Vitamin D Inhibits G1 to S Progression in LNCaP Prostate Cancer Cells through p27Kip1 Stabilization and Cdk2 Mislocalization to the Cytoplasm

Eddy S. Yang, Kerry L Burnstein

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109 Citations (Scopus)

Abstract

1,25-(OH)2 vitamin D3 (1,25-(OH)2D 3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LNCaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH) 2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D 3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr 187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH) 2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr 15 dephosphorylation. Interestingly, 1,25-(OH)2D 3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH) 2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.

Original languageEnglish
Pages (from-to)46862-46868
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number47
DOIs
StatePublished - Nov 21 2003

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Cyclin-Dependent Kinase 2
Vitamin D
Prostatic Neoplasms
Cytoplasm
Stabilization
Cells
Phosphorylation
Degradation
S-Phase Kinase-Associated Proteins
cdc25 Phosphatases
CDC2 Protein Kinase
Cyclin E
Retinoblastoma Protein
Proteins
Cyclins
Cyclin-Dependent Kinases
Confocal microscopy
Cholecalciferol
5' Untranslated Regions
3' Untranslated Regions

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{21551e31b6cc4af5a7a701f57cc21945,
title = "Vitamin D Inhibits G1 to S Progression in LNCaP Prostate Cancer Cells through p27Kip1 Stabilization and Cdk2 Mislocalization to the Cytoplasm",
abstract = "1,25-(OH)2 vitamin D3 (1,25-(OH)2D 3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LNCaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH) 2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D 3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr 187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH) 2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr 15 dephosphorylation. Interestingly, 1,25-(OH)2D 3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH) 2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.",
author = "Yang, {Eddy S.} and Burnstein, {Kerry L}",
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TY - JOUR

T1 - Vitamin D Inhibits G1 to S Progression in LNCaP Prostate Cancer Cells through p27Kip1 Stabilization and Cdk2 Mislocalization to the Cytoplasm

AU - Yang, Eddy S.

AU - Burnstein, Kerry L

PY - 2003/11/21

Y1 - 2003/11/21

N2 - 1,25-(OH)2 vitamin D3 (1,25-(OH)2D 3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LNCaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH) 2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D 3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr 187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH) 2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr 15 dephosphorylation. Interestingly, 1,25-(OH)2D 3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH) 2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.

AB - 1,25-(OH)2 vitamin D3 (1,25-(OH)2D 3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LNCaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH) 2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D 3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr 187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH) 2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr 15 dephosphorylation. Interestingly, 1,25-(OH)2D 3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH) 2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.

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