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A novel form of constitutively active farnesylated Akt1 prevents mammary epithelial cells from anoikis and suppresses chemotherapy-induced apoptosis.

Schmidt M, Hövelmann S, Beckers TL - Br. J. Cancer (2002)

Bottom Line: Enigneered MCF10A cells were rendered resistant towards apoptosis resulting from loss of cellular substrate attachment (anoikis).A profoundly decreased sensitivity towards Mitoxantrone and cisplatin was observed in cells expressing farnesylated Akt.No significant difference in sensitivity however was observed upon treatment with cell cycle specific chemotherapeutic agents like paclitaxel.

View Article: PubMed Central - PubMed

Affiliation: ASTA Medica Oncology, Weismüllerstr. 45, D-60314 Frankfurt, Germany. mathias.schmidt@altanapharma.com

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(A) Expression and constitutive activation of farnesylated Akt1 in A549 NSCLC cells and MCF10A mammary epithelial cells. Control transfected A549 and MCF10A cells, respectively or cells expressing farnesylated Akt1 (as indicated) were serum starved overnight and then stimulated for 15 min with medium containing 10% FCS (S) or 10% FCS plus a cocktail of 10 ng ml−1 each EGF, IGF-1, and PDGF (GF). Cell lysates were analysed by immunoblotting with antibodies specific for Akt1, FLAG-epitope, phospho-Akt (Thr 308) or phospho-Akt (Ser 473) as indicated. The ectopically expressed farnesylated Akt1 devoid of the PH domain migrates at a Mr of approximately 50 kDa. (B) Kinase activity of endogenous and ectopically expressed farnesylated Akt. Cells were serum starved for 16 h and then stimulated as described above. Immunoprecipitates of endogenous Akt from control transfected cells and farnesylated Akt from transfected cells were used in kinase assay reactions with GSK-3-fusion protein as substrate. Immunoblots were assayed with antibodies specific for phospho-GSK (Ser 21/9) (lower portion) and Akt (upper portion). Positions of phosphorylated GSK-fusion protein and Akt1 are indicated by an arrow.
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fig2: (A) Expression and constitutive activation of farnesylated Akt1 in A549 NSCLC cells and MCF10A mammary epithelial cells. Control transfected A549 and MCF10A cells, respectively or cells expressing farnesylated Akt1 (as indicated) were serum starved overnight and then stimulated for 15 min with medium containing 10% FCS (S) or 10% FCS plus a cocktail of 10 ng ml−1 each EGF, IGF-1, and PDGF (GF). Cell lysates were analysed by immunoblotting with antibodies specific for Akt1, FLAG-epitope, phospho-Akt (Thr 308) or phospho-Akt (Ser 473) as indicated. The ectopically expressed farnesylated Akt1 devoid of the PH domain migrates at a Mr of approximately 50 kDa. (B) Kinase activity of endogenous and ectopically expressed farnesylated Akt. Cells were serum starved for 16 h and then stimulated as described above. Immunoprecipitates of endogenous Akt from control transfected cells and farnesylated Akt from transfected cells were used in kinase assay reactions with GSK-3-fusion protein as substrate. Immunoblots were assayed with antibodies specific for phospho-GSK (Ser 21/9) (lower portion) and Akt (upper portion). Positions of phosphorylated GSK-fusion protein and Akt1 are indicated by an arrow.

Mentions: MCF10A human mammary epithelial cells and A549 human NSCLC cells were stably transfected with the expression vector encoding farnesylated Akt1 devoid of its PH domain. Clones that were tested positive for ectopic expression of Akt1 by reactivity of the lysates with the M2 anti-FLAG antibody (MCF10A-Akt1 or A549-Akt1) were used in this study. To analyse whether the membrane targeting by a farnesylation tag renders ectopically expressed Akt1 constitutively active, the phosphorylation status of threonine 308 and serine 473 was determined in transfected cells as a marker of kinase activation (Alessi and Cohen, 1998). MCF10A-Akt1 and A549-Akt1 cells were seeded out in growth medium. After attachment the cells were serum starved for 16 h and then stimulated with medium containing 10% serum or 10% serum plus growth factors (EGF, PDGF, and IGF-1). Lysates were then analysed in immunoblot assays with antibodies specific for phospho-Akt (Thr 308) or phospho-Akt (Ser 473). Cells transfected with the empty vector were used as controls. The results are shown in Figure 2AFigure 2


A novel form of constitutively active farnesylated Akt1 prevents mammary epithelial cells from anoikis and suppresses chemotherapy-induced apoptosis.

Schmidt M, Hövelmann S, Beckers TL - Br. J. Cancer (2002)

(A) Expression and constitutive activation of farnesylated Akt1 in A549 NSCLC cells and MCF10A mammary epithelial cells. Control transfected A549 and MCF10A cells, respectively or cells expressing farnesylated Akt1 (as indicated) were serum starved overnight and then stimulated for 15 min with medium containing 10% FCS (S) or 10% FCS plus a cocktail of 10 ng ml−1 each EGF, IGF-1, and PDGF (GF). Cell lysates were analysed by immunoblotting with antibodies specific for Akt1, FLAG-epitope, phospho-Akt (Thr 308) or phospho-Akt (Ser 473) as indicated. The ectopically expressed farnesylated Akt1 devoid of the PH domain migrates at a Mr of approximately 50 kDa. (B) Kinase activity of endogenous and ectopically expressed farnesylated Akt. Cells were serum starved for 16 h and then stimulated as described above. Immunoprecipitates of endogenous Akt from control transfected cells and farnesylated Akt from transfected cells were used in kinase assay reactions with GSK-3-fusion protein as substrate. Immunoblots were assayed with antibodies specific for phospho-GSK (Ser 21/9) (lower portion) and Akt (upper portion). Positions of phosphorylated GSK-fusion protein and Akt1 are indicated by an arrow.
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fig2: (A) Expression and constitutive activation of farnesylated Akt1 in A549 NSCLC cells and MCF10A mammary epithelial cells. Control transfected A549 and MCF10A cells, respectively or cells expressing farnesylated Akt1 (as indicated) were serum starved overnight and then stimulated for 15 min with medium containing 10% FCS (S) or 10% FCS plus a cocktail of 10 ng ml−1 each EGF, IGF-1, and PDGF (GF). Cell lysates were analysed by immunoblotting with antibodies specific for Akt1, FLAG-epitope, phospho-Akt (Thr 308) or phospho-Akt (Ser 473) as indicated. The ectopically expressed farnesylated Akt1 devoid of the PH domain migrates at a Mr of approximately 50 kDa. (B) Kinase activity of endogenous and ectopically expressed farnesylated Akt. Cells were serum starved for 16 h and then stimulated as described above. Immunoprecipitates of endogenous Akt from control transfected cells and farnesylated Akt from transfected cells were used in kinase assay reactions with GSK-3-fusion protein as substrate. Immunoblots were assayed with antibodies specific for phospho-GSK (Ser 21/9) (lower portion) and Akt (upper portion). Positions of phosphorylated GSK-fusion protein and Akt1 are indicated by an arrow.
Mentions: MCF10A human mammary epithelial cells and A549 human NSCLC cells were stably transfected with the expression vector encoding farnesylated Akt1 devoid of its PH domain. Clones that were tested positive for ectopic expression of Akt1 by reactivity of the lysates with the M2 anti-FLAG antibody (MCF10A-Akt1 or A549-Akt1) were used in this study. To analyse whether the membrane targeting by a farnesylation tag renders ectopically expressed Akt1 constitutively active, the phosphorylation status of threonine 308 and serine 473 was determined in transfected cells as a marker of kinase activation (Alessi and Cohen, 1998). MCF10A-Akt1 and A549-Akt1 cells were seeded out in growth medium. After attachment the cells were serum starved for 16 h and then stimulated with medium containing 10% serum or 10% serum plus growth factors (EGF, PDGF, and IGF-1). Lysates were then analysed in immunoblot assays with antibodies specific for phospho-Akt (Thr 308) or phospho-Akt (Ser 473). Cells transfected with the empty vector were used as controls. The results are shown in Figure 2AFigure 2

Bottom Line: Enigneered MCF10A cells were rendered resistant towards apoptosis resulting from loss of cellular substrate attachment (anoikis).A profoundly decreased sensitivity towards Mitoxantrone and cisplatin was observed in cells expressing farnesylated Akt.No significant difference in sensitivity however was observed upon treatment with cell cycle specific chemotherapeutic agents like paclitaxel.

View Article: PubMed Central - PubMed

Affiliation: ASTA Medica Oncology, Weismüllerstr. 45, D-60314 Frankfurt, Germany. mathias.schmidt@altanapharma.com

Show MeSH
Related in: MedlinePlus