Dual-Specificity Phosphatase

OK carried out the synthesis of compound GGTI P61A6 and helped to draft the manuscript

OK carried out the synthesis of compound GGTI P61A6 and helped to draft the manuscript. this paper, we address two preclinical issues. First, we show that GGTI P61A6 inhibits proliferation and transformed phenotypes of NSCLC cells, including the growth of xenograft tumors in mice. Second, we demonstrate the specificity of P61A6 by showing that a RhoA mutant whose biological activity NFBD1 is impartial of GGTase-I renders the cells resistant to inhibition by P61A6. Methods Cell lines and cell cultures NSCLC cell lines, H358, H23 and H1507, kindly provided by Dr. Curtis Harris (National Cancer Institute, Bethesda, MD), were maintained in RPMI 1640 medium (Cellgro, Lp-PLA2 -IN-1 Herndon, VA). The medium was supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT) and 1% penicillin/1% streptomycin stock solution (Invitrogen, Carlsbad, CA). All cells were cultured at 37C in a humidified incubator at 5% CO2. Compound GGTI P61A6 was synthesized by coupling P5-H6 [14] with an L-phenylalanamide, where the free acid L-phenylalanine is converted to an amide. A 20?mM stock solution of P61A6 in DMSO was kept at ?20C until use. Cell proliferation and cell cycle analyses Effects of P61A6 on cell proliferation were examined using the CCK-8 cell counting kit (Dojindo Molecular Technologies, Kumamoto, Japan) as described previously [14]. Briefly, cells (2.5??103) were seeded onto 96-well plates. The following day, cells were treated with the appropriate inhibitor as indicated in the physique legends. The cell proliferation assay was performed in triplicate every other day. Data of each experimental series were tested against the controls (DMSO) for statistical significance, using Students paired two-tailed test. The cell cycle profiles were analyzed by flow cytometry (UCLA Flow Cytometry Core Facilities) as described previously [24]. Western blotting Cells were treated with DMSO or P61A6 for 48?h, harvested, and lysed in lysis buffer (1% Triton X-100, 150?mM NaCl, 20?mM TrisCHCl at pH?7.5, 1?mM EDTA, and 1 protease inhibitor mixture). Proteins Lp-PLA2 -IN-1 were then resolved by 12% or 12.5% SDS-PAGE and immunoblotted with antibodies against p21CIP1/WAF1 (Millipore, Temecula, CA), p27Kip1 (rabbit, Santa Cruz Biotechnology, Inc.), RhoGDI (Santa Cruz Biotechnology, Inc.), RhoA (mouse, Santa Cruz Biotechnology), cyclin D1/2 (Millipore), the unprenylated form of Rap1 (U-Rap1; Santa cruz Biotechnology, Inc.), or actin (Calbiochem). Detection was performed using peroxidase-conjugated secondary antibodies (Biorad) and Amersham ECL Plus? Western Blotting Detection Reagents (GE Healthcare Life Sciences). Select bands were quantified using ImageJ imaging processing program (National Institutes of Health). Subcellular fractionation Cells were treated with DMSO or P61A6 for 48?h. Cells were then washed and scraped into PBS and centrifuged at 2,500?rpm for 5?min. Pellets were Lp-PLA2 -IN-1 resuspended (10?mM HEPES/KOH at ph?7.3, 10?mM KCl, 5?mM MgCl2, 0.5?mM DTT, and 1 protease inhibitor mixture), incubated on ice for 30?min, and homogenized. Homogenates were centrifuged at 1000 for 10?min to collect the cytosolic fractions (supernatant). The remaining pellets were then resuspended in buffer made up of 1% Triton X-100, 150?mM NaCl, 20?mM TrisCHCl at pH?7.5, 1?mM EDTA, and 1 protease inhibitor mixture, and centrifuged at 15,000?rpm for 15?min to collect the membrane-containing fractions (supernatant). Na+/K+ ATPase- and RhoGDI or GAPDH were used as markers for the membrane-containing fractions and Lp-PLA2 -IN-1 the cytosolic fractions, respectively. Lp-PLA2 -IN-1 GTP-bound RhoA pull-down assay Cells were serum-starved in the presence of DMSO or P61A6 for 24?h. Cells were then stimulated with 10% FBS in the presence of DMSO or P61A6 for 30?min. Whole cell lysates were collected using Mg2+-made up of buffer, and GTP-RhoA was pulled down using GST-tagged Rhotekin-RBD protein beads (Cytoskeleton). Whole cell lysates (inputs for pull-down) and pull-down were resolved on SDS-PAGE for immunoblotting analysis, using RhoA antibodies (mouse, Santa Cruz Biotechnology) to detect total RhoA and GTP-bound-RhoA. Anchorage impartial growth assay Cells were seeded at a density of 20,000 cells/well in duplicate in 6-well culture dishes in 0.4% agar over a 0.8% bottom agar layer. Various concentrations of P61A6 or DMSO were added to the top layer of cells. Cultures were re-fed and treated with the GGTI or DMSO once weekly (14?days of incubation in total). Colonies were stained with 1?mg/ml MTT (tetrazolium salt) for.