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Signaling and Regulation

Gene Expression Profiling in Prostate Cancer Cells With Akt Activation Reveals Fra-1 As an Akt-Inducible Gene¹

¹ Department of Molecular Pharmacology, Stanford University and ² Department of Pathology, Stanford University School of Medicine, Stanford, CA

Requests for reprints: Richard A. Roth, Stanford University School of Medicine, Center for Clinical Sciences Research, Room 3145B, Stanford, CA 94305-5174. Phone: (650) 723-5933; Fax: (650) 723-2253. E-mail: rroth{at}stanford.edu

To determine which genes may be regulated by Akt and participate in the transformation of cells, we have examined by microarray analyses genes turned on in the prostate cancer cell line, PC3, when Akt activity was induced. PC3 cells, which lack the lipid phosphatase PTEN, were treated overnight with a reversible inhibitor of the phosphatidylinositol 3-kinase, LY294002 (a treatment which was found to reversibly decrease Akt enzymatic activity). The inhibitor was then washed out and mRNA collected 2, 6, and 10 h later and compared by microarray analyses with mRNAs present immediately after removal of the inhibitor. One of the identified induced mRNAs, Fra-1, was further studied by transient transfections of a reporter construct containing its 5' regulatory region. This construct was found to be directly induced 4- to 5-fold by co-transfection with constitutively active Akt3 but not kinase dead Akt. The regulation by Akt3 was found to be due to two specific regions in the Fra-1 regulatory sequence which match Sp1 consensus sites. Finally, gel shift studies showed that the binding of Sp1 to one of these sites was dependent on the PI 3-kinase pathway. These results indicate that LY294002 treatment and washout is a useful method to study the activation of Akt in the context of a tumor cell. Moreover, the identification of Fra-1 as an Akt-regulated gene may have implications for the ability of Akt to transform cells since Fra-1 has been implicated in cell growth and the aggressiveness of tumors.

Key Words: transcription • Sp1 • microarray • PI 3-kinase

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