All results were measured by densitometry and displayed as relation of phosphorylated/nonphosphorylated protein and fold induction to untreated controls. A total of 168 carcinomas of the liver were retrieved from the surgical pathology files of the Department of Pathology, University Hospital Zürich, Switzerland, and the Institute of Pathology, University of Regensburg, Germany. Tumors were classified according to the World Health Organization (WHO) Classification of the Digestive System
(2000). The study was approved by the local ethics committees of Zurich (Kantonale Ethikkommission Zurich, StV 26–2005). Formalin-fixed paraffin-embedded tumor tissues were used to construct tissue microarrays (TMA) with cores of 168 HCC and 20 normal liver tissues. The TMAs were constructed as described.15 http://www.selleckchem.com/products/FK-506-(Tacrolimus).html Two tissue cores per tumor with a diameter of 0.6 mm were punched out of the donor block and transferred to the recipient block. Three-micron-thick sections of TMA blocks and formalin-fixed, paraffin-embedded tissues were mounted on glass slides (SuperFrost Plus;
Menzel), deparaffinized, rehydrated, and stained with hematoxylin-eosin using standard histological techniques. For immunohistochemical staining the Ventana Benchmark automated staining system (Ventana Medical Systems) and GW-572016 mw Ventana reagents were used. After deparaffinization in xylene, slides were rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase was blocked using the Ventana endogenous peroxidase blocking kit after a rinse with distilled water. For antigen retrieval slides were heated with cell conditioning solution (CC1, Ventana) according to the manufacturers instructions. The same primary antibodies as for western blotting were used against and adjusted to the Ventana Benchmark system after performing titrations. iVIEW-DAB was used as chromogen. Ki-67 proliferation index was defined as percentage mafosfamide of positive nuclei per 100 tumor cells and was determined on TMA tumor tissue cores. All animal experiments
were in accordance with Swiss federal animal regulations and approved by the cantonal veterinary office of Zurich. Athymic female NU/NU mice ages 6 to 8 weeks were kept on a 12-hour day/night cycle with free access to food and water. Huh7 cells were trypsinized (Invitrogen), washed in PBS, and resuspended in serum-free DMEM. Three × 106 Huh7 cells in 200 μL DMEM were injected subcutaneously in the lower back. After 3 weeks 33% of the mice bore visible tumors. When tumors reached a mean volume of 150 mm3 mice were randomized into two groups (six animals per group): Animals were treated with 0.16% DMSO in 500 μL H20 subcutaneously or 10 mg/kg BW SB204741 in 500 μL H20 twice a day.