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Departments of ¹ Radiation Oncology and ² Hematology/Oncology, Virginia Commonwealth University, Richmond, Virginia; ³ Department of Pathology, International Medical Center of Japan, Tokyo, Japan; ⁴ National Cancer Institute, Bethesda, Maryland; ⁵ Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois; and ⁶ Instituto de Medicina y Biología Experimental de Cuyo-CONICET, Mendoza, Argentina

Requests for reprints: Paul Dent, Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, 401 College Street, Richmond, VA 23298-0058. Phone: 804-628-0861; Fax: 804-828-6042. E-mail: pdent{at}hsc.vcu.edu

The abilities of mutated active RAS proteins to modulate cell survival following exposure to ionizing radiation and small molecule kinase inhibitors were examined. Homologous recombination in HCT116 cells to delete the single allele of K-RAS D13 resulted in a cell line that exhibited an 75% reduction in basal extracellular signal-regulated kinase 1/2, AKT, and c-jun-NH₂-kinase 1/2 activity. Transfection of cells lacking K-RAS D13 with H-RAS V12 restored extracellular signal-regulated kinase 1/2 and AKT activity to basal levels but did not restore c-jun-NH₂-kinase 1/2 phosphorylation. In cells expressing H-RAS V12, radiation caused prolonged intense activation of AKT. Inhibition of H-RAS V12 function, blockade of phosphatidylinositol 3-kinase (PI3K) function using small interfering RNA/small-molecule inhibitors, or expression of dominant-negative AKT abolished radiation-induced AKT activation, and radiosensitized these cells. Inhibition of PI3K function did not significantly radiosensitize parental HCT116 cells. Inhibitors of the AKT PH domain including perifosine, SH-(5, 23-25) and ml-(14-16) reduced the plating efficiency of H-RAS V12 cells in a dose-dependent fashion. Inhibition of AKT function using perifosine enhanced radiosensitivity in H-RAS V12 cells, whereas the SH and ml series of AKT PH domain inhibitors failed to promote radiation toxicity. In HCT116 H-RAS V12 cells, PI3K, PDK-1, and AKT were membrane associated, whereas in parental cells expressing K-RAS D13, only PDK-1 was membrane bound. In H-RAS V12 cells, membrane associated PDK-1 was phosphorylated at Y373/376, which was abolished by the Src family kinase inhibitor PP2. Inhibition of PDK-1 function using the PH domain inhibitor OSU-03012 or using PP2 reduced the plating efficiency of H-RAS V12 cells and profoundly increased radiosensitivity. OSU-03012 and PP2 did not radiosensitize and had modest inhibitory effects on plating efficiency in parental cells. A small interfering RNA generated against PDK1 also radiosensitized HCT116 cells expressing H-RAS V12. Collectively, our data argue that molecular inhibition of AKT and PDK-1 signaling enhances the radiosensitivity of HCT116 cells expressing H-RAS V12 but not K-RAS D13. Small-molecule inhibitory agents that blocked stimulated and/or basal PDK-1 and AKT function profoundly reduced HCT116 cell survival but had variable effects at enhancing tumor cell radiosensitivity.

Key Words: radiation • RAS • receptor • signaling • PDK-1 • DAKT

Grant support: UPHS grants R01-CA88906 and R01-DK52825 (P. Dent), P01-CA72955, R01-CA63753, and R01-CA77141 (S. Grant); Department of Defense Awards BC980148 and BC020338 (P. Dent); Leukemia Society of America grant 6405-97 (S. Grant); Department of Radiation Oncology, Virginia Commonwealth University (A. Yacoub); and Universal, Inc. Professorship in Signal Transduction Research (P. Dent).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁷ R. Carón and P. Dent, unpublished observations.

Received 9/16/04; revised 11/17/04; accepted 11/30/04.