurfaceTM) plates to keep cells in suspension. A significant portion of DsRed+ cells die (dark cells) after 3 d in suspension culture, but DNCT+ cells remain viable (bright cells). (b) Quantitation of cell viability after 5 d in suspension culture. Cells expressing DNCT, DsRed, DNCTSA, DNCT and ELA (DNCT+ELA), DNCT and ELAαC chimera (DNCT+ELAαC), or DNCTNLS were cultured in the presence (+) or absence (−) of Dox on normal tissue culture plates (control) or on ultra-low attachment plates and were assayed for their viability using the WST-1 assay. The results are expressed as a percentage of the values obtained with attached (control) cells. Values indicate the mean ± S.E.; n = 3. DNCTSA is a DNCT derivative bearing alanine substitutions of the conserved eight serine residues in the catenin-binding site, which weakens its interactions with β-catenin. DNCTNLS is a DNCT derivative bearing an SV40 NLS signal at its C-terminus. (c) Cells undergo anoikis in suspension culture; DNCT expression protected against this cell death. Cells were cultured on ultra-low attachment plates for 3 d, and then stained with FITC-annexin V. (d) DNCT+ cells show anchorage-independent growth. Cells were cultured in soft agar in the presence (+Dox) or absence (−Dox) of Dox for 21 d. The number of colonies larger than 50 μm in diameter was counted and is shown at the bottom of the pictures. Values indicate the mean ± S.E.; n = 3. Bars, 25 μm.
Mentions: We noted that suspension culture of DsRed+, but not DNCT+, cells resulted in cell death (Fig. 2a). These findings implied that DNCT could inhibit anoikis resulting from the loss of cell–substrate adhesion. To test this hypothesis, DsRed+ cells and DNCT+ cells with or without Dox treatment were cultured in specialized tissue culture plates, called PrimeSurfaceTM, which prevent cell attachment. Expression of DNCT, but not DsRed alone, promoted the proliferation of cells under suspension culture conditions as measured by the WST-1 assay (Fig. 2b). However, a Dox-induced reduction in DNCT levels decreased the rate of cell proliferation to control (DsRed+) levels. Thus, DNCT expression in T23 MDCK cells protected against anoikis.

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