Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells.
Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G(1) phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27(kip). Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (DeltaRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.
Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232, USA.
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D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002867:Chromones / Q000494:pharmacology; D019938:Cyclin D1 / Q000378:metabolism; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism*; D005434:Flow Cytometry; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology*; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D012685:Sepharose / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D000077191:Wortmannin; D018631:ras Proteins / Q000378:metabolism*
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D019938:Cyclin D1; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012685:Sepharose; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D000077191:Wortmannin