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A Novel Approach to Identification of Diagnostic Markers In A NOVEL APPROACH TO IDENTIFICATION OF DIAGNOSTIC MARKERS IN PROSTATE CANCER by INNA SHYSHYNOVA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Professor Andrei Gudkov Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2006 iii TABLE OF CONTENTS CHAPTER .................................................................................................................. PAGE A NOVEL APPROACH TO IDENTIFICATION OF DIAGNOSTIC MARKERS IN PROSTATE CANCER ...........................................................................................1 TABLE OF CONTENTS.................................................................................................. IV LIST OF FIGURES .......................................................................................................... IX LIST OF TABLES............................................................................................................ XI ACKNOWLEDMENTS .................................................................................................XIII LIST OF ABBREVIATIONS........................................................................................ XIV I. INTRODUCTION ...................................................................................................1 1. Tumor markers.............................................................................................1 2. The prostate..................................................................................................5 3. Prostate cancer .............................................................................................5 3.1 Diagnosis of prostate cancer ............................................................6 3.2 Prostate cancer treatment strategies.................................................7 4. Prostatic Acid Phosphatase (PAP)...............................................................9 5. Prostate-Specific Antigen (PSA) ...............................................................10 5.1 PSA as prostate cancer marker and its advantages ........................10 5.2 Drawbacks of PSA-based diagnostics of prostate cancer..............11 5.3 PSA is transcriptionally repressed by p53 .....................................12 6. Prostate-Specific Membrane Antigen (PSMA) .........................................15 7. The search for novel markers in prostate cancer .......................................15 7.1 The attributes of an ideal tumor marker.........................................16 7.2 Methods of prostate tumor markers search....................................19 7.3 Potential PCa markers....................................................................20 II. THE GENERAL STRATEGY OF THE STUDY.................................................26 iv III. IDENTIFICATION OF NOVEL MARKERS IN PROSTATE CANCER BY IN SILICO EXPRESSION PROFILING....................................................................29 1. Rationale ....................................................................................................29 2. Introduction................................................................................................29 2.1 General strategy of EST data mining.............................................29 2.2 EST clustering................................................................................30 2.3 Commonly used criteria of gene selection.....................................31 2.3.1 Digital Differential Display (DDD)...................................31 2.3.2 Pool specificity...................................................................32 2.3.3 Guilt By Association (GBA)..............................................32 2.4 A novel in silico profiling approach ..............................................33 3. Materials and Methods...............................................................................33 3.1 Step 1: Assignment of ESTs to their parent transcripts .................33 3.2 Step 2: cDNA library selection and tissue pool assembly .............34 3.3 Step 3: Selection of candidate genes with desired digital expression profile.............................................................................................35 3.3.1 Reconstruction of expression profiles................................35 3.3.2 Selection of potential markers ...........................................36 3.4 Software implementation of the algorithms used ..........................41 3.5 Redundancy elimination ................................................................41 3.6 The relationship between our approach and the previous work ....42 4. Results........................................................................................................43 4.1 The screening summary .................................................................43 4.2 Known cancer markers ..................................................................43 5. Discussion..................................................................................................45 6. Conclusions................................................................................................45 IV. IDENTIFICATION OF NOVEL POTENTIAL PROSTATE CANCER MARKERS BY IN VIVO EXPRESSION PROFILING .......................................47 1. Rationale ....................................................................................................47 2. Introduction................................................................................................47 2.1 Tumor-suppressor genes: human tumors and mouse models ........48 3. Materials and Methods...............................................................................49 3.1 Transgenic animals ........................................................................49 v 3.2 RNA isolation ................................................................................50 3.3 Probes preparation and microarray hybridizations ........................50 3.4 Microarray data analysis and pre-candidate gene selection...........50 4. Identification of the human homologs for the selected mouse genes: the BI-HUMANIZER software .......................................................................51 4.1 The inner workings of BI-HUMANIZER......................................52 5. Results........................................................................................................56 5.1 Genes upregulated in the murine prostate after tumor suppressor inactivation.....................................................................................56 5.2 BI-HUMANIZER results...............................................................58 5.3 Known cancer markers ..................................................................58 6. Discussion..................................................................................................60 7. Conclusions................................................................................................60 V. VERIFICATION OF THE SELECTED PRE-CANDIDATE GENES BY MICROARRAY HYBRIDIZATION....................................................................61 1. Rationale ....................................................................................................61 2. Introduction................................................................................................61 3. Materials and Methods...............................................................................62 3.1 cDNA clone selection and ordering...............................................62 3.2 Microarray printing......................................................................127 3.3 Cell culture sources for hybridization probe preparation ............127 3.4 Probe preparation and microarray hybridization .........................127 3.5 Microarray data analysis..............................................................130 3.5.1 Quality control .................................................................130 3.5.2 Normalization ..................................................................130 3.5.3 Cluster analysis ................................................................131 3.5.4 The selection of candidate genes for validation...............131 4. Results......................................................................................................131 4.1 Cluster analysis of expression across samples.............................131 4.2 Cluster analysis of expression across genes.................................132 4.3 Selection of candidates for validation of expression profiles ......134 5. Discussion................................................................................................134 6. Conclusions..............................................................................................136 vi VI. VALIDATION OF THE CANDIDATES’ EXPRESSION PATTERNS ...........138 1. Rationale ..................................................................................................138 2. Introduction..............................................................................................138 3. Semi-quantitative RT-PCR ......................................................................138 3.1 Materials and Methods.................................................................138 3.2 Results..........................................................................................139 4. Northern blot analysis..............................................................................142
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