Cell culture and reagents.
U373MG and HEK-293 cell lines were purchased from American Type Culture Collection (Rockville, Md.) and grown in Dulbecco's modified essential medium (DMEM; Gibco-BRL, Rockville, Md.) supplemented with 10% fetal bovine serum (FBS; Gemini Bioproducts, Calabasas, Calif.), 20 mM HEPES (Mediatech, Herndon, Va.), and pencillin-streptomycin (Mediatech). U373-Neo and U373-SF cell lines have been previously described and were grown in the medium described above supplemented with 300 μg of Geneticin per ml (Gibco-BRL). SNB-19 cells were a gift of J. Rao, University of Illinois College of Medicine at Peoria, and were grown in DMEM-F12 (Gibco-BRL) supplemented with 10% FBS, 20 mM HEPES, and penicillin-streptomycin. All cells were grown at 37°C in a humidified incubator with 5% CO2. Recombinant human SF/HGF was provided as a gift from Genentech, Inc. (San Francisco, Calif.).
Adenovirus vectors encoding GFP, c-Myc-hemagglutinin (HA) and MadMyc-HA constructs were provided by B. Vogelstein, Johns Hopkins University School of Medicine, Baltimore, Md., and have been previously described (5
). Adenovirus constructs were propagated in HEK-293 cells as described for the pAdEasy adenoviral generation system (15
). In vitro transductions were performed at a multiplicity of infection of 10. Transduction efficiency was judged at 24 h by expression of green fluorescent protein (GFP). pCS2, pCS2-p27, and pCS2-p27T187A constructs were provided by B. Clurman (Fred Hutchinson Cancer Center) and have been described previously (50
). Full-length cDNA probes for c-Myc and p27 Northern blots were produced by restriction digests of pBS-c-Myc (gift of C. Dang, Johns Hopkins University School of Medicine) and pCEP4p27 (gift of B. Vogelstein) followed by gel purification (Qiaex gel extraction; Qiagen, Hilden, Germany). The dominant-negative (DN) form of Cdk2 (pCMV-Cdk2DN) (kindly provided by S. van den Heuvel) (58
), and the E2F1-DN expression vector pcDNA-E2F1(1-368)/RB- (379-792) and E2F1 promoter-luciferase reporter construct (pGL2AN/Luc) (kindly supplied by W. G. Kaelin) (35
) have been previously described. Target cells were transfected with Fugene-6 transfection reagent (Roche, Indianapolis, Ind.) according to manufacturer's specifications with a 3:1 ratio of transfection reagent volume to DNA mass. To judge the efficiency of transfection, cells were cotransfected with a pCS2 plasmid encoding GFP under the control of the cytomegalovirus (CMV) promoter at a ratio of 10:1 (target gene: GFP plasmid). Efficiency of transient transfection under the conditions of these experiments was found to be typically ≥80%.
Luciferase activity was assayed as described previously (20
). Cells (2 × 10 5
) grown in six-well plates were transfected with 3 μg of pGL2AN/Luc plasmid DNA or control plasmid DNA (pGL2). Twenty-four hours after transfection, cells were stimulated with SF/HGF (100 ng/ml) for an additional 24 h, and cell extracts were prepared with reporter lysis buffer (Promega, Madison, Wis.). Luminescence was measured in 20 μl of cell extract with a luciferase assay system kit (Promega). Activity was expressed as relative light units per milligram of cellular protein as determined by Coomassie protein assay (Pierce, Rockford, Ill.).
Antibodies used for Western blotting, immunofluorescence, immunoprecipitation, and the kinase assay were obtained from the following sources: Transduction Laboratories (Lexington, Ky.), Kip1p27 (1:2,000, Western blot) and Cip1p21 (1:250, Western blot); Santa Cruz Biotechnology (Santa Cruz, Calif.), p27 (C-19; immunofluorescence, 1:100), actin (C-11; Western blot, 1:1,000), Cdk2 (M2; Western blot, 1:500; kinase assay, 1:50), Cdk4 (C-22, Western blot, 1:500), c-Myc (9E10; Western blot, 1:500), cyclin A (H-432; Western blot, 1:500), cyclin D1 (HD11; Western blot, 1:500), cyclin D2 (H-289; Western blot, 1:500), cyclin D3 (D-7; Western blot, 1:500), cyclin E (HE12; Western blot, 1:500; and C-19; immunoprecipitation, 1:50), and Rb (C-15; Western blot, 1:500); and Clontech (Palo Alto, Calif.), HA tag (Western blot, 1:1,000). Secondary antibodies conjugated to horseradish peroxidase were purchased from Jackson Immunoresearch Laboratories (West Grove, Pa.), and those conjugated to Alexa-fluor were purchased from Molecular Probes (Eugene, Oreg.).
Cells growing as a monolayer on 100-mm-diameter tissue culture dishes (Corning, Corning, N.Y.) were trypsinized (Gibco-BRL), pelleted by centrifugation, resuspended in 1 ml of ice-cold phosphate-buffered saline (PBS), and fixed by the addition of 4 ml of ice-cold ethanol under gentle vortexing. Fixed cells were stored up to 1 week in 80% ethanol at −20°C until ready for labeling. Stored cells were collected by centrifugation, resuspended in 1 ml of PBS, and treated with 20 μg of DNase-free RNase (Boehringer-Mannheim, Mannheim, Germany) for 30 min at 37°C. Cells were labeled with propidium iodide (Sigma, St. Louis, Mo.) at a final concentration of 100 μg/ml for 10 min at room temperature. Analyses were performed on a FACscan (Becton-Dickinson, Fullerton, Calif.). Raw data were gated to remove doublets and cellular debris. The resultant cell cycle histograms were analyzed with CellQuest software (Becton-Dickinson), and the area under the curve was integrated for each peak.
Tritiated thymidine incorporation.
Cells were plated (100,000 cells/well) in six-well plates in medium containing 10% FBS and allowed to adhere overnight. Cells were then switched to fresh medium containing 0.1% FBS ± 100 ng of SF/HGF per ml. After 18 h, 5 μCi of [3H]thymidine was added to each well. Cells were incubated for an additional 6 h at 37°C, and then the medium was removed and each well was washed three times with PBS. Radiolabeled cells were treated three times in 10% trichloroacetate at 1 ml per well for 20 min at ambient temperature and then lysed in 0.2 M NaOH overnight at 37°C. The extent of radiolabeling was determined by subjecting an aliquot of the cell lysate to scintillation counting (LS 6500; Beckman, Fullerton, Calif.).
Cells were plated at low density (2,000 cells per well) or high density (5,000 cells per well) in 96-well tissue culture plates in medium containing 10% FBS (full serum) or 0.1% FBS (low serum) supplemented with 100 ng of SF/HGF per ml or the equivalent amount of PBS. Seventy-two hours postplating, MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well at a final concentration of 1 mg/ml, and the cells were incubated for 2 h at 37°C. The medium was then removed, and the formazen reaction product was solubilized by addition of 100 μl of dimethyl sulfoxide (DMSO). A570 was measured with a microtiter plate reader (Dynatech, Alexandria, Va.). Absorbance values are presented as the mean of eight wells per treatment ± the standard error (SE).
Total RNA was harvested from the cells by using Qiagen RNeasy kits according to manufacturer's recommendations. Ten micrograms of RNA per sample was denatured with deionized glyoxal, combined with RNA loading buffer, and run on a 1.0% agarose gel containing ethidium bromide for 3 h at 60 V. RNA was transferred to a nylon membrane (Nytran; Schleicher & Schuell) overnight in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Membranes were prehybridized for 4 h at 42°C and then hybridized overnight at 42°C in a rotating oven. Membranes were washed three times in 1× SSC-0.1% sodium dodecyl sulfate (SDS) at 50°C and exposed to a phosphorimager cassette (Fujifilm, Tokyo, Japan). Full-length cDNA probes were generated for c-Myc, p27, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with [32P]dCTP (Amersham-Pharmacia, Piscataway, N.J.) in conjunction with a random priming labeling kit (Boehringer-Mannheim) according to manufacturer's specifications.
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed by the method of Towbin et al. (55
) with some modifications. Cells growing in a monolayer on 100-mm-diameter tissue culture dishes were washed with ice-cold PBS and then lysed by the addition of 300 μl of radioimmunoprecipitation assay (RIPA) buffer (1% Igepal, 0.5% sodium deoxycholate, and 0.1% SDS in PBS) containing fresh protease inhibitors (Calbiochem, San Diego, Calif.). Plates were scraped to recover cellular lysates that were passed through a 20-gauge needle to shear DNA, incubated on ice for 20 min, and centrifuged at 15,000 × g
at 4°C for 15 min. Supernatant protein concentrations were determined for each sample with the Coomassie protein assay (Pierce) according the manufacturer's recommendations. Aliquots of 10 μg of total protein were combined with Laemmli loading buffer containing β-mercaptoethanol and heated at 100°C for 5 min. The proteins were separated on precast SDS-PAGE gels (Invitrogen, Carlsbad, Calif.) at 125 V for 90 min. Proteins were electrophoretically transferred to nitrocellulose with a semidry transfer apparatus at 50 mA per gel for 45 min (Amersham-Pharmacia). Membranes were incubated for 1 h in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS/T) with 5% nonfat dried milk at room temperature. Incubation with primary antibody was performed overnight at 4°C in TBS/T-milk at the concentrations indicated above. Membranes were then washed three times with TBS/T, incubated with secondary antibody at 1:2,000 for 1 h in TBS/T-milk, washed three times with TBS/T, and then developed with an enhanced chemiluminescence (ECL) detection kit (Amersham-Pharmacia) per the manufacturer's instructions.
Cyclin E immunoprecipitations were performed as previously described (9
). Briefly U373MG cell monolayers were transiently cotransfected with pCS2-GFP, pCS2-p27, or pCS2-p27T187A as described above. Cells were then growth arrested in medium containing 0.1% FBS ± 100 ng of SF/HGF per ml for 24 h and then lysed on ice in a buffer containing 50 mM HEPES, 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and complete protease inhibitor cocktail (Calbiochem). Lysates were homogenized and cleared by centrifugation at 10,000 × g
for 10 min. Protein concentrations were determined in the supernatant as described above, and 100 μg of total protein was used for each immunoprecipitation. The volume of each tube was brought to 500 μl with lysis buffer, and 2 μg of cyclin E (c-19) antibody was added. The tubes were mixed continuously at 4°C for 1 h, and then 40 μl of protein A-agarose beads (Upstate Biotechnology, Lake Placid, N.Y.) was added to each tube, and the samples were mixed continuously for another hour. The beads were collected by centrifugation, washed three times with lysis buffer, heated to 100°C in Laemmli buffer, and subjected to Western blotting for p27 as described above.
Cdk2 kinase assays were performed as described previously (29
). U373MG cell monolayers were transfected with p27 and arrested in medium with 0.1% FBS as described above for immunoprecipitation. Cells were lysed on ice in a buffer containing 50 mM HEPES (ph 7.5), 150 mM NaCl, 2.5 mM EGTA, 1 mM dithiothreitol (DTT), 0.1% Tween 20, 10% glycerol, 10 mM β-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, and complete protease inhibitor cocktail (Calbiochem). Lysates were cleared by centrifugation, and the protein concentration of the supernatant was determined. Each lysate (100 μg of total protein) was immunoprecipitated with 2 μg of Cdk2 (M2) antibody for 1 h at 4°C, followed by addition of 40 μl of protein A-agarose beads (Upstate Biotechnology) for an additional hour. Immune complexes were washed three times with lysis buffer and twice with 50 mM HEPES containing 1 mM DTT, and then were incubated for 30 min at 30°C in 30 μl of buffer containing 50 mM HEPES, 10 mM MgCl2
, 1 mM DTT, 1 μg of histone H1 (Boehringer Mannheim, Indianapolis, Ind.), 2.5 mM EGTA, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM NaF, 10 μCi of [γ-32
P]ATP (100 μM ATP; Amersham-Pharmacia). After the incubation, 6× Laemmli loading buffer was added to each sample, the complexes dissociated by heating to 100°C for 5 min, and the reaction products were subjected to SDS-PAGE. The gel was subsequently dried, and the phosphorylated histone product was visualized by phosphorimaging (Fujifilm).
Cells were plated onto four-well chamber slides (Nalge-Nunc, Naperville, Ill.) in medium containing 10% FBS and allowed to adhere overnight. Fresh medium containing 0.1% FBS ± 100 ng of SF/HGF per ml was then substituted and the cells were incubated for an additional 24 h. Cells were fixed with methanol-acetone (1:1) for 2 min and then incubated for 1 h at room temperature with PBS containing 1.5% normal goat serum (Sigma). Cells were then incubated for 1 h at 4°C in PBS-goat serum containing rabbit polyclonal anti-human p27 antibody. Cells were washed three times with PBS and then incubated for 1 h with Alexa-conjugated mouse anti-rabbit secondary antibody at room temperature. Cells were washed three times with PBS, and the slides were coverslipped with Vectashield antifade solution with DAPI (Vector Laboratories, Burlingame, Calif.). Slides were observed with an Axiovision fluorescent microscope with fluorescein isothiocyanate (FITC) and DAPI filters.
U373-SF and U373-Neo cell lines were plated at 10,000 cells per well in 24-well tissue culture dishes in medium supplemented with 10% FBS. On postplating days 1, 3, 4, 5, 6, and 7, four wells from each cell line were harvested by trypsinization, and the cell number was determined with a Coulter counter (Beckman-Coulter). The mean number of cells per well and the SE were generated for each cell line at each time point.