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Loss of cellular adhesion to matrix induces p53-independent expression of PTEN tumor suppressor.

Wu RC, Blumenthal M, Li X, Schönthal AH - BMC Mol. Biol. (2002)

Bottom Line: As these findings indicated that PTEN might be involved in the regulation of anchorage-dependent cell growth, we analyzed this aspect of PTEN function in non-tumor cells with an anchorage-dependent phenotype.We found that in response to the disruption of cell-matrix interactions, expression of endogenous PTEN was transcriptionally activated, and elevated levels of PTEN protein and activity were present in the cells.In view of PTEN's potent growth-inhibitory capacity, we conclude that its induction after cell-matrix disruptions contributes to the maintenance of the anchorage-dependent phenotype of normal cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine. rwu@bcm.tmc.edu

ABSTRACT

Background: The tumor suppressor gene PTEN has been found mutated in many types of advanced tumors. When introduced into tumor cells that lack the wild-type allele of the gene, exogenous PTEN was able to suppress their ability to grow anchorage-independently, and thus reverted one of the typical characteristics of tumor cells. As these findings indicated that PTEN might be involved in the regulation of anchorage-dependent cell growth, we analyzed this aspect of PTEN function in non-tumor cells with an anchorage-dependent phenotype.

Results: We found that in response to the disruption of cell-matrix interactions, expression of endogenous PTEN was transcriptionally activated, and elevated levels of PTEN protein and activity were present in the cells. These events correlated with decreased phosphorylation of focal adhesion kinase, and occurred even in the absence of p53, a tumor suppressor protein and recently established stimulator of PTEN transcription.

Conclusions: In view of PTEN's potent growth-inhibitory capacity, we conclude that its induction after cell-matrix disruptions contributes to the maintenance of the anchorage-dependent phenotype of normal cells.

No MeSH data available.


Related in: MedlinePlus

Protein phosphatase activity of PTEN (A) In order to determine the specificity of the PTEN phosphatase assay, PTEN-negative U87 glioblastoma cells were transiently transfected with pCMV-PTEN expression vector (PTEN) or with pCMV-blue vector without PTEN cDNA insert (Vector). As a further control, non-transfected U87 cells were used in parallel (U87). 24 hours after transfection, cellular lysates were harvested and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments the quantity of phosphate released in the absence of added antigen. (B) In order to determine PTEN phosphatase activity in suspension cells, MDAH cells were transferred to suspension culture conditions for the times indicated. Total cellular lysates were prepared and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments ± sd). released in the absence of added antigen.
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Figure 4: Protein phosphatase activity of PTEN (A) In order to determine the specificity of the PTEN phosphatase assay, PTEN-negative U87 glioblastoma cells were transiently transfected with pCMV-PTEN expression vector (PTEN) or with pCMV-blue vector without PTEN cDNA insert (Vector). As a further control, non-transfected U87 cells were used in parallel (U87). 24 hours after transfection, cellular lysates were harvested and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments the quantity of phosphate released in the absence of added antigen. (B) In order to determine PTEN phosphatase activity in suspension cells, MDAH cells were transferred to suspension culture conditions for the times indicated. Total cellular lysates were prepared and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments ± sd). released in the absence of added antigen.

Mentions: We next analyzed whether the elevated quantity of PTEN protein would indeed be reflected in increased phosphatase activity in suspension culture cells. In order to establish whether PTEN protein phosphatase activity could be reliably measured in vitro, we first transfected PTEN-negative U87 cells with an expression vector harboring PTEN cDNA. As a control, the cells were also transfected with empty vector. Then, PTEN was immunoprecipitated from the respective cellular lysates and the antigen-antibody complex was analyzed for protein phosphatase activity. As shown in Figure 4A, only cells transfected with PTEN cDNA exhibited significant enzymatic activity. Non-transfected U87 cells, or cells transfected with vector alone, did not exhibit protein phosphatase activity above background levels.


Loss of cellular adhesion to matrix induces p53-independent expression of PTEN tumor suppressor.

Wu RC, Blumenthal M, Li X, Schönthal AH - BMC Mol. Biol. (2002)

Protein phosphatase activity of PTEN (A) In order to determine the specificity of the PTEN phosphatase assay, PTEN-negative U87 glioblastoma cells were transiently transfected with pCMV-PTEN expression vector (PTEN) or with pCMV-blue vector without PTEN cDNA insert (Vector). As a further control, non-transfected U87 cells were used in parallel (U87). 24 hours after transfection, cellular lysates were harvested and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments the quantity of phosphate released in the absence of added antigen. (B) In order to determine PTEN phosphatase activity in suspension cells, MDAH cells were transferred to suspension culture conditions for the times indicated. Total cellular lysates were prepared and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments ± sd). released in the absence of added antigen.
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Related In: Results  -  Collection

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Figure 4: Protein phosphatase activity of PTEN (A) In order to determine the specificity of the PTEN phosphatase assay, PTEN-negative U87 glioblastoma cells were transiently transfected with pCMV-PTEN expression vector (PTEN) or with pCMV-blue vector without PTEN cDNA insert (Vector). As a further control, non-transfected U87 cells were used in parallel (U87). 24 hours after transfection, cellular lysates were harvested and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments the quantity of phosphate released in the absence of added antigen. (B) In order to determine PTEN phosphatase activity in suspension cells, MDAH cells were transferred to suspension culture conditions for the times indicated. Total cellular lysates were prepared and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments ± sd). released in the absence of added antigen.
Mentions: We next analyzed whether the elevated quantity of PTEN protein would indeed be reflected in increased phosphatase activity in suspension culture cells. In order to establish whether PTEN protein phosphatase activity could be reliably measured in vitro, we first transfected PTEN-negative U87 cells with an expression vector harboring PTEN cDNA. As a control, the cells were also transfected with empty vector. Then, PTEN was immunoprecipitated from the respective cellular lysates and the antigen-antibody complex was analyzed for protein phosphatase activity. As shown in Figure 4A, only cells transfected with PTEN cDNA exhibited significant enzymatic activity. Non-transfected U87 cells, or cells transfected with vector alone, did not exhibit protein phosphatase activity above background levels.

Bottom Line: As these findings indicated that PTEN might be involved in the regulation of anchorage-dependent cell growth, we analyzed this aspect of PTEN function in non-tumor cells with an anchorage-dependent phenotype.We found that in response to the disruption of cell-matrix interactions, expression of endogenous PTEN was transcriptionally activated, and elevated levels of PTEN protein and activity were present in the cells.In view of PTEN's potent growth-inhibitory capacity, we conclude that its induction after cell-matrix disruptions contributes to the maintenance of the anchorage-dependent phenotype of normal cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine. rwu@bcm.tmc.edu

ABSTRACT

Background: The tumor suppressor gene PTEN has been found mutated in many types of advanced tumors. When introduced into tumor cells that lack the wild-type allele of the gene, exogenous PTEN was able to suppress their ability to grow anchorage-independently, and thus reverted one of the typical characteristics of tumor cells. As these findings indicated that PTEN might be involved in the regulation of anchorage-dependent cell growth, we analyzed this aspect of PTEN function in non-tumor cells with an anchorage-dependent phenotype.

Results: We found that in response to the disruption of cell-matrix interactions, expression of endogenous PTEN was transcriptionally activated, and elevated levels of PTEN protein and activity were present in the cells. These events correlated with decreased phosphorylation of focal adhesion kinase, and occurred even in the absence of p53, a tumor suppressor protein and recently established stimulator of PTEN transcription.

Conclusions: In view of PTEN's potent growth-inhibitory capacity, we conclude that its induction after cell-matrix disruptions contributes to the maintenance of the anchorage-dependent phenotype of normal cells.

No MeSH data available.


Related in: MedlinePlus