ABSTRACT
Pituitary tumor transforming gene binding factor (PBF) is a proto-oncogene that plays a role in many cancers; however, its involvement in prostate cancer (PCa) remains unclear. Here, we examined PBF expression in clinical specimens and investigated its regulation and function in human PCa cell lines. Immunohistochemical staining of patient tissues revealed higher PBF expression in PCa than in benign prostatic hyperplasia or adjacent normal prostate specimens. In LNCaP and 22Rv1 cells, PBF expression was upregulated by androgen treatment in a manner partially blocked by the androgen receptor (AR) antagonist bicalutamide. We identified a novel androgen response element in the PBF gene promoter and demonstrated its functional relevance using luciferase reporter assays. Androgen treatment of LNCaP cells induced binding between the endogenous AR and the androgen response element in PBF, as measured by chromatin immunoprecipitation assays. Finally, RNA interference of PBF expression significantly reduced androgen-induced LNCaP cell growth and invasion. Thus, PBF is a novel AR target gene and plays a role in androgen-induced proliferation and metastatic functions in PCa cells.
Subject(s)
Gene Expression Regulation, Neoplastic , Membrane Proteins , Neoplasm Invasiveness , Prostatic Neoplasms , Androgens/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Humans , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/physiopathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/physiopathology , Protein Binding/drug effects , Proto-Oncogene MasABSTRACT
Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.
Subject(s)
Humans , Male , Prostatic Neoplasms/metabolism , RNA Interference , Securin/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Securin/genetics , Up-RegulationABSTRACT
Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.
Subject(s)
Prostatic Neoplasms/metabolism , RNA Interference , Securin/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Male , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Proto-Oncogene Mas , Securin/genetics , Up-RegulationABSTRACT
The hepatitis C virus RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target for antiviral drug development. In this study, stable HeLa cell lines with an integrated NS5B gene were selected by G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated NS5B genes were demonstrated by RT-PCR and Western blot analysis. Further analysis demonstrated enzymatic activity of the expressed NS5B polymerase. The stable HeLa cell lines should be useful for the identification of NS5B inhibitors and for studying the mechanisms of HCV replication.
Subject(s)
Gene Expression , Hepacivirus/enzymology , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , HeLa Cells , Humans , RNA-Dependent RNA Polymerase/biosynthesis , Viral Nonstructural Proteins/biosynthesisABSTRACT
Four new ST-1435 derivatives 4-7 were synthesized. Both 4 and 5 are mixture of Z- and E-isomers. 4 was separated into Z- and E-isomers by spinning TLC. Their binding ability to progesterone receptor was examined and found to be less than ST-1435.
Subject(s)
Contraceptive Agents, Female/chemical synthesis , Norprogesterones/chemical synthesis , Animals , Breast Feeding , Female , Lactation , Norprogesterones/metabolism , Rats , Receptors, Progesterone/metabolismABSTRACT
The synthesis of peptidyl derivatives of the aminosteroid, amafalone (Am), is described. Six analogs were synthesized: the hydrochloride salts of Gly-Am (2) Ala-Gly-Am (3), D-Ala-Gly-Am (4), Pro-Am (6), Pro-Pro-Am (7), and D-Ala-Pro-Am (8). The peptide bonds were formed by the polymeric reagent method using polymeric hydroxybenzotriazole as the activating polymer. Peptidyl aminosteroids 2, 6, 7, and 8, when administered to rats intravenously, had protective antiarrhythmic effects similar to those of amafalone. By the oral route, less marked protection, in comparison to amafalone, was observed with 6, while 7 and 8 were disappointingly inactive.
