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A Novel Binding Partner of the NKX3-1 Homeodomain Protein in Prostate Cancer Cells

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A Novel Binding Partner of the NKX3-1 Homeodomain Protein in Prostate Cancer Cells FLJ22318: A Novel Binding Partner of the NKX3-1 Homeodomain Protein in Prostate Cancer Cells Linda Fiona Dawson B.Sc. (Hons) This thesis is presented for the degree of Doctor of Philosophy of Murdoch University, October 2006 School of Biological Sciences & Biotechnology, Murdoch University, Perth, Western Australia I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. .................................... Linda Fiona Dawson i Thesis Summary Prostate cancer is a frequently diagnosed malignancy which ranges from an indolent asymptomatic tumour to an aggressive, rapidly lethal systemic disease. Determination of chromosomal, genetic and epigenetic alterations associated with prostate carcinogenesis have led to the characterisation of functional consequences of these alterations, thereby elucidating pathways contributing to malignant growth that can be utilised clinically and therapeutically. FLJ22318 is a novel hypothetical protein that was identified by yeast two-hybrid analysis to interact with the prostatic homeodomain protein, NKX3-1. Expression of NKX3-1 is largely restricted to epithelial cells of the adult prostate where it is involved in maintaining the prostatic phenotype, while NKX3-1 expression is reduced or absent in prostate tumours. In contrast, FLJ22318 expression is documented in cDNA libraries derived from a variety of human adult and foetal tissues including the prostate, suggesting that it may be ubiquitously expressed and that it potentially interacts with a number of proteins in addition to NKX3-1. FLJ22318 expression is undocumented in human prostate tumours. Bioinformatic analyses have postulated multiple FLJ22318 mRNA transcripts however the proposed open reading frames encoded by these transcripts predict the FLJ22318 protein to contain three strong protein-protein interaction domains, a Lissencephaly type-1-like homology (LisH), a C-terminal to LisH (CTLH) and a CT11-RanBPM (CRA) domain. Of the 44 single nucleotide polymorphisms identified within the FLJ22318 gene, none are located within the protein coding region suggesting that FLJ22318 may be critical for cell survival and/or function. Comparison of the amino acid sequence between human FLJ22318 and its orthologues in a diverse range of mammalian species identified >97% sequence homology, providing further strong evidence of the critical cellular function of FLJ22318. To characterise the biological activity of FLJ22318 in prostate cancer cells, the FLJ22318 coding region was amplified by polymerase chain reaction (PCR) and ligated into mammalian and bacterial expression vectors to encode V5-, myc-, GFP-, HA-, and ii GST-FLJ22318 fusion proteins. Interaction between FLJ22318 and NKX3-1 was confirmed using (reverse) yeast two-hybrid, GST pull-down and co- immunoprecipitation assays. These data were supported by confocal microscopy which demonstrated the perinuclear and nuclear co-localisation of FLJ22318 and NKX3-1 in prostate cancer cells. Northern blotting identified expression of ~2Kb and ~4Kb FLJ22318 mRNA’s in prostate cancer cell lines, which was consistent with bioinformatic analyses of mRNA species. Transfection of prostate cancer cells to overexpress FLJ22318 did not alter endogenous NKX3-1 levels, however FLJ22318 exhibited transcriptional repressor function on an NKX3-1 responsive element and increased NKX3-1 transcriptional repressor activity on this element. To further investigate FLJ22318 function, additional yeast two-hybrid analyses were performed in prostate cancer cells to identify potential FLJ22318 binding proteins. These studies isolated cDNA’s encoding 33 different proteins involved in cell metabolism and apoptosis as well as transcriptional regulators associated with control of cellular proliferation. One of the candidate FLJ22318 interactors, protein kinase, interferon-inducible double stranded RNA dependent activator (PRKRA/PACT) was shown using confocal microscopy to extensively co-localise with FLJ22318 in the cytoplasm and perinuclear regions of prostate cancer cells. Preliminary co- immunoprecipitation and GST pull-down assays have provided additional evidence of the interaction of PRKRA and FLJ22318. Results of this thesis have generated important information characterising the structure of the FLJ22318 gene and protein, the interaction between FLJ22318 and NKX3-1 and the potential functions of FLJ22318 in prostate cancer cells. As the FLJ22318 gene is located on 5q35, a chromosomal region frequently disrupted in a variety of tumours, future studies of the biological activity of FLJ22318 will clarify its normal cellular functions and its contribution to tumorigenesis or malignant progression in the prostate and in other tissues. iii Table of Contents Declaration i Thesis Summary ii Table of Contents iv Acknowledgments xi Awards xii Publications xiii Index to Figures xv Index to Tables xix Abbreviations xx Chapter One – Introduction 1.1 The Prostate Gland 1 1.1.1 Anatomical Location and Development 1 1.2 Prostate Cancer 6 1.2.1 Prostate Cancer Incidence 6 1.2.2 Aetiology 9 1.2.3 Detection and Diagnosis 11 1.2.4 Grading and Staging 13 1.2.5 Treatment Options and Prognosis 13 1.2.6 Androgen Deprivation and Hormonal Therapies 15 1.2.7 Androgen Independent Prostate Cancer 19 1.2.8 Chromosomal Anomalies in Prostate Cancer 20 1.3 NKX3-1 22 1.3.1 Homeobox Genes 22 1.3.1.1 Homeodomain Proteins 22 1.3.2 NK Homeobox Genes 24 1.3.2.1 NK Homeodomain Proteins 26 1.3.3 The NKX3-1 Homeodomain Protein 29 1.3.3.1 The NKX3-1 Gene 29 1.3.3.2 Nkx3-1 Expression During Embryogenesis 32 1.3.3.3 Nkx3-1 Knockout Mice 33 1.3.3.4 Regulation of NKX3-1 Expression 35 iv 1.3.3.5 NKX3-1 DNA-binding Sites 39 1.3.3.6 NKX3-1 Co-factors 39 1.3.3.7 Isolation of NKX3-1 Co-factors 41 1.4 Summary and Statement of Aims 42 Chapter Two – Materials 2.1 Reagents 43 2.1.1 Primers 43 2.1.2 PCR 43 2.1.3 Plasmids 44 2.1.4 Yeast Assays 45 2.1.5 Tissue Culture 45 2.1.6 Western Blotting 45 2.1.7 Immunocytochemistry 46 2.1.8 Immunoprecipitation 46 2.1.9 Confocal 47 2.1.10 RNA Extraction and Northern Blotting 47 2.1.11 General Reagents 47 2.2 Commercial Kits 49 2.3 Equipment 52 Chapter Three – Methods 3.1 Polymerase Chain Reaction (PCR) 55 3.1.1 Primer Design 55 3.1.2 Reverse Transcription (RT) 55 3.1.3 Polymerase Chain Reaction 56 3.1.4 Agarose Gel Electrophoresis 56 3.1.5 Purification of PCR Products and Plasmids 56 3.1.6 Gel Purification of DNA 58 3.2 Cloning of FLJ22318 58 3.2.1 Plasmid Vectors 58 3.2.2 Restriction Enzyme Digestion 65 3.2.3 Calf Intestinal Alkaline Phosphatase Treatment 65 v 3.2.4 Ligation of PCR Products into Plasmid Vectors 65 3.2.5 Preparation of Competent Bacterial Cells 68 3.2.6 Bacterial Transformation 69 3.2.7 Preparation of Bacterial/Glycerol Stocks 70 3.3 Plasmid Purification 70 3.3.1 Small-Scale Isolation of Plasmid DNA 70 3.3.2 Large-Scale Isolation of Plasmid DNA 71 3.3.3 Spectrophotometric Quantitation of DNA/RNA 72 3.4 DNA Sequencing 72 3.4.1 Sequencing Reactions 72 3.4.2 Precipitation of Sequencing Reactions 72 3.5 Yeast Two-hybrid Analysis 73 3.5.1 Polyadenylated RNA Extraction and cDNA Synthesis 73 3.5.1.1 Polyadenylated RNA Extraction 73 3.5.1.2 First-Strand cDNA Synthesis 75 3.5.2 cDNA Library Construction 75 3.5.2.1 Long Distance-PCR (LD-PCR) 75 3.5.2.2 LD-PCR Product Purification 75 3.5.3 Preparation of Competent Yeast Cells 76 3.5.4 Small Scale LiAc Yeast Transformation 77 3.5.5 Large Scale LiAc Yeast Transformation 77 3.5.6 Library Scale LiAc Yeast Transformation 78 3.5.7 Preparation of Yeast/Glycerol Stocks 78 3.5.8 Plasmid Isolation from Yeast 79 3.5.9 Transcriptional Activation of DNA-BD Fusion Construct 79 3.5.10 Toxicity of DNA-BD Fusion Construct 79 3.5.11 Preparation of Yeast Protein Extracts 80 3.6 Tissue Culture 80 3.6.1 Cell Culture Conditions 80 3.6.2 Transfection of Cells 81 3.6.3 Cryopreservation of Cell Cultures 81 3.6.4 DHT Treatment of Cells 83 3.7 Western Blotting 83 vi 3.7.1 Protein Extraction 83 3.7.2 Polyacrylamide Gel Electrophoresis 83 3.7.3 Western Transfer 84 3.7.4 Western Blotting 84 3.8 GST Pull-Down Assays 85 3.8.1 Small-Scale Production of GST Fusion Proteins 85 3.8.2 Coomassie Blue Staining of Gels 86 3.8.3 Large-Scale Production and Purification of GST Fusion Proteins 86 3.8.4 GST Pull-Down Assays 87 3.9 Immunocytochemistry 88 3.9.1 Slide Preparation 88 3.9.2 Immunocytochemistry 88 3.10 Immunoprecipitation Assays 89 3.11 Confocal Microscopy 90 3.11.1 Immunofluorescence 90 3.11.2 Confocal Microscopy 91 3.12 Luciferase Reporter Assays 92 3.13 Northern Blotting 92 3.13.1 RNA Extraction 92 3.13.2 RNA Gel Electrophoresis 94 3.13.3 Northern Transfer 94 3.13.4 Labelling of DNA Probes 94 3.13.5 Northern Hybridisation 95 Chapter Four – Bioinformatic Analysis of FLJ22318 4.1 Introduction 96 4.2 Results 103 4.2.1 FLJ22318 Gene Structure 103 4.2.1.1 Mammalian Gene Homologues of FLJ22318 104 4.2.1.2 Transcriptional Regulation of FLJ22318 Expression 112 4.2.1.3 FLJ22318 Single Nucleotide Polymorphisms 116 4.2.2 FLJ22318 Protein Structure and Homology 116 4.3 Discussion 121 vii Chapter Five – Characterisation of FLJ22318 Interaction with NKX3-1 5.1 Introduction 133 5.2 Results 136 5.2.1 Construction of FLJ22318 Expression Vectors 136 5.2.1.1 Construction of the FLJ22318-pcDNA3.1/V5-His-TOPO Vector 136 5.2.1.1.1 PCR Optimisation with Taq Polymerase 136 5.2.1.1.2 Cloning of FLJ22318 into pcDNA3.1/V5-His-Topo 138 5.2.1.2 Construction of the pGEX2TK-FLJ22318 Vector 142 5.2.1.2.1 PCR
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