Ras-mediated loss of the pro-apoptotic response protein Par-4 is mediated by DNA hypermethylation through Raf-independent and Raf-dependent signaling cascades in epithelial cells

J Biol Chem. 2005 Jun 17;280(24):23363-70. doi: 10.1074/jbc.M503083200. Epub 2005 Apr 14.

Abstract

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Agar / chemistry
  • Alleles
  • Animals
  • Apoptosis Regulatory Proteins
  • Apoptosis*
  • Azacitidine / analogs & derivatives*
  • Azacitidine / pharmacology
  • Blotting, Northern
  • Blotting, Western
  • Butadienes / pharmacology
  • Cell Line
  • Cell Line, Tumor
  • Cell Transformation, Neoplastic
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA Methylation*
  • DNA, Complementary / metabolism
  • Decitabine
  • Down-Regulation
  • Enzyme Inhibitors / pharmacology
  • Epithelial Cells / metabolism*
  • Female
  • Fibroblasts / metabolism
  • Flavonoids / pharmacology
  • Genetic Vectors
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mice
  • Mutation
  • NIH 3T3 Cells
  • Nitriles / pharmacology
  • Ovary / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism
  • Polymerase Chain Reaction
  • RNA / metabolism
  • RNA, Messenger / metabolism
  • Rats
  • Signal Transduction*
  • Up-Regulation
  • raf Kinases / physiology*

Substances

  • Apoptosis Regulatory Proteins
  • Butadienes
  • DNA, Complementary
  • Enzyme Inhibitors
  • Flavonoids
  • Intracellular Signaling Peptides and Proteins
  • Nitriles
  • RNA, Messenger
  • U 0126
  • prostate apoptosis response-4 protein
  • RNA
  • Decitabine
  • Agar
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA methyltransferase 3A
  • DNMT1 protein, human
  • Dnmt1 protein, mouse
  • Dnmt1 protein, rat
  • Phosphatidylinositol 3-Kinases
  • raf Kinases
  • Azacitidine
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one