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CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

Zhang S, Lu Z, Mao W, Ahmed AA, Yang H, Zhou J, Jennings N, Rodriguez-Aguayo C, Lopez-Berestein G, Miranda R, Qiao W, Baladandayuthapani V, Li Z, Sood AK, Liu J, Le XF, Bast RC - PLoS ONE (2015)

Bottom Line: Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase.In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells.CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America; Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.

ABSTRACT
Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

No MeSH data available.


Related in: MedlinePlus

CDK5 knockdown increases TP53 and p27Kip1 protein half-lives, increases nuclear localization of the two proteins, decreases p27Kip1 p-T187 phosphorylation and induces p21Cip1 in a TP53-dependent manner.(A) Transfection with CDK5 siRNA increased the half-life of p53 and p27Kip1 protein. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h and then treated with 5 μg/mL of cycloheximide (CHX) for the indicated intervals. Cell lysates was subjected to Western analysis with the antibodies indicated. (B) CDK5 siRNA increased the nuclear localization of p53 and p27Kip1. HEY cells were transfected with Control siRNA or CDK5 siRNA for 24 h and subjected to fractionation into cytoplasmic and nuclear components. Cytoplasmic (CE) and nuclear (NE) extracts were analyzed by immunoblotting. (C) CDK5 siRNA decreased the phosphorylation of p27Kip1 at T187. The cell Immunoblot analysis was performed with antibodies against phosphorylated and total p27Kip1. (D) CDK5 enhanced phosphorylation of TP53 and p27Kip. A fluorescence based in vitro kinase assay was used to evaluate the interaction of CDK5 with TP53 and p27Kip1. (E) CDK5 siRNA transcriptionally regulated the expression of p21Cip1. HEY cells were transfected with CDK5 siRNA alone or in combination with TP53 siRNA and a luciferase reporter assay was used to measure p21Cip1 promoter activity. (F) CDK5, p21Cip1 and p27Kip1 knockdown decreased the expression of CDK5, p21Cip1 and p27Kip1. HEY cells were treated with control siRNA or CDK5 or/and p21 plus p27 siRNA for 48 h and then with paclitaxel (6 nM) or diluent for 24 h. Immunoblot analysis was performed with antibodies indicated. (G, H) Knockdown of p21and p27 reduced CDK5 knockdown-induced apoptosis (G) and G1 arrest (H). HEY cells were treated as in (F) and cell cycle analyzed by flow cytometry after siRNA transfection. SubG1 cells were considered apoptotic cells. Data shown are mean values from three independent experiments.
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pone.0131833.g004: CDK5 knockdown increases TP53 and p27Kip1 protein half-lives, increases nuclear localization of the two proteins, decreases p27Kip1 p-T187 phosphorylation and induces p21Cip1 in a TP53-dependent manner.(A) Transfection with CDK5 siRNA increased the half-life of p53 and p27Kip1 protein. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h and then treated with 5 μg/mL of cycloheximide (CHX) for the indicated intervals. Cell lysates was subjected to Western analysis with the antibodies indicated. (B) CDK5 siRNA increased the nuclear localization of p53 and p27Kip1. HEY cells were transfected with Control siRNA or CDK5 siRNA for 24 h and subjected to fractionation into cytoplasmic and nuclear components. Cytoplasmic (CE) and nuclear (NE) extracts were analyzed by immunoblotting. (C) CDK5 siRNA decreased the phosphorylation of p27Kip1 at T187. The cell Immunoblot analysis was performed with antibodies against phosphorylated and total p27Kip1. (D) CDK5 enhanced phosphorylation of TP53 and p27Kip. A fluorescence based in vitro kinase assay was used to evaluate the interaction of CDK5 with TP53 and p27Kip1. (E) CDK5 siRNA transcriptionally regulated the expression of p21Cip1. HEY cells were transfected with CDK5 siRNA alone or in combination with TP53 siRNA and a luciferase reporter assay was used to measure p21Cip1 promoter activity. (F) CDK5, p21Cip1 and p27Kip1 knockdown decreased the expression of CDK5, p21Cip1 and p27Kip1. HEY cells were treated with control siRNA or CDK5 or/and p21 plus p27 siRNA for 48 h and then with paclitaxel (6 nM) or diluent for 24 h. Immunoblot analysis was performed with antibodies indicated. (G, H) Knockdown of p21and p27 reduced CDK5 knockdown-induced apoptosis (G) and G1 arrest (H). HEY cells were treated as in (F) and cell cycle analyzed by flow cytometry after siRNA transfection. SubG1 cells were considered apoptotic cells. Data shown are mean values from three independent experiments.

Mentions: CDK5 knockdown in HEY cells significantly prolonged the half-life of TP53 and p27Kip1 proteins (Fig 4A). Silencing of CDK5 increased nuclear localization of TP53 and p27Kip1 proteins and cytoplasmic levels of p21Cip1 (Fig 4B). The p27Kip1 protein level is primarily regulated post-transcriptionally [20, 21]. CDK5 siRNA decreased the phosphorylation of p27Kip1 at Thr187 (Fig 4C), which is required for binding to ubiquitin ligase and leads to 26S proteasome degradation. TP53 has been reported to be a direct substrate of CDK5 [22–24]. Using purified CDK5 protein and a fluorescence-based in vitro kinase assay, we observed phosphorylation of TP53 and p27Kip1 (Fig 4D), using an unrelated protein, ARHI-NTD, as a negative control and Histone H1 as a positive control (S4 Fig) [25, 26]. Because p21Cip1 can be regulated by TP53 at the transcriptional level and CDK5 silencing also can upregulate TP53 expression, we tested the effect of CDK5 knockdown and TP53 knockdown on p21Cip1 promoter activity. When compared to control siRNA, CDK5 siRNA significantly increased promoter activity of p21Cip1, which was abolished by co-transfection with TP53 siRNA (Fig 4E). Furthermore, knockdown of p21Cip1 and p27Kip1 simultaneously (Fig 4F) reduced the CDK5 siRNA-induced increase in apoptosis (Fig 4G) and decreased the CDK5 siRNA-induced G1 cell cycle arrest in the presence or absence of paclitaxel (Fig 4H) in HEY cells. Thus, silencing of CDK5 produced post-translational upregulation of TP53 and p27Kip1 associated with TP53-dependent transcriptional induction of p21Cip1.


CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

Zhang S, Lu Z, Mao W, Ahmed AA, Yang H, Zhou J, Jennings N, Rodriguez-Aguayo C, Lopez-Berestein G, Miranda R, Qiao W, Baladandayuthapani V, Li Z, Sood AK, Liu J, Le XF, Bast RC - PLoS ONE (2015)

CDK5 knockdown increases TP53 and p27Kip1 protein half-lives, increases nuclear localization of the two proteins, decreases p27Kip1 p-T187 phosphorylation and induces p21Cip1 in a TP53-dependent manner.(A) Transfection with CDK5 siRNA increased the half-life of p53 and p27Kip1 protein. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h and then treated with 5 μg/mL of cycloheximide (CHX) for the indicated intervals. Cell lysates was subjected to Western analysis with the antibodies indicated. (B) CDK5 siRNA increased the nuclear localization of p53 and p27Kip1. HEY cells were transfected with Control siRNA or CDK5 siRNA for 24 h and subjected to fractionation into cytoplasmic and nuclear components. Cytoplasmic (CE) and nuclear (NE) extracts were analyzed by immunoblotting. (C) CDK5 siRNA decreased the phosphorylation of p27Kip1 at T187. The cell Immunoblot analysis was performed with antibodies against phosphorylated and total p27Kip1. (D) CDK5 enhanced phosphorylation of TP53 and p27Kip. A fluorescence based in vitro kinase assay was used to evaluate the interaction of CDK5 with TP53 and p27Kip1. (E) CDK5 siRNA transcriptionally regulated the expression of p21Cip1. HEY cells were transfected with CDK5 siRNA alone or in combination with TP53 siRNA and a luciferase reporter assay was used to measure p21Cip1 promoter activity. (F) CDK5, p21Cip1 and p27Kip1 knockdown decreased the expression of CDK5, p21Cip1 and p27Kip1. HEY cells were treated with control siRNA or CDK5 or/and p21 plus p27 siRNA for 48 h and then with paclitaxel (6 nM) or diluent for 24 h. Immunoblot analysis was performed with antibodies indicated. (G, H) Knockdown of p21and p27 reduced CDK5 knockdown-induced apoptosis (G) and G1 arrest (H). HEY cells were treated as in (F) and cell cycle analyzed by flow cytometry after siRNA transfection. SubG1 cells were considered apoptotic cells. Data shown are mean values from three independent experiments.
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pone.0131833.g004: CDK5 knockdown increases TP53 and p27Kip1 protein half-lives, increases nuclear localization of the two proteins, decreases p27Kip1 p-T187 phosphorylation and induces p21Cip1 in a TP53-dependent manner.(A) Transfection with CDK5 siRNA increased the half-life of p53 and p27Kip1 protein. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h and then treated with 5 μg/mL of cycloheximide (CHX) for the indicated intervals. Cell lysates was subjected to Western analysis with the antibodies indicated. (B) CDK5 siRNA increased the nuclear localization of p53 and p27Kip1. HEY cells were transfected with Control siRNA or CDK5 siRNA for 24 h and subjected to fractionation into cytoplasmic and nuclear components. Cytoplasmic (CE) and nuclear (NE) extracts were analyzed by immunoblotting. (C) CDK5 siRNA decreased the phosphorylation of p27Kip1 at T187. The cell Immunoblot analysis was performed with antibodies against phosphorylated and total p27Kip1. (D) CDK5 enhanced phosphorylation of TP53 and p27Kip. A fluorescence based in vitro kinase assay was used to evaluate the interaction of CDK5 with TP53 and p27Kip1. (E) CDK5 siRNA transcriptionally regulated the expression of p21Cip1. HEY cells were transfected with CDK5 siRNA alone or in combination with TP53 siRNA and a luciferase reporter assay was used to measure p21Cip1 promoter activity. (F) CDK5, p21Cip1 and p27Kip1 knockdown decreased the expression of CDK5, p21Cip1 and p27Kip1. HEY cells were treated with control siRNA or CDK5 or/and p21 plus p27 siRNA for 48 h and then with paclitaxel (6 nM) or diluent for 24 h. Immunoblot analysis was performed with antibodies indicated. (G, H) Knockdown of p21and p27 reduced CDK5 knockdown-induced apoptosis (G) and G1 arrest (H). HEY cells were treated as in (F) and cell cycle analyzed by flow cytometry after siRNA transfection. SubG1 cells were considered apoptotic cells. Data shown are mean values from three independent experiments.
Mentions: CDK5 knockdown in HEY cells significantly prolonged the half-life of TP53 and p27Kip1 proteins (Fig 4A). Silencing of CDK5 increased nuclear localization of TP53 and p27Kip1 proteins and cytoplasmic levels of p21Cip1 (Fig 4B). The p27Kip1 protein level is primarily regulated post-transcriptionally [20, 21]. CDK5 siRNA decreased the phosphorylation of p27Kip1 at Thr187 (Fig 4C), which is required for binding to ubiquitin ligase and leads to 26S proteasome degradation. TP53 has been reported to be a direct substrate of CDK5 [22–24]. Using purified CDK5 protein and a fluorescence-based in vitro kinase assay, we observed phosphorylation of TP53 and p27Kip1 (Fig 4D), using an unrelated protein, ARHI-NTD, as a negative control and Histone H1 as a positive control (S4 Fig) [25, 26]. Because p21Cip1 can be regulated by TP53 at the transcriptional level and CDK5 silencing also can upregulate TP53 expression, we tested the effect of CDK5 knockdown and TP53 knockdown on p21Cip1 promoter activity. When compared to control siRNA, CDK5 siRNA significantly increased promoter activity of p21Cip1, which was abolished by co-transfection with TP53 siRNA (Fig 4E). Furthermore, knockdown of p21Cip1 and p27Kip1 simultaneously (Fig 4F) reduced the CDK5 siRNA-induced increase in apoptosis (Fig 4G) and decreased the CDK5 siRNA-induced G1 cell cycle arrest in the presence or absence of paclitaxel (Fig 4H) in HEY cells. Thus, silencing of CDK5 produced post-translational upregulation of TP53 and p27Kip1 associated with TP53-dependent transcriptional induction of p21Cip1.

Bottom Line: Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase.In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells.CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America; Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.

ABSTRACT
Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

No MeSH data available.


Related in: MedlinePlus