Ng came in NIH3T3 cells No induction of p53-dependent Ngigen p21WAF and after cell cycle arrest and leads to sensitization of cells for the treatment of paclitaxel, will potentiate TACC2 surcharge drug resistance CRPC cells by modulating the functions of points controlled the cell cycle. Because erismodegib we Kernf Staining of TACC2 observed w Is during the interphase and the TACC family of proteins known to interact with the family of histone acetylation transferase, we consider that is TACC2 also be involved in the activation of the transcription machinery and chromatin remodeling in prostate cancer at both androgen-dependent ngigen and hormonrefrakt rem stages. In summary, we propose that TACC2 is a critical factor for the progression of prostate cancer to androgen-dependent Ngigen and independent Ngigen in the AR-regulated gene network. Given its transcriptional activation and modulating functions of the cell cycle progression, TACC2 a potential therapeutic target in prostate cancer, especially in CRPC. Materials and Methods Cell lines, plasmid constructs, LNCaP and DU145 cells and reagents, the cells of human prostate cancer, have been cultured in RPMI 1640, erg complements With 10% FBS, 50 U / ml penicillin, streptomycin and from ARQ 197 50g/ml. Before androgen treatment, the cells were in phenol red-free medium containing activated carbon deducted cultured 5% FBS for 48 72 h VCAP and 293T cells were grown in DMEM, erg complements With 10% FBS. Subline LTAD cells were fixed by the maintenance of LNCaP cells in phenol red-free RPMI 1640, erg complements Described with 10% FBS charcoal of more than 9 months as elsewhere stripped established. For the construction of vectors, the luciferase ARBS were ARA sequences from the Bluescript vectors, the amplified DNA chip AR prepared for sequence analysis. The amplified PCR products were inserted into pGL3-promoter. The compl length TACC2 the gene was amplified by PCR from cDNA TACC2 purchased from Invitrogen. The PCR product was inserted into pcDNA3 Flag-labeled N-terminus in the frame. LNCaP cells were transfected with expression vectors JOINT 6 reagent.
To obtain stable cell lines of pcDNA3 TACC2 flag or generate empty vectors, the cells were suspended in 0.5 mg / ml G418 selected. Antique Body against tubulin, flag, AR, ACH3, ACH4 were phospho-RNA polymerase II and RNA Pol II are used, as described above. Antique Body against tubulin and methyl were purchased from Abcam H3K4mono. Antique For TACC2 body was from Upstate Biotechnology, Inc. purchased. Antique Body was purchased from Santa for CyclinD1 Cruz Biotechnology, Inc.. R1881 was purchased from PerkinElmer. Dihydrotestosterone was purchased from Wako. Bicalutamide and nocodazole were purchased from Marbofloxacin Sigma. ChIP cloning and chip chip was performed as previously described. LNCaP cells or cells LTAD were treated with 1% formaldehyde for 5 min at room temperature. Chromatin was sheared to an average size E of 500 bp by sonication with Ultraschallger t Bioruptor. The lysates were rotated at 4 ° C overnight, anti-AR, ACH3, ACH4 ACH4 H3K4mono methyl phospho RNA polymerase II and RNA Pol II Antique Body. Protein A aga Rose added and rotated for 1 h. Washing and reversal of crosslinking at 65 ° C carried out. DNA fragments were recovered by F Precipitation with ethanol. CHIP for cloning, subtractive hybridization difference in presentation between the R1881 chip-treated DNA.