Double-stranded RNA-dependent protein kinase is involved in 2-methoxyestradiol-mediated cell death of osteosarcoma cells

Kristen L. Shogren, Russell T. Turner, Michael J. Yaszemski, Avudaiappan Maran

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

We studied the involvement of interferon-regulated, PKR on 2-ME-mediated actions in human osteosarcoma cells. Our results show that PKR is activated by 2-ME treatment and is necessary for 2-ME-mediated induction of osteosarcoma cell death. Introduction: Osteosarcoma is the most common primary bone tumor and most frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a metabolite of 17β-estradiol, induces interferon gene expression and apoptosis in human osteosarcoma cells. In this report, we studied the role of interferon-regulated double-stranded (ds)RNA-dependent protein kinase (PKR) protein on 2-ME-mediated cell death in human osteosarcoma cells. Materials and Methods: Western blot analyses were used to measure PKR protein and phosphorylation levels. Cell survival and apoptosis assays were measured using trypan blue exclusion and Hoechst dye methods, respectively. A transient transfection protocol was used to express the dominant negative PKR mutants. Results and Conclusions: PKR was increased in 2-ME-treated MG63 cells, whereas 17β-estradiol, 4-hydroxyestradiol, and 16α-hydroxyestradiol, which do not induce cell death, had no effect on PKR protein levels. Also, 2-ME treatment induced PKR kinase activity as indicated by increased autophosphorylation and phosphorylation of the endogenous substrate, eukaryotic initiation factor (eIF)-2α. dsRNA poly (I) poly (C), an activator of PKR protein, increased cell death when osteosarcoma cells were treated with a submaximal concentration of 2-ME. In contrast, a serine-threonine kinase inhibitor SB203580 and a specific PKR inhibitor 2-aminopurine (2-AP) blocked the 2-ME-induced cell death in MG63 cells. A dominant negative PKR mutant protein conferred resistance to 2-ME-induced cell death to MG63 osteosarcoma and 2-ME-mediated PKR regulation did not require interferon gene expression. PKR protein is activated in cell free extracts by 2-ME treatment, resulting in autophosphorylation and in the phosphorylation of the substrate eIF-2α. We conclude from these results that PKR is regulated by 2-ME independently of interferon and is essential for 2-ME-mediated cell death in MG63 osteosarcoma cells.

Original languageEnglish (US)
Pages (from-to)29-36
Number of pages8
JournalJournal of Bone and Mineral Research
Volume22
Issue number1
DOIs
StatePublished - Jan 1 2007

Keywords

  • Double-stranded RNA-dependent protein kinase
  • Estrogen metabolite
  • Interferon
  • MG63 cells
  • Protein kinase

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Orthopedics and Sports Medicine

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