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3. Tesi Dottorato Campana C. Frontespizio, Tesi E Index _____________________________________________________________________________________ INDEX RATIONALE .....................................................................................................................1 BACKGROUND ................................................................................................................6 1. HUMAN ADRENAL GLAND 1.1 The adrenal gland:general structure .............................................................................7 1.2 Embriology and development ......................................................................................8 1.3 Hystology ...................................................................................................................10 1.4 Adrenalcortical steroidogenesis .................................................................................11 1.5 The steroidogenic regulatory protein .........................................................................14 1.5.1 StAR structure and mechanism of action ........................................................15 1.5.2 P450scc ............................................................................................................15 1.5.3 P450c17: 17α-Hydroxylase/17,20-Lyase ........................................................16 1.5.4 Cytochrome b5 and P450c21 (Steroid 21-Hydroxylase ..................................17 1.5.5 Isozymes of P450c11 and Isozyme of 17β –Hydroxysteroid Dehydrogenase 18 1.5.6 P450arom: Aromatase .....................................................................................18 1.5.7 Isozyme of 5α-Reductase ................................................................................19 1.5.8 3βHSD ............................................................................................................19 2. ADRENOCORTICAL CANCERS 2.1 Introduction ................................................................................................................20 2.2 Epidemiology .............................................................................................................21 2.3 Pathogenesis ...............................................................................................................22 2.4 Adrenocortical adenoma ............................................................................................25 2.5 Adrenocortical carcinoma ..........................................................................................25 2.6 Role of ERα and ERβ activation on ACC development ...........................................29 3. G PROTEIN COUPLED ESTROGEN RECEPTOR: GPER AND ITS LIGAND 3.1 Introduction ................................................................................................................32 3.2 Characterization of GPER-dependent cellular functions ...........................................33 I Index _____________________________________________________________________________________ 3.3 Identification and characterization of GPER-selective ligands .................................34 3.4 Transcriptional activations mediated by GPER .........................................................37 4. CELL-BASED ASSAYS FOR SCREENING ANDROGEN RECEPTOR LIGANDS 4.1 Introduction ................................................................................................................41 4.2 Yeast-based systems using a β-galactosidase reporter ...............................................43 4.3 β-Lactamase Reporter Model .....................................................................................47 4.4 Lucifeerase Reporte Model ........................................................................................48 4.4.1 Yeast-AR luciferase models ............................................................................50 4.4.2 Mammalian cell AR luciferase models ...........................................................50 4.5 Green Fluorescent Reporter Model ............................................................................53 4.6 Fluorescent AR trans location bioassay .....................................................................55 5. MATERIALS AND METHODS 5.1 GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo .......................................................................................................................57 5.1.1 Cell cultire and tissues .....................................................................................57 5.1.2 RNA extraction, reverse transcription and real time PCR ..............................58 5.1.3 Western blot analysis ......................................................................................58 5.1.4 Histopathological and Immunohistochemical analysis ...................................59 5.1.5 Cytochrome c detection ...................................................................................59 5.1.6 Cell cycle analysis and evaluation of cell death .............................................60 5.1.7 Caspase 9 and 3/7 Activity Assay ...................................................................60 5.1.8 TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay .........................................................................................................61 5.1.9 Determination of DNA fragmentation .............................................................61 5.1.10 Assessment of cell proliferation ...................................................................61 5.1.11 Gene silencing experiments ..........................................................................62 5.1.12 Xenograft model ............................................................................................62 5.1.13 In vivo magnetic resonance analyses .............................................................63 5.1.14 Scoring system ...............................................................................................63 II Index _____________________________________________________________________________________ 5.1.15 Macroarray analysis .......................................................................................63 5.1.16 Data analysis and statistical methods.............................................................64 5.2 Development of a novel cell based androgen screening model .................................64 5.2.1 Materials ..........................................................................................................64 5.2.2 Cell line ...........................................................................................................64 5.2.3 Stable Trasduction ...........................................................................................65 5.2.4 Isolation of RNA and qPCR analysis ..............................................................65 5.2.5 Protein extraction and protein assay ................................................................66 5.2.6 Western analysis ..............................................................................................66 5.2.7 AR translocation study.....................................................................................66 5.2.8 Gaussia Luciferase analysis .............................................................................66 5.2.9 Sera ..................................................................................................................67 5.2.10 Extraction method .........................................................................................67 6. RESULTS 6.1 GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo ........................................................................................................................68 6.1.1 G-1 treatment decreases H295R cell growth in vtiro and in vivo ....................68 6.1.2 G-1 induces H295R cell cycle arrest and cell death .......................................72 6.1.3 G-1 causes cell nuclei morphological changes, DNA damage and apoptosis .73 6.1.4 G-1 treatment causes sustained ERK1/2 phosphorylation ...............................75 6.1.5 Microarray data ................................................................................................77 6.2 Development of a novel cell based androgen screening model .................................79 6.2.1 AR expression in transduced CV1 cell line .....................................................79 6.2.2 Sensitivity of CV1-ARluc cell line .................................................................81 6.2.3 Selectivity of CV1-ARluc cell line to other steroids .......................................83 6.2.4 Treatment of CV1-ARluc cell line with different C19 steroids ........................83 6.2.5 Effects of a potent anti-androgens on CV1-ARluc ..........................................84 6.2.6 Serum androgen bioactivity .............................................................................85 III Index _____________________________________________________________________________________ 7. DISCUSSION 7.1 GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo ........................................................................................................................87 7.2 Development of a novel cell based androgen screening model .................................90 REFERENCES ...............................................................................................................94
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