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J. vectors that express chimeric and exposed to paclitaxel for 16 h. Adherent and nonadherent cells were harvested, lysates were prepared, and immunoblot analysis was performed on the proteins indicated. The results are representative of three independent experiments yielding an average transfection efficiency of 40 to 45%. Flow Cytometry Flow cytometry was performed on a FACScan (BD Biosciences, San Jose, CA) using CellQuest software. For cell cycle analysis, 1 106 cells (including nonadherent cells) were fixed in 70% ethanol for at least 20 min. The cell pellets were washed in cold PBS and incubated for 30 min in PBS containing 10 g/ml propidium iodide and 1 g/ml RNase A (Boehringer Mannheim, Indianapolis, IN) at 37C. Propidium iodide fluorescence was quantified using the FL2 detector and cellular aggregates gated from all samples. For the detection of phosphatidylserine externalization, adherent and nonadherent cells were harvested by trypsinization and 1 106 cells/ml were stained with 5 L of annexin-V Fluor-488 labeled antibody Aminoacyl tRNA synthetase-IN-1 (Molecular Probes Inc., Eugene, OR) in annexin binding buffer (10 nM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) according to the manufacturers instructions and analyzed by flow cytometry on the FL1 detector. Immunoblotting Cell extracts were prepared from adherent and nonadherent cells after drug treatment by lysis in a buffer composed of 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, Aminoacyl tRNA synthetase-IN-1 1% Triton X-100, and 0.1% Nonidet P-40 containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 g/ml aprotinin, and 2 g/ml leupeptin) and phosphatase inhibitors (50 mM NaF and 1 mM sodium orthovanadate). Cellular debris was removed by centrifugation and the protein was quantified using the Bio-Rad method (Bio-Rad, Hercules, CA). Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Ponceau S staining before immunoblotting confirmed equal loading of samples. Membranes were blocked for 1 h in 3% bovine serum albumin and incubated in primary and secondary antibodies (Amersham Pharmacia Rabbit Polyclonal to TRXR2 Biotech, Piscataway, NJ) according to the manufacturers instructions. All antibodies were used at a 1:1000 dilution in 1 Tris-buffered saline containing 0.1% Tween-20 and 1 to 2% bovine serum albumin and were as follows: Bcl-2 mAb (DAKO, Glostrum, Denmark), phospho-ERK mAb, phospho-JNK mAb, JNK pAb (New England Biolabs, Beverly, MA), ERK2 pAb, MEKK1 pAb C-22, (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and PARP mAb (“type”:”entrez-protein”,”attrs”:”text”:”P76420″,”term_id”:”82583806″P76420), c-Raf-1 mAB (BD Pharmingen, San Diego, CA). An additional PARP antibody, Anti-PARP p85 fragment pAB (Promega, G7341), specific for only the 85-kDa cleavage product, was used at a dilution of 1 1:750. Membranes were developed using enhanced chemiluminescence reagent (Amersham). Multiple Drug Effect Analysis Cells were seeded in triplicate into 24-well plates and, after adherence, serial dilutions of paclitaxel, U0126, or both were added for 72 h. The drug concentrations evaluated were based on the IC50 value for each individual drug so that combinations of paclitaxel and U0126 were assessed at their equipotent ratio (i.e., at the ratio of each respective IC50 value). Drug combinations were evaluated concurrently or sequentially, wherein paclitaxel or U0126 was given for 24 h before the other drug. The effect of each drug treatment was determined by counting the number of attached viable cells after the 72-h incubation period and expressing this number as a ratio of the number of cells treated with dimethyl sulfoxide alone. This ratio was applied to the combination index (CI) method of Chou and Talalay (1984), using the software Calcusyn (Biosoft, Cambridge, UK) to analyze the nature of the interaction between paclitaxel and U0126. This software applies a mathematical model that computes a CI ratio for various levels of cytotoxicity (fractional inhibition), such that a CI value of 1 1 indicates additivity, CI 1 indicates synergism, and CI 1 indicates antagonism. For this analysis, the more conservative Aminoacyl tRNA synthetase-IN-1 assumption of mutual nonexclusion (dissimilar mechamisms of action for paclitaxel and U0126) was applied to the derivation of CI ratios. Results Activation of the Nuclear Transcription Factors Elk-1 and c-jun by Microtubule-Interacting Agents A MAPK reporter system was used to determine the ability of various microtubule-interacting drugs to phosphorylate the nuclear transcription factor Elk-1.