17-AAG (NSC 330507, NCI, Bethesda, Maryland, USA), LY294002 (Biomol, Plymouth Meeting, Pennsylvania, USA), and lactacystin (Sigma, St Louis, Missouri, USA) were dissolved in 100% DMSO. Lipids (Sigma) were stored in chloroform and sealed under nitrogen.
The human cancer lines MCF-7, MDA-MB-468 (MDA-468), SKBr-3, BT-474 (American Type Culture Collection, Manassas, Virginia, USA) were maintained in 1 : 1 mixture of DME:F12 supplemented with 2 mm glutamine, 50 units/ml penicillin, 50 units/ml streptomycin, and 10% heat inactivated fetal bovine serum (Gemini Bioproducts, Calabasa, California, USA) and incubated at 37°C in 5% CO2.
Polyclonal antibodies: Akt, P-Akt (Ser-473), P-4E-BP1 (Ser-65), P-GSK-3 (Ser-21/9), P-PDK1 (Ser-241) (Cell Signaling, Beverly, Massachusetts, USA); p85, p110α, PDK1, GSK-3 (Upstate Biotechnology, Lake Placid, New York, USA); Cyclin-D1 (m-20), 4E-BP1 (r-113), HER2 (c-18), HER3 (c- 17), P-HER2 (Santa Cruz Biotechnology, Santa Cruz, California, USA). Monoclonal antibodies: PY99 (Santa Cruz Biotechnology). HER3 (Ab-7) (Neomarkers, Fremont, California, USA) was used for immunoprecipitation.
Cells were exposed to 17-AAG or DMSO vehicle. Cells were lysed in NP-40 buffer (50 mm Tris, pH 7.5, 1% Nonidet P-40, 150 mm NaCl, 2.5 mm Na3VO4, 10mm phenylmethylsulfonyl fluoride, and 10 µm each leupeptin, aprotinin, and soybean trypsin inhibitor) and cleared by centrifugation. Protein concentration was determined by using BCA reagent (Pierce Chemical Co., Rockford, Illinois, USA). Immunoprecipitations were performed using Protein-G Sepharose (Amersham, Piscataway, New Jersey, USA). Samples were separated by 7 – 10% SDS – PAGE, transferred to nitrocellulose, immunoblotted and detected by chemiluminescence using the ECL detection reagents (Amersham).
Akt activity assay
Kinase activity was assayed using New England Biolabs Akt Kinase Kit. Akt was immunoprecipitated, washed twice with lysis buffer, then twice with kinase buffer (25 mm Tris pH 7.5, 5 mm beta-glycerolphosphate, 2 mm DTT, 0.1 mm Na3VO4, 10 mm MgCl2). Two hundred µm ATP and 1 µg substrate (paramyosin fused to a GSK-3 crosstide) were added and assays were performed at 30°C for 30 min. Reaction mixtures were separated by 10% SDS –PAGE and the P-GSK3 reaction product was detected by immunoblotting.
PI3 kinase assay
Cells were lysed in 137 mm NaCl, 20 mm Tris pH 7.5, 1 mm MgCl2, 10% v/v glycerol, 1% v/v Triton X, 10 mm phenylmethylsulfonyl fluoride, and 10 µm each leupeptin, aprotinin, and soybean trypsin inhibitor. HER3 immunoprecipitation complexes were washed three times with 1% Triton X-100 in PBS, twice with 0.5 m LiCl, 0.1 m Tris pH 7.5, and twice with 10 mm Tris pH 7.5, 100 mm NaCl, 1 mm EDTA. Complexes were incubated for 20 min at room temperature with 1 µm ATP, 5 µCi ATP (γP32), and a 0.5 µm/ml 1 : 1 lipid mix of phosphatidylinositol and phosphatidylserine in 10 mm HEPES pH 7.0, 1 mm EGTA. The reaction was quenched by 1 m HCl and lipids were extracted with 1 : 1 CHCl3 : CH3OH. The organic layer was separated by thin layer chromatography eluted with 50 : 15 : 35 1-propanol : methanol : glacial acetic acid and detected by autoradiography.
PI3 kinase (p110α) transfectants
The activated PI3k construct was provided by J Downward. The carboxy-terminal farnesylation signal from H-Ras was fused to p110 (myc
-tagged p110-CAAX in pSG5 vector) (Wennstrom and Downward, 1999
). Two million cells were transfected with 2 µg of DNA and 10 µl Lipofectin reagent (Life Technologies, Rockville, Maryland, USA). Experiments were performed 24 h after transfection.
Six-week-old athymic BALB/c female mice (NCI-Frederick Cancer Center) were maintained in pressurized ventilated cages. Experiments were carried out under an IACUC approved protocol and institutional guidelines for the proper, humane use of animals in research were followed. Prior to tumor cell inoculation, 0.72 mg SR 17B-estradiol pellets (Innovative Research of America, Sarasota, Florida, USA) were placed subcutaneously into the right flank. 1 × 107 BT-474 cells were mixed 1 : 1 with Matrigel (Collaborative Research, Bedford, Massachusetts, USA) and injected subcutaneously. Mice were randomized to treatment or control groups and treated with 17-AAG (75 mg/kg) or the egg-phospholipid (EPL) vehicle alone in cycles of 5 consecutive days. Treatment cycles were repeated 2 weeks later. Mice were weighed and tumors were measured with vernier calipers. Tumor volumes were calculated with the formula: π/6 × larger diameter × (smaller diameter)2. To analyse cellular markers, mice with established tumors were treated with a single dose of 17-AAG 50 mg/kg i.p. Mice were sacrificed pre-treatment and 1, 2, 4, 10 and 24 h post-treatment. For immunoblotting, tumor tissue was homogenized in 2% SDS lysis buffer (pH 7.4).