Liang-Yi Hung to provide pGL2-AAP plasmid and Dr

Liang-Yi Hung to provide pGL2-AAP plasmid and Dr. of Aurora-A were detected by reverse transcriptional-PCR and Western blotting, respectively. Centrosome of cells was observed by immunofluorescent staining. The transcription factor of Aurora-A was investigated by promoter activity, chromosome immunoprecipitation (ChIP), and small interfering RNA (shRNA) assays. Mouse model was utilized to confirm the relationship between arsenic and Aurora-A. Results We reveal that low dosage of arsenic treatment increased cell proliferation is associated with accumulated cell population at S phase. We also detected increased Aurora-A expression at mRNA and protein levels in immortalized bladder urothelial E7 cells exposed to low doses of arsenic. Arsenic-treated cells displayed increased multiple centrosome which is resulted from overexpressed Aurora-A. Furthermore, the transcription factor, E2F1, is responsible for Aurora-A overexpression after arsenic treatment. We further disclosed that Aurora-A expression and cell proliferation were increased in bladder and uterus tissues of the BALB/c mice after long-term arsenic (1?mg/L) exposure for 2?months. Conclusion We reveal that low dose of arsenic induced cell proliferation is through Aurora-A overexpression, which is transcriptionally regulated by E2F1 both in vitro and in vivo. Our findings disclose a new possibility that arsenic at low concentration activates Aurora-A to induce carcinogenesis. and E2F-1 [20] and selective activation of NF-kB and E2F by low concentration of arsenite in U937 human monocytic leukemia cells [21]. Aurora-A acts as a direct target of PRT-060318 E2F3 during G2/M cell cycle progression [22]. Increased E2F1 protein level accompanied with Aurora-A overexpression was detected in breast cancer specimens. Further analysis reveals that Aurora-A increased E2F1 protein stability by suppressing its degradation [23]. Currently, how arsenic-related Aurora-A dysfunctions through gene amplification or epigenic modification remain unknown. This study aimed to reveal the molecular mechanism of arsenic-induced PRT-060318 tumor development. We established an immortalized human uroepithelial cell line model system, and set up a mouse-arsenic exposure model to validate PRT-060318 our cell line investigation. Methods Cell line and culture The immortalized bladder urothelial E7 cells (ATCC, #CRL-2017) contain HPV E7 oncogene, which binds with phosporylated tumor suppressor RB protein (provided by Nan-Haw Chow; National Cheng Kong University Hospital) [24]. This cell line was maintained in F12 medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37?C in a 5% CO2 incubator. Arsenic treatment The immortalized E7 cells were treated with different amount of sodium arsenite (NaAsO2; Fluka, St. Louis, MO, USA) for various times and the protein was collected using lysis buffer (50?mM Tris-HCl, pH?7.4, 1% Nonidet P-40, 150?mM NaCl, 0.5% Sodium deoxycholate). RNA was extracted by TRizol? (Invitrogen, Carlsbad, CA, USA), and genomic DNA was extracted by the commercial kit, YGB100 (RBC Bioscience, Taipei, Taiwan). Immunofluorescent assay (IFA) E7 cells (1??105 or 5??104/well) were plated in 6-well plates. After incubation with arsenic for one week, cells were fixed with 3.7% formaldehyde for 30?min followed by washing with 1X PBS for 30?min and 0.1% Triton X-100 treatment for 30?min. Cells were washed again with 1X PBS, immersed with blocking buffer (Thermo, Rockford, IL, USA) for 30?min, and then stained with mouse anti-BrdU antibody (#RPN20AB, Amershan Biosciences, Buckigamshire, England), mouse anti-Aurora-A antibody (NCL-L-AK2, Novocastra, Bannockburn, IL, USA) and mouse anti–tubulin antibody (Sigma Chemical Co., St, Louis, MO, USA) at 4Covernight. The next day, cells were washed and stained with Fluirescein (FITC)-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1?h. To stain the nuclear DNA, cells were incubated with Propidium Iodide (PI, 5?g/ml; Sigma), or Hochest 33,258 (50?ng/ml, Sigma). Flow cytometry analysis Cell cycle distribution was determined by flow cytometry. Cells (1??105/well) were plated in 6-well plates. After incubation with different doses of arsenic for one week, cells were collected and fixed with 70% ethanol at ?20?C overnight. The cell cycle distribution was analyzed after PI (40?g/ml) staining for 1?h. Western blotting Cells were lysed in lysis buffer, and 50?g of lyset was loaded onto a SDS-PAGE followed by to PRKACA a PVDF membrane (Millipore, Billerica, MA, USA) transferring. Aurora-A, and -actin levels were determined by anti-Aurora-A (Cell signaling, Boston, MA, USA) and anti–actin (Sigma) antibodies. cDNA preparation and RT-PCR Total RNA (1?g) was used to prepare cDNA according to the manufacturers instructions PRT-060318 (Improm-IITM Reverse Transcriptase; Promega, Madison, WI, USA). PRT-060318 The cDNA (1?g) was used for PCR according to the manufacturers instructions (YEAtaq DNA polymerase; Yeastern Biotech, Taipei, Taiwan). Primers were used as follows, Aurora-A(F): GAAATTGGTCGCCCTC; Aurora-A(R): TGATGAATTTGCTGTGATCC; 18?s rRNA(F): AAACGGCTACCACATCCAAG; 18?s rRNA(R): CCTCCAATGGATCCTCGTTA. Promoter activity assay The plasmids, including pGL2-AAP (provided by Dr. Liang-Yi Hung), pRLTK (at the molar ratio of 10:1) and pCMV-E2F1 (provided by Dr. Ju-Ming Wang), were co-transfected into.