Regulation of the prostate-specific hTGP (TGM4) gene and its potential use for prostate cancer gene therapy. Guillermo Cipactl Rivera Gonzalez PhD University of York Biology Department DECEMBER 2011 Abstract Prostate cancer is a very important health problem in the UK and worldwide. Available treatments such as radiotherapy, surgery and androgen ablation have failed to significantly increase the life expectancy of prostate cancer patients. Therefore researching new treatments is paramount. A promising approach for prostate cancer is suicide gene therapy. In this work, the use of the NTR/CB1954 enzyme/prodrug system delivered by a baculovirus vector was investigated for its application in prostate cancer cell lines and prostate primary epithelial cultures. The results suggest that the NTR/CB1954 system is highly efficient in causing cell death in prostate cell lines and prostate primary epithelial cultures. The use of a baculovirus vector to deliver the NTR gene resulted in increased transduction of prostate cancer cell lines in comparison to non-malignant prostate and non-prostate cell lines. To target NTR expression to prostate cells the regulation of the hTGP promoter was dissected. hTGP expression was confirmed to be highly prostate specific and mainly regulated by retinoic acid, androgens, retinoic acid receptor gamma and the androgen receptor. This work presents the first evidence of an interaction between these nuclear receptors and challenges the current model for prostate specific expression. Finally a baculovirus encoding the NTR gene under the control of the hTGP promoter was tested in prostate cancer cell lines resulting in moderate cell death. These findings are very encouraging but in order to use the hTGP promoter for gene therapy there needs to be further manipulation of the sequence to optimise its potency while maintaining its prostate specificity The use of NTR and baculovirus coupled to the targeting controlled provided by the hTGP promoter could develop into a potent and specific approach to treat prostate cancer. 2 List of contents ABSTRACT ........................................................................................................ 2 LIST OF CONTENTS .......................................................................................... 3 LIST OF FIGURES AND TABLES ..................................................................... 8 ACKNOWLEDGEMENTS ................................................................................. 10 AUTHOR’S DECLARATION ............................................................................ 11 1. INTRODUCTION ........................................................................................... 12 1.1 PROSTATE ANATOMY AND PHYSIOLOGY ........................................................ 12 1.1.1 Prostate development ....................................................................... 15 1.1.2 Regulation of prostate development by hormones ........................... 15 1.2 STEROID RECEPTORS IN THE PROSTATE ....................................................... 17 1.2.1 Androgen receptor ............................................................................ 17 1.2.2 Retinoic acid receptors ..................................................................... 20 1.3 PROSTATE CANCER .................................................................................... 23 1.3.1 Risk factors for prostate cancer ........................................................ 23 1.3.2 Genes involved in prostate cancer ................................................... 25 1.4 AR AND CASTRATION-RESISTANT PROSTATE CANCER .................................... 29 1.5 PROSTATE CANCER STEM CELLS .................................................................. 32 1.6 CURRENT TREATMENTS FOR PROSTATE CANCER. ......................................... 35 1.6.1 Radical prostatectomy ...................................................................... 35 1.6.2 Radiotherapy ..................................................................................... 36 1.6.3 Androgen deprivation therapy ........................................................... 36 3 1.6.4 Chemotherapy .................................................................................. 37 1.7 Gene therapy as an alternative treatment for prostate cancer ............ 38 1.7.1 Viral vectors for gene therapy ........................................................... 39 1.8 GENE THERAPY STRATEGIES FOR PROSTATE CANCER ................................. 51 1.8.1 Corrective Gene therapy ................................................................... 51 1.8.3 Oncolytic Gene Therapy ................................................................... 52 1.8.4 Suicide Gene Therapy ...................................................................... 53 1.9 TARGETING VIRAL TROPISM ........................................................................ 57 1.9.1 Pseudotyping .................................................................................... 57 1.9.2 Adaptor Proteins ............................................................................... 57 1.9.3 Genetic Incorporation ........................................................................ 58 1.9.4 Transcriptional targeting ................................................................... 60 1.9.5 Human prostate-specific transglutaminase ....................................... 63 2. AIMS OF RESEARCH .................................................................................. 64 3. MATERIALS AND METHODS ..................................................................... 66 3.1 CELL CULTURE .......................................................................................... 66 3.1.1 Insect cell culture .............................................................................. 66 3.1.2 Human cell culture ............................................................................ 66 3.2 FOETAL CALF SERUM HORMONE DEPLETION ................................................. 67 3.3 SDS-PAGE AND WESTERN BLOT ............................................................... 68 3.4 RNA ISOLATION AND CDNA SYNTHESIS ....................................................... 69 3.5 GENERATION OF RECOMBINANT BACULOVIRUS ............................................. 70 3.6 VIRUS TITRATION ........................................................................................ 71 3.7 BACULOVIRUS AMPLIFICATION ..................................................................... 72 3.8 VIRAL DNA EXTRACTION ............................................................................ 72 4 3.9 VIRUS CONCENTRATION .............................................................................. 73 3.10 BACULOVIRUS TRANSDUCTION OF HUMAN CELLS ......................................... 73 3.11 RT-PCR AND RT-QPCR .......................................................................... 73 3.12 FLOW CYTOMETRY ................................................................................... 74 3.13 GENE EXPRESSION PROFILE IN HUMAN TISSUES .......................................... 75 3.14 BACTERIAL TRANSFORMATION ................................................................... 75 3.15 BACTERIAL CULTURES, PLASMID ISOLATION AND PURIFICATION .................... 75 3.16 GENERATION OF HTGP PROMOTER CONSTRUCTS ...................................... 76 3.17 IMMUNOFLUORESCENCE ........................................................................... 77 3.18 PLASMID TRANSFECTION INTO HUMAN CELL LINES ....................................... 78 3.19 PLASMID TRANSFECTION INTO SF9 INSECT CELLS ....................................... 79 3.20 LUCIFERASE ASSAY .................................................................................. 79 3.21 MTS ASSAY ............................................................................................. 79 3.22 SIRNA TRANSFECTION ............................................................................. 80 3.23 NTR HALF-LIFE ........................................................................................ 80 3.24 CHROMATIN IMMUNOPRECIPITATION (CHIP) ............................................... 81 4. RESULTS ..................................................................................................... 83 4.1 EFFICIENCY OF THE NTR/CB1954 SYSTEM IN PROSTATE CELLS .................... 83 4.1.1 Determining prostate cell lines’ sensitivity to CB1954 ...................... 83 4.1.2 NTR expression in prostate cancer cells triggers cell death after CB1954 treatment. ..................................................................................... 85 4.2 BACULOVIRUS AS A VECTOR FOR PROSTATE CANCER GENE THERAPY ............. 87 4.2.1 Baculovirus effectively transduces prostate cancer cell lines ........... 87 4.2.2 Baculovirus can efficiently transduce cultured cells derived from patients. ..................................................................................................... 91 5 4.3 HTGP PROMOTER CHARACTERIZATION ........................................................ 93 4.3.1 hTGP expression is highly prostate specific ..................................... 93 4.3.2 hTGP expression in prostate cell lines is controlled by retinoic acid 95 4.3.3 Prostate cell lines have different abilities to activate transcription following atRA treatment ............................................................................ 99 4.3.4 hTGP expression is repressed by androgens