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The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E - J. Cell Biol. (2001)

Bottom Line: In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin.PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins.Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U410, Faculté de Médecine Bichat, 75018 Paris, France.

ABSTRACT
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

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Effects of PTEN expression on the invasive phenotype and cell aggregation of the MDCKts-src and MDCKras-f cell lines. (A) Invasion of type I collagen. MDCKts-src, MDCKras-f cells, and their derivatives transfected by wild-type or mutant PTEN were seeded on type I collagen gel at the restrictive or permissive temperature for Src activity (MDCKts-src) or at 37°C (MDCKras-f cells), and the number and depth of cells inside the gel were measured after 24 h. The dependence of this process on E-cadherin function was assessed by the inactivation of E-cadherin with DECMA-1 mAb. (B) Fast aggregation assay. The activity of junctional complexes in MDCKts-src cells and their derivatives was assessed by the distribution of the size of cell aggregates evaluated at t0 (□) or after a 30-min incubation (t30). Aggregation of parental cells for 30 min at 40°C yielded curves corresponding to particles with large size (left panel, ▪). The same was observed for PTEN (wild-type or mutant) transfectants at 40°C (unpublished data). Src activation at 35°C impaired aggregation of the parental MDCKts-src cells and derivatives expressing mutant PTEN (PTEN Δ237–239/71, PTEN Δ55–70/3, and unpublished data) yielding curves coinciding with the t0 curves (similar results were obtained with the MDCKts-srcΔ55–70 /9 and MDCKts-srcC124A/11 cells; unpublished data). In contrast, aggregation of MDCKts-srcPTENwt3 cells for 30 min at 35°C yielded a curve corresponding to large cell aggregates, which can be superimposed on the one measured at 40°C (unpublished data). Treatment with mAb DECMA-1 against E-cadherin at the restrictive (▴) or the permissive (▵) temperature for Src activity abolished aggregation in all cell types.
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fig2: Effects of PTEN expression on the invasive phenotype and cell aggregation of the MDCKts-src and MDCKras-f cell lines. (A) Invasion of type I collagen. MDCKts-src, MDCKras-f cells, and their derivatives transfected by wild-type or mutant PTEN were seeded on type I collagen gel at the restrictive or permissive temperature for Src activity (MDCKts-src) or at 37°C (MDCKras-f cells), and the number and depth of cells inside the gel were measured after 24 h. The dependence of this process on E-cadherin function was assessed by the inactivation of E-cadherin with DECMA-1 mAb. (B) Fast aggregation assay. The activity of junctional complexes in MDCKts-src cells and their derivatives was assessed by the distribution of the size of cell aggregates evaluated at t0 (□) or after a 30-min incubation (t30). Aggregation of parental cells for 30 min at 40°C yielded curves corresponding to particles with large size (left panel, ▪). The same was observed for PTEN (wild-type or mutant) transfectants at 40°C (unpublished data). Src activation at 35°C impaired aggregation of the parental MDCKts-src cells and derivatives expressing mutant PTEN (PTEN Δ237–239/71, PTEN Δ55–70/3, and unpublished data) yielding curves coinciding with the t0 curves (similar results were obtained with the MDCKts-srcΔ55–70 /9 and MDCKts-srcC124A/11 cells; unpublished data). In contrast, aggregation of MDCKts-srcPTENwt3 cells for 30 min at 35°C yielded a curve corresponding to large cell aggregates, which can be superimposed on the one measured at 40°C (unpublished data). Treatment with mAb DECMA-1 against E-cadherin at the restrictive (▴) or the permissive (▵) temperature for Src activity abolished aggregation in all cell types.

Mentions: Because PTEN interfered with Src-induced cell scattering, we further investigated the invasive properties of parental and PTEN-transfected MDCKts-src cells in type I collagen gels. As shown in Fig. 2 A, the parental MDCKts-src cells and those derivatives that did not express exogenous PTEN, MDCKts-src11 and MDCKts-src31, became invasive after a temperature shift to 35°C. In contrast, the six MDCKts-srcPTENwt clones that overexpressed wild-type PTEN remained noninvasive at the permissive temperature (Fig. 2 A). Overexpression of PTEN mutants in MDCKts-src cells did not revert the invasive phenotype induced by Src at 35°C, demonstrating the critical role of the PTEN lipid phosphatase activity in the control of the invasive phenotype. Besides the lipid phosphatase activity of PTEN, invasion suppression clearly involved the E-cadherin system, as inactivation of E-cadherin by DECMA-1 mAb induced the invasive phenotype in MDCKts-srcPTENwt, both at the restrictive and the permissive temperature for Src activity (Fig. 2 A). Likewise, PTEN could not counteract the invasive phenotype of MDCKras-f cells that poorly expressed E-cadherins (Vleminckx et al., 1991).


The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E - J. Cell Biol. (2001)

Effects of PTEN expression on the invasive phenotype and cell aggregation of the MDCKts-src and MDCKras-f cell lines. (A) Invasion of type I collagen. MDCKts-src, MDCKras-f cells, and their derivatives transfected by wild-type or mutant PTEN were seeded on type I collagen gel at the restrictive or permissive temperature for Src activity (MDCKts-src) or at 37°C (MDCKras-f cells), and the number and depth of cells inside the gel were measured after 24 h. The dependence of this process on E-cadherin function was assessed by the inactivation of E-cadherin with DECMA-1 mAb. (B) Fast aggregation assay. The activity of junctional complexes in MDCKts-src cells and their derivatives was assessed by the distribution of the size of cell aggregates evaluated at t0 (□) or after a 30-min incubation (t30). Aggregation of parental cells for 30 min at 40°C yielded curves corresponding to particles with large size (left panel, ▪). The same was observed for PTEN (wild-type or mutant) transfectants at 40°C (unpublished data). Src activation at 35°C impaired aggregation of the parental MDCKts-src cells and derivatives expressing mutant PTEN (PTEN Δ237–239/71, PTEN Δ55–70/3, and unpublished data) yielding curves coinciding with the t0 curves (similar results were obtained with the MDCKts-srcΔ55–70 /9 and MDCKts-srcC124A/11 cells; unpublished data). In contrast, aggregation of MDCKts-srcPTENwt3 cells for 30 min at 35°C yielded a curve corresponding to large cell aggregates, which can be superimposed on the one measured at 40°C (unpublished data). Treatment with mAb DECMA-1 against E-cadherin at the restrictive (▴) or the permissive (▵) temperature for Src activity abolished aggregation in all cell types.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199329&req=5

fig2: Effects of PTEN expression on the invasive phenotype and cell aggregation of the MDCKts-src and MDCKras-f cell lines. (A) Invasion of type I collagen. MDCKts-src, MDCKras-f cells, and their derivatives transfected by wild-type or mutant PTEN were seeded on type I collagen gel at the restrictive or permissive temperature for Src activity (MDCKts-src) or at 37°C (MDCKras-f cells), and the number and depth of cells inside the gel were measured after 24 h. The dependence of this process on E-cadherin function was assessed by the inactivation of E-cadherin with DECMA-1 mAb. (B) Fast aggregation assay. The activity of junctional complexes in MDCKts-src cells and their derivatives was assessed by the distribution of the size of cell aggregates evaluated at t0 (□) or after a 30-min incubation (t30). Aggregation of parental cells for 30 min at 40°C yielded curves corresponding to particles with large size (left panel, ▪). The same was observed for PTEN (wild-type or mutant) transfectants at 40°C (unpublished data). Src activation at 35°C impaired aggregation of the parental MDCKts-src cells and derivatives expressing mutant PTEN (PTEN Δ237–239/71, PTEN Δ55–70/3, and unpublished data) yielding curves coinciding with the t0 curves (similar results were obtained with the MDCKts-srcΔ55–70 /9 and MDCKts-srcC124A/11 cells; unpublished data). In contrast, aggregation of MDCKts-srcPTENwt3 cells for 30 min at 35°C yielded a curve corresponding to large cell aggregates, which can be superimposed on the one measured at 40°C (unpublished data). Treatment with mAb DECMA-1 against E-cadherin at the restrictive (▴) or the permissive (▵) temperature for Src activity abolished aggregation in all cell types.
Mentions: Because PTEN interfered with Src-induced cell scattering, we further investigated the invasive properties of parental and PTEN-transfected MDCKts-src cells in type I collagen gels. As shown in Fig. 2 A, the parental MDCKts-src cells and those derivatives that did not express exogenous PTEN, MDCKts-src11 and MDCKts-src31, became invasive after a temperature shift to 35°C. In contrast, the six MDCKts-srcPTENwt clones that overexpressed wild-type PTEN remained noninvasive at the permissive temperature (Fig. 2 A). Overexpression of PTEN mutants in MDCKts-src cells did not revert the invasive phenotype induced by Src at 35°C, demonstrating the critical role of the PTEN lipid phosphatase activity in the control of the invasive phenotype. Besides the lipid phosphatase activity of PTEN, invasion suppression clearly involved the E-cadherin system, as inactivation of E-cadherin by DECMA-1 mAb induced the invasive phenotype in MDCKts-srcPTENwt, both at the restrictive and the permissive temperature for Src activity (Fig. 2 A). Likewise, PTEN could not counteract the invasive phenotype of MDCKras-f cells that poorly expressed E-cadherins (Vleminckx et al., 1991).

Bottom Line: In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin.PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins.Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U410, Faculté de Médecine Bichat, 75018 Paris, France.

ABSTRACT
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

Show MeSH
Related in: MedlinePlus